@article {1469585,
	title = {A human organoid system that self-organizes to recapitulate growth and differentiation of a benign mammary tumor},
	journal = {Proc Natl Acad Sci U S A},
	volume = {116},
	number = {23},
	year = {2019},
	month = {2019 Jun 04},
	pages = {11444-11453},
	abstract = {As 3D culture has become central to investigation of tissue biology, mammary epithelial organoids have emerged as powerful tools for investigation of epithelial cell polarization and carcinogenesis. However, most current protocols start from single cells suspended in Matrigel, which can also restrict cell differentiation and behavior. Here, we show that the noncancerous mammary cell line HMT-3522 S1, when allowed to spontaneously form cell aggregates ("spheroids") in medium without Matrigel, switches to a collective growth mode that recapitulates many attributes of "usual ductal hyperplasia" (UDH), a common benign mammary lesion. Interestingly, these spheroids undergo a complex maturation process reminiscent of embryonic development: solid-cell cords form their own basement membrane, grow on the surface of initially homogeneous cell aggregates, and form asymmetric lumina lined by two distinct cell types that express basal and luminal cytokeratins. This sequence of events provides a cellular mechanism that explains how the characteristic crescent-shaped, asymmetrical lumina form in UDH. Our results suggest that HMT-3522 S1 spheroids are useful as an in vitro model system to study UDH biology, glandular lumen formation, and stem cell biology of the mammary gland.},
	issn = {1091-6490},
	doi = {10.1073/pnas.1702372116},
	author = {Florian, Stefan and Iwamoto, Yoshiko and Coughlin, Margaret and Weissleder, Ralph and Mitchison, Timothy J.}
}
@article {1433283,
	title = {Targeted degradation of aberrant tau in frontotemporal dementia patient-derived neuronal cell models},
	journal = {Elife},
	volume = {8},
	year = {2019},
	month = {2019 Mar 25},
	abstract = {Tauopathies are neurodegenerative diseases characterized by aberrant forms of tau protein accumulation leading to neuronal death in focal brain areas. Positron emission tomography (PET) tracers that bind to pathological tau are used in diagnosis, but there are no current therapies to eliminate these tau species. We employed targeted protein degradation technology to convert a tau PET-probe into a functional degrader of pathogenic tau. The hetero-bifunctional molecule QC-01-175 was designed to engage both tau and Cereblon (CRBN), a substrate-receptor for the E3-ubiquitin ligase CRL4, to trigger tau ubiquitination and proteasomal degradation. QC-01-175 effected clearance of tau in frontotemporal dementia (FTD) patient-derived neuronal cell models, with minimal effect on tau from neurons of healthy controls, indicating specificity for disease-relevant forms. QC-01-175 also rescued stress vulnerability in FTD neurons, phenocopying CRISPR-mediated -knockout. This work demonstrates that aberrant tau in FTD patient-derived neurons is amenable to targeted degradation, representing an important advance for therapeutics.},
	issn = {2050-084X},
	doi = {10.7554/eLife.45457},
	author = {Silva, M Catarina and Ferguson, Fleur M and Cai, Quan and Donovan, Katherine A and Nandi, Ghata and Patnaik, Debasis and Zhang, Tinghu and Huang, Hai-Tsang and Lucente, Diane E and Dickerson, Bradford C and Mitchison, Timothy J. and Fischer, Eric S and Gray, Nathanael S and Haggarty, Stephen J}
}
@article {1433282,
	title = {Tubulin mRNA stability is sensitive to change in microtubule dynamics caused by multiple physiological and toxic cues},
	journal = {PLoS Biol},
	volume = {17},
	number = {4},
	year = {2019},
	month = {2019 Apr},
	pages = {e3000225},
	abstract = {The localization, mass, and dynamics of microtubules are important in many processes. Cells may actively monitor the state of their microtubules and respond to perturbation, but how this occurs outside mitosis is poorly understood. We used gene-expression analysis in quiescent cells to analyze responses to subtle and strong perturbation of microtubules. Genes encoding α-, β, and γ-tubulins (TUBAs, TUBBs, and TUBGs), but not δ- or ε-tubulins (TUBDs or TUBEs), exhibited the strongest differential expression response to microtubule-stabilizing versus destabilizing drugs. Quantitative PCR of exon versus intron sequences confirmed that these changes were caused by regulation of tubulin mRNA stability and not transcription. Using tubulin mRNA stability as a signature to query the Gene Expression Omnibus (GEO) database, we find that tubulin genes respond to toxins known to damage microtubules. Importantly, we find many other experimental perturbations, including multiple signaling and metabolic inputs that trigger tubulin differential expression, suggesting their novel, to our knowledge, role in the regulation of the microtubule cytoskeleton. Mechanistic follow-up confirms that one important physiological signal, phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) activity, indeed regulates tubulin mRNA stability via changes in microtubule dynamics. We propose that tubulin gene expression is regulated as part of many coordinated biological responses, with wide implications in physiology and toxicology. Furthermore, we present a new way to discover microtubule regulation using transcriptomics.},
	issn = {1545-7885},
	doi = {10.1371/journal.pbio.3000225},
	author = {Gasic, Ivana and Boswell, Sarah A and Mitchison, Timothy J.}
}
@article {1423874,
	title = {Unsupervised identification of disease states from high-dimensional physiological and histopathological profiles},
	journal = {Mol Syst Biol},
	volume = {15},
	number = {2},
	year = {2019},
	month = {2019 Feb 19},
	pages = {e8636},
	abstract = {The liver and kidney in mammals play central roles in protecting the organism from xenobiotics and are at high risk of xenobiotic-induced injury. Xenobiotic-induced tissue injury has been extensively studied from both classical histopathological and biochemical perspectives. Here, we introduce a machine-learning approach to analyze toxicological response. Unsupervised characterization of physiological and histological changes in a large toxicogenomic dataset revealed nine discrete toxin-induced disease states, some of which correspond to known pathology, but others were novel. Analysis of dynamics revealed transitions between disease states at constant toxin exposure, mostly toward decreased pathology, implying induction of tolerance. Tolerance correlated with induction of known xenobiotic defense genes and decrease of novel ferroptosis sensitivity biomarkers, suggesting ferroptosis as a druggable driver of tissue pathophysiology. Lastly, mechanism of body weight decrease, a known primary marker for xenobiotic toxicity, was investigated. Combined analysis of food consumption, body weight, and molecular biomarkers indicated that organ injury promotes cachexia by whole-body signaling through Gdf15 and Igf1, suggesting strategies for therapeutic intervention that may be broadly relevant to human disease.},
	issn = {1744-4292},
	doi = {10.15252/msb.20188636},
	author = {Shimada, Kenichi and Mitchison, Timothy J.}
}
@article {1420456,
	title = {Autoregulation and repair in microtubule homeostasis},
	journal = {Curr Opin Cell Biol},
	volume = {56},
	year = {2019},
	month = {2019 Feb},
	pages = {80-87},
	abstract = {Even in the face of damaging insults, most cells maintain stability over time through multiple homeostatic pathways, including maintenance of the microtubule cytoskeleton that is fundamental to numerous cellular processes. The dynamic instability-perpetual growth and shrinkage-is the best-known microtubule regulatory pathway, which allows rapid rebuilding of the microtubule cytoskeleton in response to internal or external cues. Much less investigated is homeostatic regulation through availability of α-β tubulin heterodimers-microtubules{\textquoteright} main building blocks-which influences total mass and dynamic behavior of microtubules. Finally, the most recently discovered is microtubule homeostasis through self-repair, where new GTP-bound tubulin heterodimers replace the lost ones in the microtubule lattice. In this review we try to integrate our current knowledge on how dynamic instability, regulation of tubulin mass, and self-repair work together to achieve microtubule homeostasis.},
	issn = {1879-0410},
	doi = {10.1016/j.ceb.2018.10.003},
	author = {Gasic, Ivana and Mitchison, Timothy J.}
}
@article {1420458,
	title = {Colloid osmotic parameterization and measurement of subcellular crowding},
	journal = {Mol Biol Cell},
	volume = {30},
	number = {2},
	year = {2019},
	month = {2019 Jan 15},
	pages = {173-180},
	abstract = {Crowding of the subcellular environment by macromolecules is thought to promote protein aggregation and phase separation. A challenge is how to parameterize the degree of crowding of the cell interior or artificial solutions that is relevant to these reactions. Here I review colloid osmotic pressure as a crowding metric. This pressure is generated by solutions of macromolecules in contact with pores that are permeable to water and ions but not macromolecules. It generates depletion forces that push macromolecules together in crowded solutions and thus promotes aggregation and phase separation. I discuss measurements of colloid osmotic pressure inside cells using the nucleus, the cytoplasmic gel, and fluorescence resonant energy transfer (FRET) biosensors as osmometers, which return a range of values from 1 to 20 kPa. I argue for a low value, 1-2 kPa, in frog eggs and perhaps more generally. This value is close to the linear range on concentration-pressure curves and is thus not crowded from an osmotic perspective. I discuss the implications of a low crowding pressure inside cells for phase separation biology, buffer design, and proteome evolution. I also discuss a pressure-tension model for nuclear shape, where colloid osmotic pressure generated by nuclear protein import inflates the nucleus.},
	issn = {1939-4586},
	doi = {10.1091/mbc.E18-09-0549},
	author = {Mitchison, T. J.}
}
@article {1420457,
	title = {Site occupancy calibration of taxane pharmacology in live cells and tissues},
	journal = {Proc Natl Acad Sci U S A},
	volume = {115},
	number = {48},
	year = {2018},
	month = {2018 11 27},
	pages = {E11406-E11414},
	abstract = {Drug receptor site occupancy is a central pharmacology parameter that quantitatively relates the biochemistry of drug binding to the biology of drug action. Taxanes and epothilones bind to overlapping sites in microtubules (MTs) and stabilize them. They are used to treat cancer and are under investigation for neurodegeneration. In cells, they cause concentration-dependent inhibition of MT dynamics and perturbation of mitosis, but the degree of site occupancy required to trigger different effects has not been measured. We report a live cell assay for taxane-site occupancy, and relationships between site occupancy and biological effects across four drugs and two cell lines. By normalizing to site occupancy, we were able to quantitatively compare drug activities and cell sensitivities independent of differences in drug affinity and uptake/efflux kinetics. Across all drugs and cells tested, we found that inhibition of MT dynamics, postmitotic micronucleation, and mitotic arrest required successively higher site occupancy. We also found interesting differences between cells and drugs, for example, insensitivity of the spindle assembly checkpoint to site occupancy. By extending our assay to a mouse xenograft tumor model, we estimated the initial site occupancy required for paclitaxel to completely prevent tumor growth as 80\%. The most important cellular action of taxanes for cancer treatment may be formation of micronuclei, which occurs over a broad range of site occupancies.},
	issn = {1091-6490},
	doi = {10.1073/pnas.1800047115},
	author = {Pineda, Javier J and Miller, Miles A and Song, Yuyu and Kuhn, Hallie and Mikula, Hannes and Naren Tallapragada and Weissleder, Ralph and Mitchison, Timothy J.}
}
@article {1345567,
	title = {Assembly of Spindles and Asters in  Egg Extracts},
	journal = {Cold Spring Harb Protoc},
	volume = {2018},
	number = {6},
	year = {2018},
	month = {2018 Jun 01},
	pages = {pdb.prot099796},
	abstract = {Here, we provide methods for assembly of mitotic spindles and interphase asters in  egg extract, and compare them to spindles and asters in the egg and zygote. Classic "cycled" spindles are made by adding sperm nuclei to metaphase-arrested cytostatic factor (CSF) extract and inducing entry into interphase extract to promote nucleus formation and DNA replication. Interphase nuclei are then converted to cycled spindles arrested in metaphase by addition of CSF extract. Kinetochores assemble in this reaction and these spindles can segregate chromosomes. CSF spindles are made by addition of sperm nuclei to CSF extract. They are less physiological and lack functional kinetochores but suffice for some applications. Large interphase asters are prepared by addition of artificial centrosomes or sperm nuclei to actin-intact egg extract. These asters grow rapidly to hundreds of microns in radius by branching microtubule nucleation at the periphery, so the aster as a whole is a network of short, dynamic microtubules. They resemble the sperm aster after fertilization, and the asters that grow out of the poles of the mitotic spindle at anaphase. When interphase asters grow into each other they interact and assemble aster interaction zones at their shared boundary. These zones consist of a line (in extract) or disc (in zygotes) of antiparallel microtubule bundles coated with cytokinesis midzone proteins. Interaction zones block interpenetration of microtubules from the two asters, and signal to the cortex to induce cleavage furrows. Their reconstitution in extract allows dissection of the biophysics of spatially regulated cytokinesis signaling.},
	issn = {1559-6095},
	doi = {10.1101/pdb.prot099796},
	author = {Field, Christine M and Mitchison, Timothy J.}
}
@article {1345566,
	title = {A PDGFRα-driven mouse model of glioblastoma reveals a stathmin1-mediated mechanism of sensitivity to vinblastine},
	journal = {Nat Commun},
	volume = {9},
	number = {1},
	year = {2018},
	month = {2018 08 06},
	pages = {3116},
	abstract = {Glioblastoma multiforme (GBM) is an aggressive primary brain cancer that includes focal amplification of PDGFRα and for which there are no effective therapies. Herein, we report the development of a genetically engineered mouse model of GBM based on autocrine, chronic stimulation of overexpressed PDGFRα, and the analysis of GBM signaling pathways using proteomics. We discover the tubulin-binding protein Stathmin1 (STMN1) as a PDGFRα phospho-regulated target, and that this mis-regulation confers sensitivity to vinblastine (VB) cytotoxicity. Treatment of PDGFRα-positive mouse and a patient-derived xenograft (PDX) GBMs with VB in mice prolongs survival and is dependent on STMN1. Our work reveals a previously unconsidered link between PDGFRα activity and STMN1, and highlight an STMN1-dependent cytotoxic effect of VB in GBM.},
	keywords = {Animals, Antineoplastic Agents, Apoptosis, Breast Neoplasms, Cell Cycle, Cell Survival, Cells, Cultured, Computational Biology, Disease Models, Animal, Drug Resistance, Neoplasm, Female, Gene Expression Regulation, Neoplastic, Glioblastoma, Humans, Magnetic Resonance Imaging, Male, Mice, Neoplasm Transplantation, Phosphorylation, Proteomics, Receptor, Platelet-Derived Growth Factor beta, Signal Transduction, Stathmin, Vinblastine},
	issn = {2041-1723},
	doi = {10.1038/s41467-018-05036-4},
	author = {Jun, Hyun Jung and Appleman, Vicky A and Wu, Hua-Jun and Rose, Christopher M and Pineda, Javier J and Yeo, Alan T and Delcuze, Bethany and Lee, Charlotte and Gyuris, Aron and Haihao Zhu and Woolfenden, Steve and Bronisz, Agnieszka and Nakano, Ichiro and Chiocca, Ennio A and Bronson, Roderick T. and Ligon, Keith L and Sarkaria, Jann N and Gygi, Steve P and Michor, Franziska and Mitchison, Timothy J. and Charest, Al}
}
@article {1345570,
	title = {Prc1E and Kif4A control microtubule organization within and between large  egg asters},
	journal = {Mol Biol Cell},
	volume = {29},
	number = {3},
	year = {2018},
	month = {2018 Feb 01},
	pages = {304-316},
	abstract = {The cleavage furrow in  zygotes is positioned by two large microtubule asters that grow out from the poles of the first mitotic spindle. Where these asters meet at the midplane, they assemble a disk-shaped interaction zone consisting of anti-parallel microtubule bundles coated with chromosome passenger complex (CPC) and centralspindlin that instructs the cleavage furrow. Here we investigate the mechanism that keeps the two asters separate and forms a distinct boundary between them, focusing on the conserved cytokinesis midzone proteins Prc1 and Kif4A. Prc1E, the egg orthologue of Prc1, and Kif4A were recruited to anti-parallel bundles at interaction zones between asters in  egg extracts. Prc1E was required for Kif4A recruitment but not vice versa. Microtubule plus-end growth slowed and terminated preferentially within interaction zones, resulting in a block to interpenetration that depended on both Prc1E and Kif4A. Unexpectedly, Prc1E and Kif4A were also required for radial order of large asters growing in isolation, apparently to compensate for the direction-randomizing influence of nucleation away from centrosomes. We propose that Prc1E and Kif4, together with catastrophe factors, promote "anti-parallel pruning" that enforces radial organization within asters and generates boundaries to microtubule growth between asters.},
	issn = {1939-4586},
	doi = {10.1091/mbc.E17-09-0540},
	author = {Nguyen, P A and Field, C. M. and Mitchison, T. J.}
}
@article {1345568,
	title = {Quantitative Analysis of NAD Synthesis-Breakdown Fluxes},
	journal = {Cell Metab},
	volume = {27},
	number = {5},
	year = {2018},
	month = {2018 May 01},
	pages = {1067-1080.e5},
	abstract = {The redox cofactor nicotinamide adenine dinucleotide (NAD) plays a central role in metabolism and is a substrate for signaling enzymes including poly-ADP-ribose-polymerases (PARPs) and sirtuins. NAD concentration falls during aging, which has triggered intense interest in strategies to boost NAD levels. A limitation in understanding NAD metabolism has been reliance on concentration measurements. Here, we present isotope-tracer methods for NAD flux quantitation. In cell lines, NAD was made from nicotinamide and consumed largely by PARPs and sirtuins. In~vivo, NAD was made from tryptophan selectively in the liver, which then excreted nicotinamide. NAD fluxes varied widely across tissues, with high flux in the small intestine and spleen and low flux in the skeletal muscle. Intravenous administration of nicotinamide riboside or mononucleotide delivered intact molecules to multiple tissues, but the same agents given orally were metabolized to nicotinamide in the liver. Thus, flux analysis can reveal tissue-specific NAD metabolism.},
	issn = {1932-7420},
	doi = {10.1016/j.cmet.2018.03.018},
	author = {Liu, Ling and Su, Xiaoyang and Quinn, William J and Sheng Hui and Krukenberg, Kristin and Frederick, David W and Redpath, Philip and Zhan, Le and Chellappa, Karthikeyani and White, Eileen and Migaud, Marie and Mitchison, Timothy J. and Baur, Joseph A and Rabinowitz, Joshua D}
}
@article {1105441,
	title = {Increased lateral microtubule contact at the cell cortex is sufficient to drive mammalian spindle elongation},
	journal = {Mol Biol Cell},
	year = {2017},
	month = {2017 May 03},
	abstract = {The spindle is a dynamic structure that changes its architecture and size in response to biochemical and physical cues. For example, a simple physical change, cell confinement, can trigger centrosome separation and increase spindle steady-state length at metaphase. How this occurs is not understood, and is the question we pose here. We find that metaphase and anaphase spindles elongate at the same rate when confined, suggesting that similar elongation forces can be generated independent of biochemical and spindle structural differences. Furthermore, this elongation does not require bipolar spindle architecture or dynamic microtubules. Rather, confinement increases numbers of astral microtubules laterally contacting the cortex, shifting contact geometry from {\textquoteright}end-on{\textquoteright} to {\textquoteright}side-on{\textquoteright}. Astral microtubules engage cortically anchored motors along their length, as demonstrated by outward sliding and buckling after ablation-mediated release from the centrosome. We show that dynein is required for confinement-induced spindle elongation, and chemical and physical centrosome removal demonstrate that astral microtubules are required for such spindle elongation and its maintenance. Together, the data suggest that promoting lateral cortex-microtubule contacts increases dynein-mediated force generation and is sufficient to drive spindle elongation. More broadly, changes in microtubule-to-cortex contact geometry could offer a mechanism for translating changes in cell shape into dramatic intracellular remodeling.},
	issn = {1939-4586},
	doi = {10.1091/mbc.E17-03-0171},
	author = {Guild, Joshua and Ginzberg, Miriam B and Hueschen, Christina L and Mitchison, Timothy J. and Dumont, Sophie}
}
@article {1105436,
	title = {Vimentin fibers orient traction stress},
	journal = {Proc Natl Acad Sci U S A},
	volume = {114},
	number = {20},
	year = {2017},
	month = {2017 May 16},
	pages = {5195-5200},
	abstract = {The intermediate filament vimentin is required for cells to transition from the epithelial state to the mesenchymal state and migrate as single cells; however, little is known about the specific role of vimentin in the regulation of mesenchymal migration. Vimentin is known to have a significantly greater ability to resist stress without breaking in vitro compared with actin or microtubules, and also to increase cell elasticity in vivo. Therefore, we hypothesized that the presence of vimentin could support the anisotropic mechanical strain of single-cell migration. To study this, we fluorescently labeled vimentin with an mEmerald tag using TALEN genome editing. We observed vimentin architecture in migrating human foreskin fibroblasts and found that network organization varied from long, linear bundles, or "fibers," to shorter fragments with a mesh-like organization. We developed image analysis tools employing steerable filtering and iterative graph matching to characterize the fibers embedded in the surrounding mesh. Vimentin fibers were aligned with fibroblast branching and migration direction. The presence of the vimentin network was correlated with 10-fold slower local actin retrograde flow rates, as well as spatial homogenization of actin-based forces transmitted to the substrate. Vimentin fibers coaligned with and were required for the anisotropic orientation of traction stresses. These results indicate that the vimentin network acts as a load-bearing superstructure capable of integrating and reorienting actin-based forces. We propose that vimentin{\textquoteright}s role in cell motility is to govern the alignment of traction stresses that permit single-cell migration.},
	issn = {1091-6490},
	doi = {10.1073/pnas.1614610114},
	author = {Costigliola, Nancy and Ding, Liya and Burckhardt, Christoph J and Han, Sangyoon J and Gutierrez, Edgar and Mota, Andressa and Groisman, Alex and Mitchison, Timothy J. and Danuser, Gaudenz}
}
@article {1062471,
	title = {27 T ultra-high static magnetic field changes orientation and morphology of mitotic spindles in human cells},
	journal = {Elife},
	volume = {6},
	year = {2017},
	month = {2017 Feb 28},
	abstract = {Purified microtubules have been shown to align along the static magnetic field (SMF) in vitro because of their diamagnetic anisotropy. However, whether mitotic spindle in cells can be aligned by magnetic field has not been experimentally proved. In particular, the biological effects of SMF of above 20 T (Tesla) have never been reported. Here we found that in both CNE-2Z and RPE1 human cells spindle orients in 27 T SMF. The direction of spindle alignment depended on the extent to which chromosomes were aligned to form a planar metaphase plate. Our results show that the magnetic torque acts on both microtubules and chromosomes, and the preferred direction of spindle alignment relative to the field depends more on chromosome alignment than microtubules. In addition, spindle morphology was also perturbed by 27 T SMF. This is the first reported study that investigated the cellular responses to ultra-high magnetic field of above 20 T. Our study not only found that ultra-high magnetic field can change the orientation and morphology of mitotic spindles, but also provided a tool to probe the role of spindle orientation and perturbation in developmental and cancer biology.},
	issn = {2050-084X},
	doi = {10.7554/eLife.22911},
	author = {Zhang, Lei and Hou, Yubin and Li, Zhiyuan and Ji, Xinmiao and Wang, Ze and Wang, Huizhen and Xiaofei Tian and Yu, Fazhi and Yang, Zhenye and Pi, Li and Mitchison, Timothy J. and Lu, Qingyou and Zhang, Xin}
}
@article {1062466,
	title = {Cell death response to anti-mitotic drug treatment in cell culture, mouse tumor model and the clinic},
	journal = {Endocr Relat Cancer},
	year = {2017},
	month = {2017 Mar 01},
	abstract = {Anti-mitotic cancer drugs include classic microtubule-targeting drugs, such as taxanes and vinca alkaloids, and the newer spindle-targeting drugs, such as inhibitors of the motor protein, Kinesin-5 (aka KSP, Eg5, KIF11), and Aurora-A, Aurora-B and Polo-like kinases. Microtubule-targeting drugs are among the first line of chemotherapies for a wide spectrum of cancers, but patient responses vary greatly. We still lack understanding of how these drugs achieve a favorable therapeutic index, and why individual patient responses vary. Spindle-targeting drugs have so far shown disappointing results in the clinic, but it is possible that certain patients could benefit if we understand their mechanism of action better. Pre-clinical data from both cell culture and mouse tumor models showed that the cell death response is the most variable point of the drug action. Hence, in this review we focus on current mechanistic understanding of the cell death response to anti-mitotics. We first draw on extensive results from cell culture studies, and then cross-examine them with the more limited data from animal tumor models and the clinic. We end by discussing how cell-type variation in cell death response might be harnessed to improve anti-mitotic chemotherapy by better patient stratification, new drug combinations and identification of novel targets for drug development.},
	issn = {1479-6821},
	doi = {10.1530/ERC-17-0003},
	author = {Shi, Jue and Mitchison, Timothy J.}
}
@article {1062461,
	title = {Chromosomal passenger complex hydrodynamics suggests chaperoning of the inactive state by nucleoplasmin/nucleophosmin},
	journal = {Mol Biol Cell},
	year = {2017},
	month = {2017 Apr 12},
	abstract = {The chromosomal passenger complex (CPC) is a conserved, essential regulator of cell division. As such, significant anti-cancer drug development efforts have been focused on targeting it, most notably by inhibiting its AURKB kinase subunit. The CPC is activated by AURKB-catalyzed autophosphorylation on multiple subunits, but how this regulates CPC interactions with other mitotic proteins remains unclear. We investigated the hydrodynamic behavior of the CPC in Xenopus laevis egg cytosol using sucrose gradient sedimentation and in HeLa cells using fluorescence correlation spectroscopy (FCS). We found that autophosphorylation of the CPC decreases its sedimentation coefficient (S-value) in egg cytosol and increases its diffusion coefficient in live cells, indicating a decrease in mass. Using immunoprecipitation coupled with mass spectrometry and immunoblots, we discovered that inactive, unphosphorylated CPC interacts with nucleophosmin/nucleoplasmin proteins, which are known to oligomerize into pentamers and decamers. Autophosphorylation of the CPC causes it to dissociate from nucleophosmin/nucleoplasmin. We propose nucleophosmin/nucleoplasmin complexes serve as chaperones that negatively regulate the CPC and/or stabilize its inactive form, preventing CPC autophosphorylation and recruitment to chromatin and microtubules in mitosis.},
	issn = {1939-4586},
	doi = {10.1091/mbc.E16-12-0860},
	author = {Hanley, Mariah L and Yoo, Tae Yeon and Sonnett, Matthew and Needleman, Daniel J and Mitchison, Timothy J.}
}
@article {1062476,
	title = {Xenopus extract approaches to studying microtubule organization and signaling in cytokinesis},
	journal = {Methods Cell Biol},
	volume = {137},
	year = {2017},
	month = {2017},
	pages = {395-435},
	abstract = {We report optimized methods for preparing actin-intact Xenopus egg extract. This extract is minimally perturbed, undiluted egg cytoplasm where the cell cycle can be experimentally controlled. It contains abundant organelles and glycogen~and supports active metabolism and cytoskeletal dynamics that closely mimic egg physiology. The concentration of the most abundant \~{}11,000 proteins is known from mass spectrometry. Actin-intact egg extract can be used for analysis of actin dynamics and interaction of actin with other cytoplasmic systems, as well as microtubule organization. It can be spread as thin layers~and naturally depletes oxygen though mitochondrial metabolism, which makes it ideal for fluorescence imaging. When combined with artificial lipid bilayers, it allows reconstitution and analysis of the spatially controlled signaling that positions the cleavage furrow during early cytokinesis. Actin-intact extract is generally useful for probing the biochemistry and biophysics of the large Xenopus egg. Protocols are provided for preparation of actin-intact egg extract, control of the cell cycle, fluorescent probes for cytoskeleton and cytoskeleton-dependent signaling, preparation of glass surfaces for imaging experiments, and immunodepletion to probe the role of specific proteins and protein complexes. We also describe methods for adding supported lipid bilayers to mimic the plasma membrane~and for confining in microfluidic droplets to explore size scaling issues.},
	issn = {0091-679X},
	doi = {10.1016/bs.mcb.2016.04.014},
	author = {Field, C. M. and Pelletier, J F and Mitchison, T. J.}
}
@article {957066,
	title = {The balbiani body and the concept of physiological amyloids},
	journal = {Cell Cycle},
	volume = {16},
	number = {2},
	year = {2017},
	month = {2017 Jan 17},
	pages = {153-154},
	issn = {1551-4005},
	doi = {10.1080/15384101.2016.1241605},
	author = {Boke, Elvan and Mitchison, Timothy J.}
}
@article {957081,
	title = {Amyloid-like Self-Assembly of a Cellular Compartment},
	journal = {Cell},
	volume = {166},
	number = {3},
	year = {2016},
	month = {2016 Jul 28},
	pages = {637-50},
	abstract = {Most vertebrate oocytes contain a Balbiani body, a large, non-membrane-bound compartment packed with RNA, mitochondria, and other organelles. Little is known about this compartment, though it specifies germline identity in many non-mammalian vertebrates. We show Xvelo, a disordered protein with an N-terminal prion-like domain, is an abundant constituent of Xenopus Balbiani bodies. Disruption of the prion-like domain of Xvelo, or substitution with a prion-like domain from an unrelated protein, interferes with its incorporation into Balbiani bodies in~vivo. Recombinant Xvelo forms amyloid-like networks in~vitro. Amyloid-like assemblies of Xvelo recruit both RNA and mitochondria in binding assays. We propose that Xenopus Balbiani bodies form by amyloid-like assembly of Xvelo, accompanied by co-recruitment of mitochondria and RNA. Prion-like domains are found in germ plasm organizing proteins~in other species, suggesting that Balbiani body formation by amyloid-like assembly could be a conserved mechanism that helps oocytes function as long-lived germ cells.},
	keywords = {Amyloid, Animals, Female, Fluorescent Dyes, Mitochondria, Oocytes, Organelle Biogenesis, Organelles, Prions, Protein Domains, Protein Transport, Recombinant Fusion Proteins, RNA, Messenger, Sf9 Cells, T-Box Domain Proteins, Thiazoles, Xenopus laevis, Xenopus Proteins, Zebrafish},
	issn = {1097-4172},
	doi = {10.1016/j.cell.2016.06.051},
	author = {Boke, Elvan and Ruer, Martine and W{\"u}hr, Martin and Coughlin, Margaret and Lemaitre, Regis and Gygi, Steven P and Alberti, Simon and Drechsel, David and Hyman, Anthony a and Mitchison, Timothy J.}
}
@article {957086,
	title = {Anti-Microtubule Drugs},
	journal = {Methods Mol Biol},
	volume = {1413},
	year = {2016},
	month = {2016},
	pages = {403-21},
	abstract = {Small molecule drugs that target microtubules (MTs), many of them natural products, have long been important tools in the MT field. Indeed, tubulin (Tb) was discovered, in part, as the protein binding partner of colchicine. Several anti-MT drug classes also have important medical uses, notably colchicine, which is used to treat gout, familial Mediterranean fever (FMF), and pericarditis, and the vinca alkaloids and taxanes, which are used to treat cancer. Anti-MT drugs have in common that they bind specifically to Tb in the dimer, MT or some other form. However, their effects on polymerization dynamics and on the human body differ markedly. Here we briefly review the most-studied molecules, and comment on their uses in basic research and medicine. Our focus is on practical applications of different anti-MT drugs in the laboratory, and key points that users should be aware of when designing experiments. We also touch on interesting unsolved problems, particularly in the area of medical applications. In our opinion, the mechanism by which any MT drug cures or treats any disease is still unsolved, despite decades of research. Solving this problem for particular drug-disease combinations might open new uses for old drugs, or provide insights into novel routes for treatment.},
	issn = {1940-6029},
	doi = {10.1007/978-1-4939-3542-0_25},
	author = {Florian, Stefan and Mitchison, Timothy J.}
}
@article {957071,
	title = {Computational imaging reveals mitochondrial morphology as a biomarker of cancer phenotype and drug response},
	journal = {Sci Rep},
	volume = {6},
	year = {2016},
	month = {2016 Sep 09},
	pages = {32985},
	abstract = {Mitochondria, which are essential organelles in resting and replicating cells, can vary in number, mass and shape. Past research has primarily focused on short-term molecular mechanisms underlying fission/fusion. Less is known about longer-term mitochondrial behavior such as the overall makeup of cell populations{\textquoteright} morphological patterns and whether these patterns can be used as biomarkers of drug response in human cells. We developed an image-based analytical technique to phenotype mitochondrial morphology in different cancers, including cancer cell lines and patient-derived cancer cells. We demonstrate that (i) cancer cells of different origins, including patient-derived xenografts, express highly diverse mitochondrial phenotypes; (ii) a given phenotype is characteristic of a cell population and fairly constant over time; (iii) mitochondrial patterns correlate with cell metabolic measurements and (iv) therapeutic interventions can alter mitochondrial phenotypes in drug-sensitive cancers as measured in pre- versus post-treatment fine needle aspirates in mice. These observations shed light on the role of mitochondrial dynamics in the biology and drug response of cancer cells. On the basis of these findings, we propose that image-based mitochondrial phenotyping can provide biomarkers for assessing cancer phenotype and drug response.},
	issn = {2045-2322},
	doi = {10.1038/srep32985},
	author = {Giedt, Randy J and Fumene Feruglio, Paolo and Pathania, Divya and Yang, Katherine S and Kilcoyne, Aoife and Vinegoni, Claudio and Mitchison, Timothy J. and Weissleder, Ralph}
}
@article {957076,
	title = {Mitotic MELK-eIF4B signaling controls protein synthesis and tumor cell survival},
	journal = {Proc Natl Acad Sci U S A},
	volume = {113},
	number = {35},
	year = {2016},
	month = {2016 Aug 30},
	pages = {9810-5},
	abstract = {The protein kinase maternal and embryonic leucine zipper kinase (MELK) is critical for mitotic progression of cancer cells; however, its mechanisms of action remain largely unknown. By combined approaches of immunoprecipitation/mass spectrometry and peptide library profiling, we identified the eukaryotic translation initiation factor 4B (eIF4B) as a MELK-interacting protein during mitosis and a bona fide substrate of MELK. MELK phosphorylates eIF4B at Ser406, a modification found to be most robust in the mitotic phase of the cell cycle. We further show that the MELK-eIF4B signaling axis regulates protein synthesis during mitosis. Specifically, synthesis of myeloid cell leukemia 1 (MCL1), an antiapoptotic protein known to play a role in cancer cell survival during cell division, depends on the function of MELK-elF4B. Inactivation of MELK or eIF4B results in reduced protein synthesis of MCL1, which, in turn, induces apoptotic cell death of cancer cells. Our study thus defines a MELK-eIF4B signaling axis that regulates protein synthesis during mitosis, and consequently influences cancer cell survival.},
	issn = {1091-6490},
	doi = {10.1073/pnas.1606862113},
	author = {Wang, Yubao and Begley, Michael and Li, Qing and Huang, Hai-Tsang and Lako, Ana and Eck, Michael J and Gray, Nathanael S and Mitchison, Timothy J. and Cantley, Lewis C and Zhao, Jean J}
}
@article {957061,
	title = {Physical basis of large microtubule aster growth},
	journal = {Elife},
	volume = {5},
	year = {2016},
	month = {2016 Nov 28},
	abstract = {Microtubule asters - radial arrays of microtubules organized by centrosomes - play a fundamental role in the spatial coordination of animal cells. The standard model of aster growth assumes a fixed number of microtubules originating from the centrosomes. However, aster morphology in this model does not scale with cell size, and we recently found evidence for non-centrosomal microtubule nucleation. Here, we combine autocatalytic nucleation and polymerization dynamics to develop a biophysical model of aster growth. Our model predicts that asters expand as traveling waves and recapitulates all major aspects of aster growth. With increasing nucleation rate, the model predicts an explosive transition from stationary to growing asters with a discontinuous jump of the aster velocity to a nonzero value. Experiments in frog egg extract confirm the main theoretical predictions. Our results suggest that asters observed in large fish and amphibian eggs are a meshwork of short, unstable microtubules maintained by autocatalytic nucleation and provide a paradigm for the assembly of robust and evolvable polymer networks.},
	issn = {2050-084X},
	doi = {10.7554/eLife.19145},
	author = {Ishihara, Keisuke and Korolev, Kirill S and Mitchison, Timothy J.}
}
@article {957091,
	title = {Purification and Fluorescent Labeling of Tubulin from Xenopus laevis Egg Extracts.},
	journal = {Methods Mol Biol},
	volume = {1413},
	year = {2016},
	month = {2016},
	pages = {35-45},
	abstract = {For many years, microtubule research has depended on tubulin purified from cow and pig brains, which may not be ideal for experiments using proteins or extracts from non-brain tissues and cold-blooded organisms. Here, we describe a method to purify functional tubulin from the eggs of the frog, Xenopus laevis. This tubulin has many benefits for the study of microtubules and microtubule based structures assembled in vitro at room temperature. Frog tubulin lacks many of the highly stabilizing posttranslational modifications present in pig brain-derived tubulin, and polymerizes efficiently at room temperature. In addition, fluorescently labeled frog egg tubulin incorporates into meiotic spindles assembled in egg extract more efficiently than brain tubulin, and is thus superior as a probe for Xenopus egg extract experiments. Frog egg tubulin will provide excellent opportunities to identify active nucleation complexes and revisit microtubule polymerization dynamics in vitro.},
	issn = {1940-6029},
	doi = {10.1007/978-1-4939-3542-0_3},
	author = {Groen, Aaron C and Mitchison, Timothy J.}
}
@article {572301,
	title = {Extracellular poly(ADP-ribose) is a pro-inflammatory signal for macrophages.},
	journal = {Chem Biol},
	volume = {22},
	number = {4},
	year = {2015},
	month = {2015 Apr 23},
	pages = {446-52},
	abstract = {Poly(ADP-ribose) polymerase 1 (PARP1) synthesizes poly(ADP-ribose) (PAR), an essential post-translational modification whose function is important in many cellular processes including DNA damage signaling, cell death, and inflammation. All known PAR biology is intracellular, but we suspected it might also play a role in cell-to-cell communication during inflammation. We found that PAR activated cytokine release in human and mouse macrophages, a hallmark of innate immune activation, and determined structure-activity relationships. PAR was rapidly internalized by murine macrophages, while the monomer, ADP-ribose, was not. Inhibitors of Toll-like receptor 2 (TLR2) and TLR4 signaling blocked macrophage responses to PAR, and PAR induced TLR2 and TLR4 signaling in reporter cell lines suggesting it was recognized by these TLRs, much like bacterial pathogens. We propose that PAR acts as an extracellular damage associated molecular pattern that drives inflammatory signaling.},
	issn = {1879-1301},
	doi = {10.1016/j.chembiol.2015.03.007},
	author = {Krukenberg, Kristin A and Kim, Sujeong and Tan, Edwin S and Maliga, Zoltan and Mitchison, Timothy J.}
}
@article {572296,
	title = {In vivo cell-cycle profiling in xenograft tumors by quantitative intravital microscopy.},
	journal = {Nat Methods},
	volume = {12},
	number = {6},
	year = {2015},
	month = {2015 Jun},
	pages = {577-85},
	abstract = {Quantification of cell-cycle state at a single-cell level is essential to understand fundamental three-dimensional (3D) biological processes such as tissue development and cancer. Analysis of 3D in vivo images, however, is very challenging. Today{\textquoteright}s best practice, manual annotation of select image events, generates arbitrarily sampled data distributions, which are unsuitable for reliable mechanistic inferences. Here, we present an integrated workflow for quantitative in vivo cell-cycle profiling. It combines image analysis and machine learning methods for automated 3D segmentation and cell-cycle state identification of individual cell-nuclei with widely varying morphologies embedded in complex tumor environments. We applied our workflow to quantify cell-cycle effects of three antimitotic cancer drugs over 8 d in HT-1080 fibrosarcoma xenografts in living mice using a data set of 38,000 cells and compared the induced phenotypes. In contrast to results with 2D culture, observed mitotic arrest was relatively low, suggesting involvement of additional mechanisms in their antitumor effect in vivo.},
	keywords = {Animals, Cell Cycle, Gene Expression Regulation, Neoplastic, Image Processing, Computer-Assisted, Mice, Microscopy, Neoplasms, Experimental, Transcriptome},
	issn = {1548-7105},
	doi = {10.1038/nmeth.3363},
	author = {Chittajallu, Deepak R and Florian, Stefan and Kohler, Rainer H and Iwamoto, Yoshiko and Orth, James D and Weissleder, Ralph and Danuser, Gaudenz and Mitchison, Timothy J.}
}
@article {572271,
	title = {The Nuclear Proteome of a Vertebrate.},
	journal = {Curr Biol},
	volume = {25},
	number = {20},
	year = {2015},
	month = {2015 Oct 19},
	pages = {2663-71},
	abstract = {The composition of the nucleoplasm determines the behavior of key processes such as transcription, yet there is still no reliable and quantitative resource of nuclear proteins. Furthermore, it is still unclear how the distinct nuclear and cytoplasmic compositions are maintained. To describe the nuclear proteome quantitatively, we isolated the large nuclei of frog oocytes via microdissection and measured the nucleocytoplasmic partitioning of \~{}9,000 proteins by mass spectrometry. Most proteins localize entirely to either nucleus or cytoplasm; only \~{}17\% partition equally. A protein{\textquoteright}s native size in a complex, but not polypeptide molecular weight, is predictive of localization: partitioned proteins exhibit native sizes larger than \~{}100~kDa, whereas natively smaller proteins are equidistributed. To evaluate the role of nuclear export in maintaining localization, we inhibited Exportin 1. This resulted in the expected re-localization of proteins toward the nucleus, but only 3\% of the proteome was affected. Thus, complex assembly and passive retention, rather than continuous active transport, is the dominant mechanism for the maintenance of nuclear and cytoplasmic proteomes.},
	issn = {1879-0445},
	doi = {10.1016/j.cub.2015.08.047},
	author = {W{\"u}hr, Martin and G{\"u}ttler, Thomas and Peshkin, Leonid and McAlister, Graeme C and Sonnett, Matthew and Ishihara, Keisuke and Groen, Aaron C and Presler, Marc and Erickson, Brian K and Mitchison, Timothy J. and Kirschner, Marc W and Gygi, Steven P}
}
@article {572311,
	title = {Quantitative comparison of a human cancer cell surface proteome between interphase and mitosis.},
	journal = {EMBO J},
	volume = {34},
	number = {2},
	year = {2015},
	month = {2015 Jan 13},
	pages = {251-65},
	abstract = {The cell surface is the cellular compartment responsible for communication with the environment. The interior of mammalian cells undergoes dramatic reorganization when cells enter mitosis. These changes are triggered by activation of the CDK1 kinase and have been studied extensively. In contrast, very little is known of the cell surface changes during cell division. We undertook a quantitative proteomic comparison of cell surface-exposed proteins in human cancer cells that were tightly synchronized in mitosis or interphase. Six hundred and twenty-eight surface and surface-associated proteins in HeLa cells were identified; of these, 27 were significantly enriched at the cell surface in mitosis and 37 in interphase. Using imaging techniques, we confirmed the mitosis-selective cell surface localization of protocadherin PCDH7, a member of a family with anti-adhesive roles in embryos. We show that PCDH7 is required for development of full mitotic rounding pressure at the onset of mitosis. Our analysis provided basic information on how cell cycle progression affects the cell surface. It also provides potential pharmacodynamic biomarkers for anti-mitotic cancer chemotherapy.},
	keywords = {Biological Markers, Biotinylation, Cadherins, Chromatography, Affinity, HeLa Cells, Humans, Interphase, MCF-7 Cells, Membrane Proteins, Mitosis, Proteome, Proteomics, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization},
	issn = {1460-2075},
	doi = {10.15252/embj.201385162},
	author = {{\"O}zl{\"u}, Nurhan and Qureshi, Mohammad H and Toyoda, Yusuke and Renard, Bernhard Y and Mollaoglu, G{\"u}rkan and {\"O}zkan, Nazl{\i} E and Bulbul, Selda and Poser, Ina and Timm, Wiebke and Hyman, Anthony a and Mitchison, Timothy J. and Steen, Judith A}
}
@article {572286,
	title = {Size Scaling of Microtubule Assemblies in Early Xenopus Embryos.},
	journal = {Cold Spring Harb Perspect Biol},
	volume = {7},
	number = {10},
	year = {2015},
	month = {2015},
	abstract = {The first 12 cleavage divisions in Xenopus embryos provide a natural experiment in size scaling, as cell radius decreases \~{}16-fold with little change in biochemistry. Analyzing both natural cleavage and egg extract partitioned into droplets revealed that mitotic spindle size scales with cell size, with an upper limit in very large cells. We discuss spindle-size scaling in the small- and large-cell regimes with a focus on the "limiting-component" hypotheses. Zygotes and early blastomeres show a scaling mismatch between spindle and cell size. This problem is solved, we argue, by interphase asters that act to position the spindle and transport chromosomes to the center of daughter cells. These tasks are executed by the spindle in smaller cells. We end by discussing possible mechanisms that limit mitotic aster size and promote interphase aster growth to cell-spanning dimensions.},
	issn = {1943-0264},
	doi = {10.1101/cshperspect.a019182},
	author = {Mitchison, Timothy J. and Ishihara, Keisuke and Nguyen, Phuong and W{\"u}hr, Martin}
}
@article {572276,
	title = {Spindle-to-cortex communication in cleaving, polyspermic Xenopus eggs.},
	journal = {Mol Biol Cell},
	volume = {26},
	number = {20},
	year = {2015},
	month = {2015 Oct 15},
	pages = {3628-40},
	abstract = {Mitotic spindles specify cleavage planes in early embryos by communicating their position and orientation to the cell cortex using microtubule asters that grow out from the spindle poles during anaphase. Chromatin also plays a poorly understood role. Polyspermic fertilization provides a natural experiment in which aster pairs from the same spindle (sister asters) have chromatin between them, whereas asters pairs from different spindles (nonsisters) do not. In frogs, only sister aster pairs induce furrows. We found that only sister asters recruited two conserved furrow-inducing signaling complexes, chromosome passenger complex (CPC) and Centralspindlin, to a plane between them. This explains why only sister pairs induce furrows. We then investigated factors that influenced CPC recruitment to microtubule bundles in intact eggs and a cytokinesis extract system. We found that microtubule stabilization, optimal starting distance between asters, and proximity to chromatin all favored CPC recruitment. We propose a model in which proximity to chromatin biases initial CPC recruitment to microtubule bundles between asters from the same spindle. Next a positive feedback between CPC recruitment and microtubule stabilization promotes lateral growth of a plane of CPC-positive microtubule bundles out to the cortex to position the furrow.},
	issn = {1939-4586},
	doi = {10.1091/mbc.E15-04-0233},
	author = {Field, Christine M and Groen, Aaron C and Nguyen, Phuong A and Mitchison, Timothy J.}
}
@article {572291,
	title = {Using supported bilayers to study the spatiotemporal organization of membrane-bound proteins.},
	journal = {Methods Cell Biol},
	volume = {128},
	year = {2015},
	month = {2015},
	pages = {223-41},
	abstract = {Cell division in prokaryotes and eukaryotes is commonly initiated by the well-controlled binding of proteins to the cytoplasmic side of the cell membrane. However, a precise characterization of the spatiotemporal dynamics of membrane-bound proteins is often difficult to achieve in vivo. Here, we present protocols for the use of supported lipid bilayers to rebuild the cytokinetic machineries of cells with greatly different dimensions: the bacterium Escherichia coli and eggs of the vertebrate Xenopus laevis. Combined with total internal reflection fluorescence microscopy, these experimental setups allow for precise quantitative analyses of membrane-bound proteins. The protocols described to obtain glass-supported membranes from bacterial and vertebrate lipids can be used as starting points for other reconstitution experiments. We believe that similar biochemical assays will be instrumental to study the biochemistry and biophysics underlying a variety of complex cellular tasks, such as signaling, vesicle trafficking, and cell motility.},
	issn = {0091-679X},
	doi = {10.1016/bs.mcb.2015.01.007},
	author = {Nguyen, Phuong A and Field, Christine M and Groen, Aaron C and Mitchison, Timothy J. and Loose, Martin}
}
@article {572381,
	title = {The bacterial cell division proteins FtsA and FtsZ self-organize into dynamic cytoskeletal patterns.},
	journal = {Nat Cell Biol},
	volume = {16},
	number = {1},
	year = {2014},
	month = {2014 Jan},
	pages = {38-46},
	abstract = {Bacterial cytokinesis is commonly initiated by the Z-ring, a cytoskeletal structure that assembles at the site of division. Its primary component is FtsZ, a tubulin superfamily GTPase, which is recruited to the membrane by the actin-related protein FtsA. Both proteins are required for the formation of the Z-ring, but if and how they influence each other{\textquoteright}s assembly dynamics is not known.~Here, we reconstituted FtsA-dependent recruitment of FtsZ polymers to supported membranes, where both proteins self-organize into complex patterns, such as fast-moving filament bundles and chirally rotating rings. Using fluorescence microscopy and biochemical perturbations, we found that these large-scale rearrangements of FtsZ emerge from its polymerization dynamics and a dual, antagonistic role of FtsA: recruitment of FtsZ filaments to the membrane and negative regulation of FtsZ organization. Our findings provide a model for the initial steps of bacterial cell division and illustrate how dynamic polymers can self-organize into large-scale structures.},
	keywords = {Bacterial Proteins, Cell Division, Cell Membrane, Cytoskeletal Proteins, Cytoskeleton, Escherichia coli K12, Escherichia coli Proteins, Models, Biological, Polymerization, Protein Binding},
	issn = {1476-4679},
	doi = {10.1038/ncb2885},
	author = {Loose, Martin and Mitchison, Timothy J.}
}
@article {572371,
	title = {Electron microscopy of microtubule cytoskeleton assembly in vitro.},
	journal = {Methods Mol Biol},
	volume = {1117},
	year = {2014},
	month = {2014},
	pages = {259-71},
	abstract = {Cell-free cytoplasm isolated from meiotic Xenopus egg extracts reconstitutes microtubule phenomena in vitro. These crude extracts assemble bipolar meiotic spindles and are readily fractionated for biochemical assays, providing a good tool to dissect molecular mechanism. We developed techniques for immunoelectron microscopy of microtubule structures assembled in perfusion chambers and in solution.},
	keywords = {Animals, Cytoskeleton, Histocytological Preparation Techniques, Microscopy, Electron, Microtubules, Ovum, Xenopus},
	issn = {1940-6029},
	doi = {10.1007/978-1-62703-776-1_12},
	author = {Coughlin, Margaret and Groen, Aaron C and Mitchison, Timothy J.}
}
@article {572341,
	title = {Basal activity of a PARP1-NuA4 complex varies dramatically across cancer cell lines.},
	journal = {Cell Rep},
	volume = {8},
	number = {6},
	year = {2014},
	month = {2014 Sep 25},
	pages = {1808-18},
	abstract = {Poly(ADP-ribose) polymerases (PARPs) catalyze poly(ADP-ribose) addition onto proteins, an important posttranslational modification involved in transcription, DNA damage repair, and stem cell identity. Previous studies established the activation of PARP1 in response to DNA damage, but little is known about PARP1 regulation outside of DNA repair. We developed an assay for measuring PARP activity in cell lysates and found that the basal activity of PARP1 was highly variable across breast cancer cell lines, independent of DNA damage. Sucrose gradient fractionation demonstrated that PARP1 existed in at least three biochemically distinct states in both high- and low-activity lines. A discovered complex containing the NuA4 chromatin-remodeling complex and PARP1 was responsible for high basal PARP1 activity, and NuA4 subunits were required for this activity. These findings present a pathway for PARP1 activation and a direct link between PARP1 and chromatin remodeling outside of the DNA damage response.},
	keywords = {Breast Neoplasms, Cell Line, Tumor, Female, Histone Acetyltransferases, Humans, MCF-7 Cells, Poly(ADP-ribose) Polymerases, Protein Binding, Protein Subunits, RNA Interference, RNA, Small Interfering},
	issn = {2211-1247},
	doi = {10.1016/j.celrep.2014.08.009},
	author = {Krukenberg, Kristin A and Jiang, Ruomu and Steen, Judith A and Mitchison, Timothy J.}
}
@article {572351,
	title = {The engine of microtubule dynamics comes into focus.},
	journal = {Cell},
	volume = {157},
	number = {5},
	year = {2014},
	month = {2014 May 22},
	pages = {1008-10},
	abstract = {In this issue, Alushin et al. report high-resolution structures of three states of the microtubule lattice: GTP-bound, which is stable to depolymerization; unstable GDP-bound; and stable Taxol and GDP-bound. By comparing these structures at near-atomic resolution, they are able to propose a detailed model for how GTP hydrolysis destabilizes the microtubule and thus powers dynamic instability and chromosome movement. Destabilization of cytoskeleton filaments by nucleotide hydrolysis is an important general principle in cell dynamics, and this work represents a major step forward on a problem with a long history.},
	keywords = {Animals, Guanosine Triphosphate, Humans, Microtubules, Tubulin},
	issn = {1097-4172},
	doi = {10.1016/j.cell.2014.05.001},
	author = {Mitchison, T. J.}
}
@article {572361,
	title = {Glycogen-supplemented mitotic cytosol for analyzing Xenopus egg microtubule organization.},
	journal = {Methods Enzymol},
	volume = {540},
	year = {2014},
	month = {2014},
	pages = {417-33},
	abstract = {Undiluted cytoplasmic extract prepared from unfertilized Xenopus laevis eggs by low-speed centrifugation (CSF extracts) is useful for reconstitution of egg microtubule dynamics and meiosis-II spindle organization, but it suffers limitations for biochemical analysis due to abundant particulates. Here, we describe preparation and the use of fully clarified, undiluted mitotic cytosol derived from CSF extract. Addition of glycogen improves the ability of this cytosol to reconstitute microtubule organization, in part through improved energy metabolism. Using fully clarified, glycogen-supplemented mitotic cytosol, we reconstituted (i) stimulation of microtubule polymerization by Ran.GTP (Groen, Coughlin, \& Mitchison, 2011; Ohba, Nakamura, Nishitani, \& Nishimoto, 1999) and (ii) self-organization of highly regular bipolar arrays of taxol-stabilized microtubules that we termed "pineapples" (Mitchison, Nguyen, Coughlin, \& Groen, 2013). Both systems will be useful for biochemical dissection of spindle assembly mechanisms. We also describe reliable small-scale methods for preparing fluorescent antibody probes that can be used for live imaging in egg extract systems as well as standard immunofluorescence.},
	keywords = {Animals, Cytosol, Glycogen, Microscopy, Fluorescence, Microtubules, Mitosis, Ovum, Polymerization, Xenopus laevis},
	issn = {1557-7988},
	doi = {10.1016/B978-0-12-397924-7.00023-6},
	author = {Groen, Aaron C and Ngyuen, Phuong A and Field, Christine M and Ishihara, Keisuke and Mitchison, Timothy J.}
}
@article {572326,
	title = {Hydrolysis of 2{\textquoteright}3{\textquoteright}-cGAMP by ENPP1 and design of nonhydrolyzable analogs.},
	journal = {Nat Chem Biol},
	volume = {10},
	number = {12},
	year = {2014},
	month = {2014 Dec},
	pages = {1043-8},
	abstract = {Agonists of mouse STING (TMEM173) shrink and even cure solid tumors by activating innate immunity; human STING (hSTING) agonists are needed to test this therapeutic hypothesis in humans. The endogenous STING agonist is 2{\textquoteright}3{\textquoteright}-cGAMP, a second messenger that signals the presence of cytosolic double-stranded DNA. We report activity-guided partial purification and identification of ecto-nucleotide pyrophosphatase/phosphodiesterase (ENPP1) to be the dominant 2{\textquoteright}3{\textquoteright}-cGAMP hydrolyzing activity in cultured cells. The hydrolysis activity of ENPP1 was confirmed using recombinant protein and was depleted in tissue extracts and plasma from Enpp1(-/-) mice. We synthesized a hydrolysis-resistant bisphosphothioate analog of 2{\textquoteright}3{\textquoteright}-cGAMP (2{\textquoteright}3{\textquoteright}-cG(s)A(s)MP) that has similar affinity for hSTING in vitro and is ten times more potent at inducing IFN-β secretion from human THP1 monocytes. Studies in mouse Enpp1(-/-) lung fibroblasts indicate that resistance to hydrolysis contributes substantially to its higher potency. 2{\textquoteright}3{\textquoteright}-cG(s)A(s)MP is therefore improved over natural 2{\textquoteright}3{\textquoteright}-cGAMP as a model agonist and has potential as a vaccine adjuvant and cancer therapeutic.},
	keywords = {Animals, Antineoplastic Agents, Cell Line, Tumor, Gene Expression Regulation, Neoplastic, Humans, Hydrolysis, Interferon-beta, Membrane Proteins, Mice, Monocytes, Nucleotides, Cyclic, Organothiophosphorus Compounds, Phosphoric Diester Hydrolases, Pyrophosphatases, Recombinant Proteins, Second Messenger Systems, Signal Transduction},
	issn = {1552-4469},
	doi = {10.1038/nchembio.1661},
	author = {Li, Lingyin and Yin, Qian and Kuss, Pia and Maliga, Zoltan and Mill{\'a}n, Jos{\'e} L and Wu, Hao and Mitchison, Timothy J.}
}
@article {572356,
	title = {MELK is an oncogenic kinase essential for mitotic progression in basal-like breast cancer cells.},
	journal = {Elife},
	volume = {3},
	year = {2014},
	month = {2014},
	pages = {e01763},
	abstract = {Despite marked advances in breast cancer therapy, basal-like breast cancer (BBC), an aggressive subtype of breast cancer usually lacking estrogen and progesterone receptors, remains difficult to treat. In this study, we report the identification of MELK as a novel oncogenic kinase from an in vivo tumorigenesis screen using a kinome-wide open reading frames (ORFs) library. Analysis of clinical data reveals a high level of MELK overexpression in BBC, a feature that is largely dependent on FoxM1, a master mitotic transcription factor that is also found to be highly overexpressed in BBC. Ablation of MELK selectively impairs proliferation of basal-like, but not luminal breast cancer cells both in vitro and in vivo. Mechanistically, depletion of MELK in BBC cells induces caspase-dependent cell death, preceded by defective mitosis. Finally, we find that Melk is not required for mouse development and physiology. Together, these data indicate that MELK is a normally non-essential kinase, but is critical for BBC and thus represents a promising selective therapeutic target for the most aggressive subtype of breast cancer.DOI: http://dx.doi.org/10.7554/eLife.01763.001.},
	issn = {2050-084X},
	doi = {10.7554/eLife.01763},
	author = {Wang, Yubao and Lee, Young-Mi and Baitsch, Lukas and Huang, Alan and Xiang, Yi and Tong, Haoxuan and Lako, Ana and Von, Thanh and Choi, Christine and Lim, Elgene and Min, Junxia and Li, Li and Stegmeier, Frank and Schlegel, Robert and Eck, Michael J and Gray, Nathanael S and Mitchison, Timothy J. and Zhao, Jean J}
}
@article {572346,
	title = {Organization of early frog embryos by chemical waves emanating from centrosomes.},
	journal = {Philos Trans R Soc Lond B Biol Sci},
	volume = {369},
	number = {1650},
	year = {2014},
	month = {2014 Sep 5},
	abstract = {The large cells in early vertebrate development face an extreme physical challenge in organizing their cytoplasm. For example, amphibian embryos have to divide cytoplasm that spans hundreds of micrometres every 30 min according to a precise geometry, a remarkable accomplishment given the extreme difference between molecular and cellular scales in this system. How do the biochemical reactions occurring at the molecular scale lead to this emergent behaviour of the cell as a whole? Based on recent findings, we propose that the centrosome plays a crucial role by initiating two autocatalytic reactions that travel across the large cytoplasm as chemical waves. Waves of mitotic entry and exit propagate out from centrosomes using the Cdk1 oscillator to coordinate the timing of cell division. Waves of microtubule-stimulated microtubule nucleation propagate out to assemble large asters that position spindles for the following mitosis and establish cleavage plane geometry. By initiating these chemical waves, the centrosome rapidly organizes the large cytoplasm during the short embryonic cell cycle, which would be impossible using more conventional mechanisms such as diffusion or nucleation by structural templating. Large embryo cells provide valuable insights to how cells control chemical waves, which may be a general principle for cytoplasmic organization.},
	keywords = {Animals, Anura, Body Patterning, CDC2 Protein Kinase, Cell Cycle, Centrosome, Embryo, Nonmammalian, Microtubules, Models, Biological, Signal Transduction},
	issn = {1471-2970},
	doi = {10.1098/rstb.2013.0454},
	author = {Ishihara, Keisuke and Nguyen, Phuong A and W{\"u}hr, Martin and Groen, Aaron C and Field, Christine M and Mitchison, Timothy J.}
}
@article {572336,
	title = {Quantitative analysis of resistance to natural killer attacks reveals stepwise killing kinetics.},
	journal = {Integr Biol (Camb)},
	volume = {6},
	number = {12},
	year = {2014},
	month = {2014 Dec},
	pages = {1153-61},
	abstract = {Molecular mechanisms can protect cancer cells from immune attacks. At the level of bulk tissue, these survival mechanisms are often indistinguishable and simply appear as reduced cell death. However, by tracking individual cell survival and death times, we found broad variation in the kinetics of immune evasion. In response to attacks by natural killer cells, we observed that some cancer lines exhibited exponential survival time distributions. Slowly killed cancer lines had reduced exponential rate constants. In contrast, a line engineered to express the serpin protein PI-9, which is known to promote resistance to immune killing, exhibited a markedly nonexponential survival time distribution. By following the histories of individual cancer cells with multiplexed reporters, we obtained evidence that two or more immune attacks are likely required to kill serpin-expressing cells. Thus, resistance is a finite and measurable quantity, with a distinct kinetic signature. A quantitative model based on independently measured parameters is consistent with our conclusions.},
	keywords = {Apoptosis, Cell Communication, Cell Survival, Computer Simulation, HeLa Cells, Humans, Immunity, Cellular, Killer Cells, Natural, Kinetics, Models, Immunological, Models, Statistical, Serpins},
	issn = {1757-9708},
	doi = {10.1039/c4ib00096j},
	author = {Choi, Paul J and Mitchison, Timothy J.}
}
@article {572321,
	title = {Single-cell pharmacokinetic imaging reveals a therapeutic strategy to overcome drug resistance to the microtubule inhibitor eribulin.},
	journal = {Sci Transl Med},
	volume = {6},
	number = {261},
	year = {2014},
	month = {2014 Nov 5},
	pages = {261ra152},
	abstract = {Eribulin mesylate was developed as a potent microtubule-targeting cytotoxic agent to treat taxane-resistant cancers, but recent clinical trials have shown that it eventually fails in many patient subpopulations for unclear reasons. To investigate its resistance mechanisms, we developed a fluorescent analog of eribulin with pharmacokinetic (PK) properties and cytotoxic activity across a human cell line panel that are sufficiently similar to the parent drug to study its cellular PK and tissue distribution. Using intravital imaging and automated tracking of cellular dynamics, we found that resistance to eribulin and the fluorescent analog depended directly on the multidrug resistance protein 1 (MDR1). Intravital imaging allowed for real-time analysis of in vivo PK in tumors that were engineered to be spatially heterogeneous for taxane resistance, whereby an MDR1-mApple fusion protein distinguished resistant cells fluorescently. In vivo, MDR1-mediated drug efflux and the three-dimensional tumor vascular architecture were discovered to be critical determinants of drug accumulation in tumor cells. We furthermore show that standard intravenous administration of a third-generation MDR1 inhibitor, HM30181, failed to rescue drug accumulation; however, the same MDR1 inhibitor encapsulated within a nanoparticle delivery system reversed the multidrug-resistant phenotype and potentiated the eribulin effect in vitro and in vivo in mice. Our work demonstrates that in vivo assessment of cellular PK of an anticancer drug is a powerful strategy for elucidating mechanisms of drug resistance in heterogeneous tumors and evaluating strategies to overcome this resistance.},
	keywords = {Animals, Antineoplastic Agents, Benzopyrans, Cell Line, Cell Survival, Chemistry, Pharmaceutical, Dose-Response Relationship, Drug, Drug Carriers, Drug Resistance, Neoplasm, Female, Furans, Humans, Isoquinolines, Ketones, Mice, Nude, Microscopy, Confocal, Microscopy, Fluorescence, Nanoparticles, Neoplasms, P-Glycoproteins, Recombinant Fusion Proteins, Tetrazoles, Transfection, Tubulin Modulators, Xenograft Model Antitumor Assays},
	issn = {1946-6242},
	doi = {10.1126/scitranslmed.3009318},
	author = {Laughney, Ashley M and Kim, Eunha and Sprachman, Melissa M and Miller, Miles A and Kohler, Rainer H and Yang, Katy S and Orth, James D and Mitchison, Timothy J. and Weissleder, Ralph}
}
@article {572331,
	title = {Spatial organization of cytokinesis signaling reconstituted in a cell-free system.},
	journal = {Science},
	volume = {346},
	number = {6206},
	year = {2014},
	month = {2014 Oct 10},
	pages = {244-7},
	abstract = {During animal cell division, the cleavage furrow is positioned by microtubules that signal to the actin cortex at the cell midplane. We developed a cell-free system to recapitulate cytokinesis signaling using cytoplasmic extract from Xenopus eggs. Microtubules grew out as asters from artificial centrosomes and met to organize antiparallel overlap zones. These zones blocked the interpenetration of neighboring asters and recruited cytokinesis midzone proteins, including the chromosomal passenger complex (CPC) and centralspindlin. The CPC was transported to overlap zones, which required two motor proteins, Kif4A and a Kif20A paralog. Using supported lipid bilayers to mimic the plasma membrane, we observed the recruitment of cleavage furrow markers, including an active RhoA reporter, at microtubule overlaps. This system opens further approaches to understanding the biophysics of cytokinesis signaling.},
	keywords = {Animals, Cell Membrane, Cell-Free System, Centrosome, Cytokinesis, DNA-Binding Proteins, Guanosine Triphosphate, Kinesin, Lipid Bilayers, Microtubules, Models, Biological, Nuclear Proteins, rhoA GTP-Binding Protein, Signal Transduction, Xenopus laevis},
	issn = {1095-9203},
	doi = {10.1126/science.1256773},
	author = {Nguyen, Phuong A and Groen, Aaron C and Loose, Martin and Ishihara, Keisuke and W{\"u}hr, Martin and Field, Christine M and Mitchison, Timothy J.}
}
@article {572366,
	title = {Xenopus egg cytoplasm with intact actin.},
	journal = {Methods Enzymol},
	volume = {540},
	year = {2014},
	month = {2014},
	pages = {399-415},
	abstract = {We report optimized methods for preparing Xenopus egg extracts without cytochalasin D, that we term "actin-intact egg extract." These are undiluted egg cytoplasm that contains abundant organelles, and glycogen which supplies energy, and represents the least perturbed cell-free cytoplasm preparation we know of. We used this system to probe cell cycle regulation of actin and myosin-II dynamics (Field et al., 2011), and to reconstitute the large, interphase asters that organize early Xenopus embryos (Mitchison et al., 2012; W{\"u}hr, Tan, Parker, Detrich, \& Mitchison, 2010). Actin-intact Xenopus egg extracts are useful for analysis of actin dynamics, and interaction of actin with other cytoplasmic systems, in a cell-free system that closely mimics egg physiology, and more generally for probing the biochemistry and biophysics of the egg, zygote, and early embryo. Detailed protocols are provided along with assays used to check cell cycle state and tips for handling and storing undiluted egg extracts.},
	keywords = {Actins, Animals, Cell Cycle, Cell-Free System, Cytoplasm, Microscopy, Fluorescence, Ovum, Xenopus laevis},
	issn = {1557-7988},
	doi = {10.1016/B978-0-12-397924-7.00022-4},
	author = {Field, Christine M and Nguyen, Phuong A and Ishihara, Keisuke and Groen, Aaron C and Mitchison, Timothy J.}
}
@article {572316,
	title = {Microtubule nucleation remote from centrosomes may explain how asters span large cells.},
	journal = {Proc Natl Acad Sci U S A},
	volume = {111},
	number = {50},
	year = {2014},
	month = {2014 Dec 16},
	pages = {17715-22},
	abstract = {A major challenge in cell biology is to understand how nanometer-sized molecules can organize micrometer-sized cells in space and time. One solution in many animal cells is a radial array of microtubules called an aster, which is nucleated by a central organizing center and spans the entire cytoplasm. Frog (here Xenopus laevis) embryos are more than 1 mm in diameter and divide with a defined geometry every 30 min. Like smaller cells, they are organized by asters, which grow, interact, and move to precisely position the cleavage planes. It has been unclear whether asters grow to fill the enormous egg by the same mechanism used in smaller somatic cells, or whether special mechanisms are required. We addressed this question by imaging growing asters in a cell-free system derived from eggs, where asters grew to hundreds of microns in diameter. By tracking marks on the lattice, we found that microtubules could slide outward, but this was not essential for rapid aster growth. Polymer treadmilling did not occur. By measuring the number and positions of microtubule ends over time, we found that most microtubules were nucleated away from the centrosome and that interphase egg cytoplasm supported spontaneous nucleation after a time lag. We propose that aster growth is initiated by centrosomes but that asters grow by propagating a wave of microtubule nucleation stimulated by the presence of preexisting microtubules.},
	keywords = {Animals, Cell Size, Cell-Free System, Centrosome, Embryo, Nonmammalian, Microscopy, Fluorescence, Microtubules, Models, Biological, Rheology, Xenopus laevis},
	issn = {1091-6490},
	doi = {10.1073/pnas.1418796111},
	author = {Ishihara, Keisuke and Nguyen, Phuong A and Groen, Aaron C and Field, Christine M and Mitchison, Timothy J.}
}
@article {572431,
	title = {Branching microtubule nucleation in Xenopus egg extracts mediated by augmin and TPX2.},
	journal = {Cell},
	volume = {152},
	number = {4},
	year = {2013},
	month = {2013 Feb 14},
	pages = {768-77},
	abstract = {The microtubules that comprise mitotic spindles in animal cells are nucleated at centrosomes and by spindle assembly factors that are activated in the vicinity of chromatin. Indirect evidence has suggested that microtubules also might be nucleated from pre-existing microtubules throughout the spindle, but this process has not been observed directly. Here, we demonstrate microtubule nucleation from the sides of existing microtubules in meiotic Xenopus egg extracts. Daughter microtubules grow at a low branch angle and with the same polarity as mother filaments. Branching microtubule nucleation requires γ-tubulin and augmin and is stimulated by factors previously implicated in chromatin-stimulated nucleation, guanosine triphosphate(GTP)-bound Ran and its effector, TPX2. Because of the rapid amplification of microtubule numbers and the preservation of microtubule polarity, microtubule-dependent microtubule nucleation is well suited for spindle assembly and maintenance.},
	keywords = {Animals, Cell Cycle Proteins, Meiosis, Microscopy, Microtubule-Associated Proteins, Microtubules, Nuclear Proteins, Ovum, Phosphoproteins, Xenopus laevis, Xenopus Proteins},
	issn = {1097-4172},
	doi = {10.1016/j.cell.2012.12.044},
	author = {Petry, Sabine and Groen, Aaron C and Ishihara, Keisuke and Mitchison, Timothy J. and Vale, Ronald D}
}
@article {572421,
	title = {Maintenance of mitochondrial oxygen homeostasis by cosubstrate compensation.},
	journal = {Biophys J},
	volume = {104},
	number = {6},
	year = {2013},
	month = {2013 Mar 19},
	pages = {1338-48},
	abstract = {Mitochondria maintain a constant rate of aerobic respiration over a wide range of oxygen levels. However, the control strategies underlying oxygen homeostasis are still unclear. Using mathematical modeling, we found that the mitochondrial electron transport chain (ETC) responds to oxygen level changes by undergoing compensatory changes in reduced electron carrier levels. This emergent behavior, which we named cosubstrate compensation (CSC), enables the ETC to maintain homeostasis over a wide of oxygen levels. When performing CSC, our ETC models recapitulated a classic scaling relationship discovered by Chance [Chance B (1965) J. Gen. Physiol. 49:163-165] relating the extent of oxygen homeostasis to the kinetics of mitochondrial electron transport. Analysis of an in silico mitochondrial respiratory system further showed evidence that CSC constitutes the dominant control strategy for mitochondrial oxygen homeostasis during active respiration. Our findings indicate that CSC constitutes a robust control strategy for homeostasis and adaptation in cellular biochemical networks.},
	keywords = {Cell Respiration, Computational Biology, Electron Transport, Homeostasis, Mitochondria, Models, Biological, Oxygen},
	issn = {1542-0086},
	doi = {10.1016/j.bpj.2013.01.030},
	author = {Kueh, Hao Yuan and Niethammer, Philipp and Mitchison, Timothy J.}
}
@article {572426,
	title = {Self-organization of stabilized microtubules by both spindle and midzone mechanisms in Xenopus egg cytosol.},
	journal = {Mol Biol Cell},
	volume = {24},
	number = {10},
	year = {2013},
	month = {2013 May},
	pages = {1559-73},
	abstract = {Previous study of self-organization of Taxol-stabilized microtubules into asters in Xenopus meiotic extracts revealed motor-dependent organizational mechanisms in the spindle. We revisit this approach using clarified cytosol with glycogen added back to supply energy and reducing equivalents. We added probes for NUMA and Aurora B to reveal microtubule polarity. Taxol and dimethyl sulfoxide promote rapid polymerization of microtubules that slowly self-organize into assemblies with a characteristic morphology consisting of paired lines or open circles of parallel bundles. Minus ends align in NUMA-containing foci on the outside, and plus ends in Aurora B-containing foci on the inside. Assemblies have a well-defined width that depends on initial assembly conditions, but microtubules within them have a broad length distribution. Electron microscopy shows that plus-end foci are coated with electron-dense material and resemble similar foci in monopolar midzones in cells. Functional tests show that two key spindle assembly factors, dynein and kinesin-5, act during assembly as they do in spindles, whereas two key midzone assembly factors, Aurora B and Kif4, act as they do in midzones. These data reveal the richness of self-organizing mechanisms that operate on microtubules after they polymerize in meiotic cytoplasm and provide a biochemically tractable system for investigating plus-end organization in midzones.},
	keywords = {Animals, Aurora Kinase B, Cell-Free System, Cytokinesis, Cytosol, Dyneins, Kinesin, Meiosis, Microtubules, Oocytes, Protein Stability, Spindle Apparatus, Time-Lapse Imaging, Xenopus laevis, Xenopus Proteins},
	issn = {1939-4586},
	doi = {10.1091/mbc.E12-12-0850},
	author = {Mitchison, Timothy J. and Nguyen, Phuong and Coughlin, Margaret and Groen, Aaron C}
}
@article {572411,
	title = {Anticancer flavonoids are mouse-selective STING agonists.},
	journal = {ACS Chem Biol},
	volume = {8},
	number = {7},
	year = {2013},
	month = {2013 Jul 19},
	pages = {1396-401},
	abstract = {The flavonoids FAA and DMXAA showed impressive activity against solid tumors in mice but failed clinical trials. They act on a previously unknown molecular target(s) to trigger cytokine release from leukocytes, which causes tumor-specific vascular damage and other antitumor effects. We show that DMXAA is a competitive agonist ligand for mouse STING (stimulator of interferon genes), a receptor for the bacterial PAMP cyclic-di-GMP (c-di-GMP) and an endogenous second messenger cyclic-GMP-AMP. In our structure-activity relationship studies, STING binding affinity and pathway activation activity of four flavonoids correlated with activity in a mouse tumor model measured previously. We propose that STING agonist activity accounts for the antitumor effects of FAA and DMXAA in mice. Importantly, DMXAA does not bind to human STING, which may account for its lack of efficacy or mechanism-related toxicity in man. We propose that STING is a druggable target for a novel innate immune activation mechanism of chemotherapy.},
	keywords = {Animals, Antineoplastic Agents, Flavonoids, Humans, Immunity, Innate, Membrane Proteins, Mice, Molecular Targeted Therapy, Protein Binding, Structure-Activity Relationship, Xanthones},
	issn = {1554-8937},
	doi = {10.1021/cb400264n},
	author = {Kim, Sujeong and Li, Lingyin and Maliga, Zoltan and Yin, Qian and Wu, Hao and Mitchison, Timothy J.}
}
@article {572376,
	title = {Biased migration of confined neutrophil-like cells in asymmetric hydraulic environments.},
	journal = {Proc Natl Acad Sci U S A},
	volume = {110},
	number = {52},
	year = {2013},
	month = {2013 Dec 24},
	pages = {21006-11},
	abstract = {Cells integrate multiple measurement modalities to navigate their environment. Soluble and substrate-bound chemical gradients and physical cues have all been shown to influence cell orientation and migration. Here we investigate the role of asymmetric hydraulic pressure in directional sensing. Cells confined in microchannels identified and chose a path of lower hydraulic resistance in the absence of chemical cues. In a bifurcating channel with asymmetric hydraulic resistances, this choice was preceded by the elaboration of two leading edges with a faster extension rate along the lower resistance channel. Retraction of the "losing" edge appeared to precipitate a final choice of direction. The pressure differences altering leading edge protrusion rates were small, suggesting weak force generation by leading edges. The response to the physical asymmetry was able to override a dynamically generated chemical cue. Motile cells may use this bias as a result of hydraulic resistance, or "barotaxis," in concert with chemotaxis to navigate complex environments.},
	keywords = {Cell Line, Tumor, Cell Movement, Chemotaxis, Humans, Hydrodynamics, Microfluidic Analytical Techniques, Microscopy, Fluorescence, Microspheres, Pressure},
	issn = {1091-6490},
	doi = {10.1073/pnas.1317441110},
	author = {Prentice-Mott, Harrison V and Chang, Chi-Han and Mahadevan, L and Mitchison, Timothy J. and Irimia, Daniel and Shah, Jagesh V}
}
@article {572406,
	title = {Differential determinants of cancer cell insensitivity to antimitotic drugs discriminated by a one-step cell imaging assay.},
	journal = {J Biomol Screen},
	volume = {18},
	number = {9},
	year = {2013},
	month = {2013 Oct},
	pages = {1062-71},
	abstract = {Cancer cells can be drug resistant due to genetic variation at multiple steps in the drug response pathway, including drug efflux pumping, target mutation, and blunted apoptotic response. These are not discriminated by conventional cell survival assays. Here, we report a rapid and convenient high-content cell-imaging assay that measures multiple physiological changes in cells responding to antimitotic small-molecule drugs. Our one-step, no-wash assay uses three dyes to stain living cells and is much more accurate for scoring weakly adherent mitotic and apoptotic cells than conventional antibody-based assays. We profiled responses of 33 cell lines to 8 antimitotic drugs at multiple concentrations and time points using this assay and deposited our data and assay protocols into a public database (http://lincs.hms.harvard.edu/). Our data discriminated between alternative mechanisms that compromise drug sensitivity to paclitaxel and revealed an unexpected bell-shaped dose-response curve for BI2536, a highly selective inhibitor of Polo-like kinases. Our approach can be generalized, is scalable, and should therefore facilitate identification of molecular biomarkers for mechanisms of drug insensitivity in high-throughput screens and other assays.},
	keywords = {Antimitotic Agents, Apoptosis, Benzamides, Cell Cycle Proteins, Cell Line, Tumor, Cell Survival, Dose-Response Relationship, Drug, Drug Discovery, Drug Resistance, Neoplasm, Fluorescent Dyes, Heterocyclic Compounds, 2-Ring, High-Throughput Screening Assays, Humans, Molecular Imaging, Paclitaxel, Protein-Serine-Threonine Kinases, Proto-Oncogene Proteins, Pteridines, Small Molecule Libraries, Structure-Activity Relationship},
	issn = {1552-454X},
	doi = {10.1177/1087057113493804},
	author = {Tang, Yangzhong and Xie, Tiao and Florian, Stefan and Moerke, Nathan and Shamu, Caroline and Benes, Cyril and Mitchison, Timothy J.}
}
@article {572416,
	title = {Imaging burst kinetics and spatial coordination during serial killing by single natural killer cells.},
	journal = {Proc Natl Acad Sci U S A},
	volume = {110},
	number = {16},
	year = {2013},
	month = {2013 Apr 16},
	pages = {6488-93},
	abstract = {Cytotoxic lymphocytes eliminate virus-infected and cancerous cells by immune recognition and killing through the perforin-granzyme pathway. Traditional killing assays measure average target cell lysis at fixed times and high effector:target ratios. Such assays obscure kinetic details that might reveal novel physiology. We engineered target cells to report on granzyme activity, used very low effector:target ratios to observe potential serial killing, and performed low magnification time-lapse imaging to reveal time-dependent statistics of natural killer (NK) killing at the single-cell level. Most kills occurred during serial killing, and a single NK cell killed up to 10 targets over a 6-h assay. The first kill was slower than subsequent kills, especially on poor targets, or when NK signaling pathways were partially inhibited. Spatial analysis showed that sequential kills were usually adjacent. We propose that NK cells integrate signals from the previous and current target, possibly by simultaneous contact. The resulting burst kinetics and spatial coordination may control the activity of NK cells in tissues.},
	keywords = {Cell Death, Cell Engineering, Fluorescence Resonance Energy Transfer, Granzymes, HeLa Cells, Humans, Killer Cells, Natural, Kinetics, MCF-7 Cells, Microscopy, Time-Lapse Imaging},
	issn = {1091-6490},
	doi = {10.1073/pnas.1221312110},
	author = {Choi, Paul J and Mitchison, Timothy J.}
}
@article {572396,
	title = {Micron-scale coherence in interphase chromatin dynamics.},
	journal = {Proc Natl Acad Sci U S A},
	volume = {110},
	number = {39},
	year = {2013},
	month = {2013 Sep 24},
	pages = {15555-60},
	abstract = {Chromatin structure and dynamics control all aspects of DNA biology yet are poorly understood, especially at large length scales. We developed an approach, displacement correlation spectroscopy based on time-resolved image correlation analysis, to map chromatin dynamics simultaneously across the whole nucleus in cultured human cells. This method revealed that chromatin movement was coherent across large regions (4-5 {\textmu}m) for several seconds. Regions of coherent motion extended beyond the boundaries of single-chromosome territories, suggesting elastic coupling of motion over length scales much larger than those of genes. These large-scale, coupled motions were ATP dependent and unidirectional for several seconds, perhaps accounting for ATP-dependent directed movement of single genes. Perturbation of major nuclear ATPases such as DNA polymerase, RNA polymerase II, and topoisomerase II eliminated micron-scale coherence, while causing rapid, local movement to increase; i.e., local motions accelerated but became uncoupled from their neighbors. We observe similar trends in chromatin dynamics upon inducing a direct DNA damage; thus we hypothesize that this may be due to DNA damage responses that physically relax chromatin and block long-distance communication of forces.},
	keywords = {Chromatin, Green Fluorescent Proteins, HeLa Cells, Histones, Humans, Interphase, Models, Molecular, Spectrum Analysis},
	issn = {1091-6490},
	doi = {10.1073/pnas.1220313110},
	author = {Zidovska, Alexandra and Weitz, David A and Mitchison, Timothy J.}
}
@article {572386,
	title = {Multiple mechanisms determine ER network morphology during the cell cycle in Xenopus egg extracts.},
	journal = {J Cell Biol},
	volume = {203},
	number = {5},
	year = {2013},
	month = {2013 Dec 9},
	pages = {801-14},
	abstract = {In metazoans the endoplasmic reticulum (ER) changes during the cell cycle, with the nuclear envelope (NE) disassembling and reassembling during mitosis and the peripheral ER undergoing extensive remodeling. Here we address how ER morphology is generated during the cell cycle using crude and fractionated Xenopus laevis egg extracts. We show that in interphase the ER is concentrated at the microtubule (MT)-organizing center by dynein and is spread by outward extension of ER tubules through their association with plus ends of growing MTs. Fusion of membranes into an ER network is dependent on the guanosine triphosphatase atlastin (ATL). NE assembly requires fusion by both ATL and ER-soluble N-ethyl-maleimide-sensitive factor adaptor protein receptors. In mitotic extracts, the ER converts into a network of sheets connected by ER tubules and loses most of its interactions with MTs. Together, these results indicate that fusion of ER membranes by ATL and interaction of ER with growing MT ends and dynein cooperate to generate distinct ER morphologies during the cell cycle.},
	keywords = {Animals, Cell Cycle, Cell Fractionation, Dyneins, Endoplasmic Reticulum, GTP Phosphohydrolases, Interphase, Intracellular Membranes, Membrane Fusion, Microtubule-Organizing Center, Nuclear Envelope, Xenopus laevis},
	issn = {1540-8140},
	doi = {10.1083/jcb.201308001},
	author = {Wang, Songyu and Romano, Fabian B and Field, Christine M and Mitchison, Tim J and Rapoport, Tom A}
}
@article {572391,
	title = {A question of taste.},
	journal = {Mol Biol Cell},
	volume = {24},
	number = {21},
	year = {2013},
	month = {2013 Nov},
	pages = {3278-80},
	abstract = {A career in science is shaped by many factors, one of the most important being our tastes in research. These typically form early and are shaped by subsequent successes and failures. My tastes run to microscopes, chemistry, and spatial organization of cytoplasm. I will try to identify where they came from, how they shaped my career, and how they continue to evolve. My hope is to inspire young scientists to identify and celebrate their own unique tastes.},
	keywords = {Chemistry, Cytoplasm, History, 20th Century, History, 21st Century, Microscopy, Research, Science, United States},
	issn = {1939-4586},
	doi = {10.1091/mbc.E13-07-0410},
	author = {Mitchison, T. J.}
}
@article {572401,
	title = {Visualization of poly(ADP-ribose) bound to PARG reveals inherent balance between exo- and endo-glycohydrolase activities.},
	journal = {Nat Commun},
	volume = {4},
	year = {2013},
	month = {2013},
	pages = {2164},
	abstract = {Poly-ADP-ribosylation is a post-translational modification that regulates processes involved in genome stability. Breakdown of the poly(ADP-ribose) (PAR) polymer is catalysed by poly(ADP-ribose) glycohydrolase (PARG), whose endo-glycohydrolase activity generates PAR fragments. Here we present the crystal structure of PARG incorporating the PAR substrate. The two terminal ADP-ribose units of the polymeric substrate are bound in exo-mode. Biochemical and modelling studies reveal that PARG acts predominantly as an exo-glycohydrolase. This preference is linked to Phe902 (human numbering), which is responsible for low-affinity binding of the substrate in endo-mode. Our data reveal the mechanism of poly-ADP-ribosylation reversal, with ADP-ribose as the dominant product, and suggest that the release of apoptotic PAR fragments occurs at unusual PAR/PARG ratios.},
	keywords = {Biocatalysis, Conserved Sequence, Crystallography, X-Ray, Glutamic Acid, Glycoside Hydrolases, Humans, Hydrolysis, Models, Molecular, Molecular Dynamics Simulation, Mutagenesis, Poly Adenosine Diphosphate Ribose, Substrate Specificity, Tetrahymena thermophila},
	issn = {2041-1723},
	doi = {10.1038/ncomms3164},
	author = {Barkauskaite, Eva and Brassington, Amy and Tan, Edwin S and Warwicker, Jim and Dunstan, Mark S and Banos, Benito and Lafite, Pierre and Ahel, Marijan and Mitchison, Timothy J. and Ahel, Ivan and Leys, David}
}
@article {572476,
	title = {Midbody assembly and its regulation during cytokinesis.},
	journal = {Mol Biol Cell},
	volume = {23},
	number = {6},
	year = {2012},
	month = {2012 Mar},
	pages = {1024-34},
	abstract = {The midbody is a transient structure that connects two daughter cells at the end of cytokinesis, with the principal function being to localize the site of abscission, which physically separates two daughter cells. Despite its importance, understanding of midbody assembly and its regulation is still limited. Here we describe how the structural composition of the midbody changes during progression throughout cytokinesis and explore the functional implications of these changes. Deriving from midzones, midbodies are organized by a set of microtubule interacting proteins that colocalize to a zone of microtubule overlap in the center. We found that these proteins split into three subgroups that relocalize to different parts of the midbody: the bulge, the dark zone, and the flanking zone. We characterized these relocalizations and defined domain requirements for three key proteins: MKLP1, KIF4, and PRC1. Two cortical proteins-anillin and RhoA-localized to presumptive abscission sites in mature midbodies, where they may regulate the endosomal sorting complex required for transport machinery. Finally, we characterized the role of Plk1, a key regulator of cytokinesis, in midbody assembly. Our findings represent the most detailed description of midbody assembly and maturation to date and may help elucidate how abscission sites are positioned and regulated.},
	keywords = {Cell Division, Cytokinesis, Endosomal Sorting Complexes Required for Transport, HeLa Cells, Humans, Kinesin, Microtubules, Plant Cells},
	issn = {1939-4586},
	doi = {10.1091/mbc.E11-08-0721},
	author = {Hu, Chi-Kuo and Coughlin, Margaret and Mitchison, Timothy J.}
}
@article {572471,
	title = {Plk1 negatively regulates PRC1 to prevent premature midzone formation before cytokinesis.},
	journal = {Mol Biol Cell},
	volume = {23},
	number = {14},
	year = {2012},
	month = {2012 Jul},
	pages = {2702-11},
	abstract = {To achieve mitosis and cytokinesis, microtubules must assemble into distinct structures at different stages of cell division-mitotic spindles to segregate the chromosomes before anaphase and midzones to keep sister genomes apart and guide the cleavage furrow after anaphase. This temporal regulation is believed to involve Cdk1 kinase, which is inactivated in a switch-like way after anaphase. We found that inhibiting Plk1 caused premature assembly of midzones in cells still in metaphase, breaking the temporal regulation of microtubules. The antiparallel microtubule-bundling protein PRC1 plays a key role in organizing the midzone complex. We found that Plk1 negatively regulates PRC1 through phosphorylation of a single site, Thr-602, near the C-terminus of PRC1. We also found that microtubules stimulated Thr-602 phosphorylation by Plk1. This creates a potential negative feedback loop controlling PRC1 activity. It also made the extent of Thr-602 phosphorylation during mitotic arrest dependent on the mechanism of the arresting drug. Unexpectedly, we could not detect a preanaphase regulatory role for Cdk1 sites on PRC1. We suggest that PRC1 is regulated by Plk1, rather than Cdk1 as previously proposed, because its activity must be spatiotemporally regulated both preanaphase and postanaphase, and Cdk1 activity is too binary for this purpose.},
	keywords = {CDC2 Protein Kinase, Cell Cycle Proteins, Cell Line, Tumor, Cytokinesis, HeLa Cells, Humans, Microtubules, Mitosis, Phosphorylation, Protein-Serine-Threonine Kinases, Proto-Oncogene Proteins, Spindle Apparatus},
	issn = {1939-4586},
	doi = {10.1091/mbc.E12-01-0058},
	author = {Hu, Chi-Kuo and Ozl{\"u}, Nurhan and Coughlin, Margaret and Steen, Judith J and Mitchison, Timothy J.}
}
@article {572481,
	title = {The proliferation rate paradox in antimitotic chemotherapy.},
	journal = {Mol Biol Cell},
	volume = {23},
	number = {1},
	year = {2012},
	month = {2012 Jan},
	pages = {1-6},
	abstract = {Cytotoxic cancer chemotherapy drugs are believed to gain selectivity by targeting cells that proliferate rapidly. However, the proliferation rate is low in many chemosensitive human cancers, and it is not clear how a drug that only kills dividing cells could promote tumor regression. Four potential solutions to this "proliferation rate paradox" are discussed for the microtubule-stabilizing drug paclitaxel: drug retention in tumors, killing of quiescent cells, targeting of noncancer cells in the tumor, and bystander effects. Testing these potential mechanisms of drug action will facilitate rational improvement of antimitotic chemotherapy and perhaps cytotoxic chemotherapy more generally.},
	keywords = {Animals, Antimitotic Agents, Bystander Effect, Cell Cycle, Cell Death, Cell Line, Tumor, Cell Proliferation, Humans, Neoplasms},
	issn = {1939-4586},
	doi = {10.1091/mbc.E10-04-0335},
	author = {Mitchison, Timothy J.}
}
@article {572486,
	title = {Prolonged mitotic arrest triggers partial activation of apoptosis, resulting in DNA damage and p53 induction.},
	journal = {Mol Biol Cell},
	volume = {23},
	number = {4},
	year = {2012},
	month = {2012 Feb},
	pages = {567-76},
	abstract = {Mitotic arrest induced by antimitotic drugs can cause apoptosis or p53-dependent cell cycle arrest. It can also cause DNA damage, but the relationship between these events has been unclear. Live, single-cell imaging in human cancer cells responding to an antimitotic kinesin-5 inhibitor and additional antimitotic drugs revealed strong induction of p53 after cells slipped from prolonged mitotic arrest into G1. We investigated the cause of this induction. We detected DNA damage late in mitotic arrest and also after slippage. This damage was inhibited by treatment with caspase inhibitors and by stable expression of mutant, noncleavable inhibitor of caspase-activated DNase, which prevents activation of the apoptosis-associated nuclease caspase-activated DNase (CAD). These treatments also inhibited induction of p53 after slippage from prolonged arrest. DNA damage was not due to full apoptosis, since most cytochrome C was still sequestered in mitochondria when damage occurred. We conclude that prolonged mitotic arrest partially activates the apoptotic pathway. This partly activates CAD, causing limited DNA damage and p53 induction after slippage. Increased DNA damage via caspases and CAD may be an important aspect of antimitotic drug action. More speculatively, partial activation of CAD may explain the DNA-damaging effects of diverse cellular stresses that do not immediately trigger apoptosis.},
	keywords = {Antimitotic Agents, Apoptosis, Cell Cycle Checkpoints, Cell Line, Tumor, Deoxyribonucleases, DNA Damage, Humans, Kinesin, Mitosis, Quinolines, Tumor Suppressor Protein p53},
	issn = {1939-4586},
	doi = {10.1091/mbc.E11-09-0781},
	author = {Orth, James D and Loewer, Alexander and Lahav, Galit and Mitchison, Timothy J.}
}
@article {572461,
	title = {Deformations within moving kinetochores reveal different sites of active and passive force generation.},
	journal = {Science},
	volume = {337},
	number = {6092},
	year = {2012},
	month = {2012 Jul 20},
	pages = {355-8},
	abstract = {Kinetochores mediate chromosome segregation at mitosis. They are thought to contain both active, force-producing and passive, frictional interfaces with microtubules whose relative locations have been unclear. We inferred mechanical deformation within single kinetochores during metaphase oscillations by measuring average separations between fluorescently labeled kinetochore subunits in living cells undergoing mitosis. Inter-subunit distances were shorter in kinetochores moving toward poles than in those moving away. Inter-subunit separation decreased abruptly when kinetochores switched to poleward movement and decreased further when pulling force increased, suggesting that active force generation during poleward movement compresses kinetochores. The data revealed an active force-generating interface within kinetochores and a separate passive frictional interface located at least 20 nanometers away poleward. Together, these interfaces allow persistent attachment with intermittent active force generation.},
	keywords = {Animals, Cell Cycle Proteins, Chromosomal Proteins, Non-Histone, Chromosome Segregation, Kinetochores, Mechanical Processes, Metaphase, Motion, Nuclear Proteins, Potoroidae},
	issn = {1095-9203},
	doi = {10.1126/science.1221886},
	author = {Dumont, Sophie and Salmon, E D and Mitchison, Timothy J.}
}
@article {572451,
	title = {Dopamine receptor D3 regulates endocytic sorting by a Prazosin-sensitive interaction with the coatomer COPI.},
	journal = {Proc Natl Acad Sci U S A},
	volume = {109},
	number = {31},
	year = {2012},
	month = {2012 Jul 31},
	pages = {12485-90},
	abstract = {Macromolecules enter cells by endocytosis and are sorted to different cellular destinations in early/sorting endosomes. The mechanism and regulation of sorting are poorly understood, although transitions between vesicular and tubular endosomes are important. We found that the antihypertensive drug Prazosin inhibits endocytic sorting by an off-target perturbation of the G protein-coupled receptor dopamine receptor D(3) (DRD3). Prazosin is also a potent cytokinesis inhibitor, likely as a consequence of its effects on endosomes. Prazosin stabilizes a normally transient interaction between DRD3 and the coatomer COPI, a complex involved in membrane transport, and shifts endosomal morphology entirely to tubules, disrupting cargo sorting. RNAi depletion of DRD3 alone also inhibits endocytic sorting, indicating a noncanonical role for a G protein-coupled receptor. Prazosin is a powerful tool for rapid and reversible perturbation of endocytic dynamics.},
	keywords = {Adrenergic alpha-1 Receptor Antagonists, Biological Transport, Active, Coat Protein Complex I, Endocytosis, Endosomes, HeLa Cells, Humans, Prazosin, Protein Stability, Receptors, Dopamine D3},
	issn = {1091-6490},
	doi = {10.1073/pnas.1207821109},
	author = {Zhang, Xin and Wang, Wenchao and Bedigian, Anne V and Coughlin, Margaret L and Mitchison, Timothy J. and Eggert, Ulrike S}
}
@article {572441,
	title = {The first World Cell Race.},
	journal = {Curr Biol},
	volume = {22},
	number = {17},
	year = {2012},
	month = {2012 Sep 11},
	pages = {R673-5},
	keywords = {Animals, Cell Movement, Cells, Cultured, Humans, Mice, Rats},
	issn = {1879-0445},
	doi = {10.1016/j.cub.2012.07.052},
	author = {Maiuri, Paolo and Terriac, Emmanuel and Paul-Gilloteaux, Perrine and Vignaud, Timoth{\'e}e and McNally, Krista and Onuffer, James and Thorn, Kurt and Nguyen, Phuong A and Georgoulia, Nefeli and Soong, Daniel and Jayo, Asier and Beil, Nina and Beneke, J{\"u}rgen and Lim, Joleen Chooi Hong and Sim, Chloe Pei-Ying and Chu, Yeh-Shiu and Jim{\'e}nez-Dalmaroni, Andrea and Joanny, Jean-Fran{\c c}ois and Thiery, Jean-Paul and Erfle, Holger and Parsons, Maddy and Mitchison, Timothy J. and Lim, Wendell A and Lennon-Dum{\'e}nil, Ana-Maria and Piel, Matthieu and Th{\'e}ry, Manuel}
}
@article {572436,
	title = {Graduate education. Interdisciplinary graduate training in teaching labs.},
	journal = {Science},
	volume = {338},
	number = {6114},
	year = {2012},
	month = {2012 Dec 21},
	pages = {1542-3},
	keywords = {Curriculum, Education, Graduate, Faculty, Interdisciplinary Communication, Laboratories, Massachusetts, Physiology, Research, Teaching},
	issn = {1095-9203},
	doi = {10.1126/science.1216570},
	author = {Vale, Ronald D and DeRisi, Joseph and Phillips, Rob and Mullins, R Dyche and Waterman, Clare and Mitchison, Timothy J.}
}
@article {572456,
	title = {Large-scale preparation and characterization of poly(ADP-ribose) and defined length polymers.},
	journal = {Anal Biochem},
	volume = {428},
	number = {2},
	year = {2012},
	month = {2012 Sep 15},
	pages = {126-36},
	abstract = {Poly(ADP-ribose) (pADPr) is a large, structurally complex polymer of repeating ADP-ribose units. It is biosynthesized from NAD$^{+}$ by poly(ADP-ribose) polymerases (PARPs) and degraded to ADP-ribose by poly(ADP-ribose) glycohydrolase. pADPr is involved in many cellular processes and exerts biological function through covalent modification and noncovalent binding to specific proteins. Very little is known about molecular recognition and structure-activity relationships for noncovalent interaction between pADPr and its binding proteins, in part because of lack of access to the polymer on a large scale and to units of defined lengths. We prepared polydisperse pADPr from PARP1 and tankyrase 1 at the hundreds of milligram scale by optimizing enzymatic synthesis and scaling up chromatographic purification methods. We developed and calibrated an anion exchange chromatography method to assign pADPr size and scaled it up to purify defined length polymers on the milligram scale. Furthermore, we present a pADPr profiling method to characterize the polydispersity of pADPr produced by PARPs under different reaction conditions and find that substrate proteins affect the pADPr size distribution. These methods will facilitate structural and biochemical studies of pADPr and its binding proteins.},
	keywords = {Animals, Biocatalysis, Biochemistry, Biopolymers, Calibration, Cattle, Chemical Fractionation, Chromatography, High Pressure Liquid, Glycoside Hydrolases, Humans, Poly Adenosine Diphosphate Ribose, Poly(ADP-ribose) Polymerases, Polymerization, Protein Structure, Tertiary, Tankyrases},
	issn = {1096-0309},
	doi = {10.1016/j.ab.2012.06.015},
	author = {Tan, Edwin S and Krukenberg, Kristin A and Mitchison, Timothy J.}
}
@article {572466,
	title = {Pronuclear migration: no attachment? No union, but a futile cycle!},
	journal = {Curr Biol},
	volume = {22},
	number = {10},
	year = {2012},
	month = {2012 May 22},
	pages = {R409-11},
	abstract = {How do pronuclei migrate towards each other? The zebrafish futile cycle gene is shown to encode a maternally expressed membrane protein required for nuclear attachment and migration along the sperm aster.},
	keywords = {Animals, Cell Nucleus, Centrosome, Female, Male, Membrane Proteins, Zebrafish, Zebrafish Proteins, Zygote},
	issn = {1879-0445},
	doi = {10.1016/j.cub.2012.03.062},
	author = {Nguyen, Phuong A and Ishihara, Keisuke and W{\"u}hr, Martin and Mitchison, Timothy J.}
}
@article {572446,
	title = {Vascular disrupting agent drug classes differ in effects on the cytoskeleton.},
	journal = {PLoS One},
	volume = {7},
	number = {7},
	year = {2012},
	month = {2012},
	pages = {e40177},
	abstract = {Vascular disrupting agents (VDAs), anti-cancer drugs that target established tumor blood vessels, fall into two main classes: microtubule targeting drugs, exemplified by combretastatin A4 (CA4), and flavonoids, exemplified by 5,6-dimethylxanthenone-4-acetic acid (DMXAA). Both classes increase permeability of tumor vasculature in mouse models, and DMXAA in particular can cause massive tumor necrosis. The molecular target of CA4 is clearly microtubules. The molecular target(s) of DMXAA remains unclear. It is thought to promote inflammatory signaling in leukocytes, and has been assumed to not target microtubules, though it is not clear from the literature how carefully this assumption has been tested. An earlier flavone analog, flavone acetic acid, was reported to promote mitotic arrest suggesting flavones might possess anti-microtubule activity, and endothelial cells are sensitive to even mild disruption of microtubules. We carefully investigated whether DMXAA directly affects the microtubule or actin cytoskeletons of endothelial cells by comparing effects of CA4 and DMXAA on human umbilical vein endothelial cells (HUVEC) using time-lapse imaging and assays for cytoskeleton integrity. CA4 caused retraction of the cell margin, mitotic arrest and microtubule depolymerization, while DMXAA, up to 500 {\textmu}M, showed none of these effects. DMXAA also had no effect on pure tubulin nucleation and polymerization, unlike CA4. We conclude that DMXAA exhibits no direct anti-microtubule action and thus cleanly differs from CA4 in its mechanism of action at the molecular level.},
	keywords = {Animals, Antineoplastic Agents, Phytogenic, Capillary Permeability, Cell Line, Dose-Response Relationship, Drug, Human Umbilical Vein Endothelial Cells, Humans, Mice, Microtubules, Neoplasms, Experimental, Neovascularization, Pathologic, Stilbenes, Xanthones},
	issn = {1932-6203},
	doi = {10.1371/journal.pone.0040177},
	author = {Kim, Sujeong and Peshkin, Leonid and Mitchison, Timothy J.}
}
@article {572526,
	title = {Active liquid-like behavior of nucleoli determines their size and shape in Xenopus laevis oocytes.},
	journal = {Proc Natl Acad Sci U S A},
	volume = {108},
	number = {11},
	year = {2011},
	month = {2011 Mar 15},
	pages = {4334-9},
	abstract = {For most intracellular structures with larger than molecular dimensions, little is known about the connection between underlying molecular activities and higher order organization such as size and shape. Here, we show that both the size and shape of the amphibian oocyte nucleolus ultimately arise because nucleoli behave as liquid-like droplets of RNA and protein, exhibiting characteristic viscous fluid dynamics even on timescales of < 1 min. We use these dynamics to determine an apparent nucleolar viscosity, and we show that this viscosity is ATP-dependent, suggesting a role for active processes in fluidizing internal contents. Nucleolar surface tension and fluidity cause their restructuring into spherical droplets upon imposed mechanical deformations. Nucleoli exhibit a broad distribution of sizes with a characteristic power law, which we show is a consequence of spontaneous coalescence events. These results have implications for the function of nucleoli in ribosome subunit processing and provide a physical link between activity within a macromolecular assembly and its physical properties on larger length scales.},
	keywords = {Actins, Adenosine Triphosphate, Animals, Cell Nucleolus, Hydrodynamics, Oocytes, Organelle Shape, Organelle Size, Xenopus laevis},
	issn = {1091-6490},
	doi = {10.1073/pnas.1017150108},
	author = {Brangwynne, Clifford P and Mitchison, Timothy J. and Hyman, Anthony a}
}
@article {572521,
	title = {Navitoclax (ABT-263) accelerates apoptosis during drug-induced mitotic arrest by antagonizing Bcl-xL.},
	journal = {Cancer Res},
	volume = {71},
	number = {13},
	year = {2011},
	month = {2011 Jul 1},
	pages = {4518-26},
	abstract = {Combining microtubule-targeting antimitotic drugs with targeted apoptosis potentiators is a promising new chemotherapeutic strategy to treat cancer. In this study, we investigate the cellular mechanism by which navitoclax (previously called ABT-263), a Bcl-2 family inhibitor, potentiates apoptosis triggered by paclitaxel and an inhibitor of kinesin-5 (K5I, also called a KSP inhibitor), across a panel of epithelial cancer lines. By using time-lapse microscopy, we showed that navitoclax has little effect on cell death during interphase, but strongly accelerates apoptosis during mitotic arrest, and greatly increases the fraction of apoptosis-resistant cells that die. By systematically knocking down individual Bcl-2 proteins, we determined that Mcl-1 and Bcl-xL are the primary negative regulators of apoptosis during prolonged mitotic arrest. Mcl-1 levels decrease during mitotic arrest because of an imbalance between synthesis and turnover, and turnover depends in part on the MULE/HUWE1 E3 ligase. The combination of Mcl-1 loss with inhibition of Bcl-xL by navitoclax causes rapid apoptosis in all lines tested. Variation in expression levels of Mcl-1 and Bcl-xL largely determines variation in response to antimitotics alone, and antimitotics combined with navitoclax, across our panel. We concluded that Bcl-xL is a critical target of Bcl-2 family inhibitors for enhancing the lethality of antimitotic drugs in epithelial cancers, and combination treatment with navitoclax and a spindle specific antimitotic, such as a K5I, might be more effective than paclitaxel alone.},
	keywords = {Aniline Compounds, Antineoplastic Combined Chemotherapy Protocols, Apoptosis, Apoptosis Regulatory Proteins, bcl-X Protein, Cell Line, Tumor, Drug Synergism, Gene Knockdown Techniques, HeLa Cells, Humans, Membrane Proteins, Mitosis, Myeloid Cell Leukemia Sequence 1 Protein, Paclitaxel, Proto-Oncogene Proteins, Proto-Oncogene Proteins c-bcl-2, Sulfonamides},
	issn = {1538-7445},
	doi = {10.1158/0008-5472.CAN-10-4336},
	author = {Shi, Jue and Zhou, Yuan and Huang, Hsiao-Chun and Mitchison, Timothy J.}
}
@article {572506,
	title = {Actin behavior in bulk cytoplasm is cell cycle regulated in early vertebrate embryos.},
	journal = {J Cell Sci},
	volume = {124},
	number = {Pt 12},
	year = {2011},
	month = {2011 Jun 15},
	pages = {2086-95},
	abstract = {The mechanical properties of cells change as they proceed through the cell cycle, primarily owing to regulation of actin and myosin II. Most models for cell mechanics focus on actomyosin in the cortex and ignore possible roles in bulk cytoplasm. We explored cell cycle regulation of bulk cytoplasmic actomyosin in Xenopus egg extracts, which is almost undiluted cytoplasm from unfertilized eggs. We observed dramatic gelation-contraction of actomyosin in mitotic (M phase) extract where Cdk1 activity is high, but not in interphase (I-phase) extract. In spread droplets, M-phase extract exhibited regular, periodic pulses of gelation-contraction a few minutes apart that continued for many minutes. Comparing actin nucleation, disassembly and myosin II activity between M-phase and I-phase extracts, we conclude that regulation of nucleation is likely to be the most important for cell cycle regulation. We then imaged F-actin in early zebrafish blastomeres using a GFP-Utrophin probe. Polymerization in bulk cytoplasm around vesicles increased dramatically during mitosis, consistent with enhanced nucleation. We conclude that F-actin polymerization in bulk cytoplasm is cell cycle regulated in early vertebrate embryos and discuss possible biological functions of this regulation.},
	keywords = {Actins, Actomyosin, Animals, Cell Cycle, Cell Division, Cell Movement, Cytoplasm, Cytoskeleton, Interphase, Mitosis, Vertebrates, Xenopus},
	issn = {1477-9137},
	doi = {10.1242/jcs.082263},
	author = {Field, Christine M and W{\"u}hr, Martin and Anderson, Graham A and Kueh, Hao Yuan and Strickland, Devin and Mitchison, Timothy J.}
}
@article {572501,
	title = {Analysis of mitosis and antimitotic drug responses in tumors by in vivo microscopy and single-cell pharmacodynamics.},
	journal = {Cancer Res},
	volume = {71},
	number = {13},
	year = {2011},
	month = {2011 Jul 1},
	pages = {4608-16},
	abstract = {Cancer relies upon frequent or abnormal cell division, but how the tumor microenvironment affects mitotic processes in vivo remains unclear, largely due to the technical challenges of optical access, spatial resolution, and motion. We developed high-resolution in vivo microscopy methods to visualize mitosis in a murine xenograft model of human cancer. Using these methods, we determined whether the single-cell response to the antimitotic drug paclitaxel (Ptx) was the same in tumors as in cell culture, observed the impact of Ptx on the tumor response as a whole, and evaluated the single-cell pharmacodynamics (PD) of Ptx (by in vivo PD microscopy). Mitotic initiation was generally less frequent in tumors than in cell culture, but subsequently it proceeded normally. Ptx treatment caused spindle assembly defects and mitotic arrest, followed by slippage from mitotic arrest, multinucleation, and apoptosis. Compared with cell culture, the peak mitotic index in tumors exposed to Ptx was lower and the tumor cells survived longer after mitotic arrest, becoming multinucleated rather than dying directly from mitotic arrest. Thus, the tumor microenvironment was much less proapoptotic than cell culture. The morphologies associated with mitotic arrest were dose and time dependent, thereby providing a semiquantitative, single-cell measure of PD. Although many tumor cells did not progress through Ptx-induced mitotic arrest, tumor significantly regressed in the model. Our findings show that in vivo microscopy offers a useful tool to visualize mitosis during tumor progression, drug responses, and cell fate at the single-cell level.},
	keywords = {Animals, Antineoplastic Agents, Cell Line, Tumor, Humans, Mice, Mice, Nude, Microscopy, Mitosis, Paclitaxel, Single-Cell Analysis, Tubulin Modulators, Xenograft Model Antitumor Assays},
	issn = {1538-7445},
	doi = {10.1158/0008-5472.CAN-11-0412},
	author = {Orth, James D and Kohler, Rainer H and Foijer, Floris and Sorger, Peter K and Weissleder, Ralph and Mitchison, Timothy J.}
}
@article {572511,
	title = {KIF4 regulates midzone length during cytokinesis.},
	journal = {Curr Biol},
	volume = {21},
	number = {10},
	year = {2011},
	month = {2011 May 24},
	pages = {815-24},
	abstract = {BACKGROUND: Midzones, also called central spindles, are an array of antiparallel microtubules that form during cytokinesis between the separated chromosomes. Midzones can be considered to be platforms that recruit specific proteins and orchestrate cytokinetic events, such as sister nuclei being kept apart, furrow ingression, and abscission. Despite this important role, many aspects of midzone biology remain unknown, including the dynamic organization of midzone microtubules. Investigating midzone microtubule dynamics has been difficult in part because their plus ends are interdigitated and buried in a dense matrix, making them difficult to observe.

RESULT: We employed monopolar cytokinesis to reveal that midzone plus ends appear to be nondynamic. We identified the chromokinesin KIF4 as a negative regulator of midzone plus-end dynamics whose activity controls midzone length but not stability. KIF4 is required to terminate midzone elongation in late anaphase. In the absence of KIF4, midzones elongate abnormally, and their overlap regions are unfocused. Electron-dense material and midbodies are both absent from the elongated midzones, and actin filaments from the furrow cortex are not disassembled after ingression.

CONCLUSION: KIF4-mediated midzone length regulation appears to occur by terminating midzone elongation at a specific time during cytokinesis, making midzones and mitotic spindles differ in their dynamics and length-regulating mechanisms.},
	keywords = {Cytokinesis, HeLa Cells, Humans, Kinesin, Microscopy, Electron, Transmission, Microscopy, Fluorescence, Microtubules, Spindle Apparatus},
	issn = {1879-0445},
	doi = {10.1016/j.cub.2011.04.019},
	author = {Hu, Chi-Kuo and Coughlin, Margaret and Field, Christine M and Mitchison, Timothy J.}
}
@article {572516,
	title = {Live imaging of the cytoskeleton in early cleavage-stage zebrafish embryos.},
	journal = {Methods Cell Biol},
	volume = {101},
	year = {2011},
	month = {2011},
	pages = {1-18},
	abstract = {The large and transparent cells of cleavage-stage zebrafish embryos provide unique opportunities to study cell division and cytoskeletal dynamics in very large animal cells. Here, we summarize recent progress, from our laboratories and others, on live imaging of the microtubule and actin cytoskeletons during zebrafish embryonic cleavage. First, we present simple protocols for extending the breeding competence of zebrafish mating ensembles throughout the day, which ensures a steady supply of embryos in early cleavage, and for mounting these embryos for imaging. Second, we describe a transgenic zebrafish line [Tg(bactin2:HsENSCONSIN17-282-3xEGFP)hm1] that expresses the green fluorescent protein (GFP)-labeled microtubule-binding part of ensconsin (EMTB-3GFP). We demonstrate that the microtubule-based structures of the early cell cycles can be imaged live, with single microtubule resolution and with high contrast, in this line. Microtubules are much more easily visualized using this tagged binding protein rather than directly labeled tubulin (injected Alexa-647-labeled tubulin), presumably due to lower background from probe molecules not attached to microtubules. Third, we illustrate live imaging of the actin cytoskeleton by injection of the actin-binding fragment of utrophin fused to GFP. Fourth, we compare epifluorescence-, spinning-disc-, laser-scanning-, and two-photon-microscopic modalities for live imaging of the microtubule cytoskeleton in early embryos of our EMTB-3GFP-expressing transgenic line. Finally, we discuss future applications and extensions of our methods.},
	keywords = {Actins, Animals, Animals, Genetically Modified, Cytoskeleton, Humans, Microscopy, Fluorescence, Microtubules, Zebrafish},
	issn = {0091-679X},
	doi = {10.1016/B978-0-12-387036-0.00001-3},
	author = {W{\"u}hr, M and Obholzer, N D and Megason, S G and Detrich, H. W. and Mitchison, T. J.}
}
@article {572496,
	title = {Microtubule assembly in meiotic extract requires glycogen.},
	journal = {Mol Biol Cell},
	volume = {22},
	number = {17},
	year = {2011},
	month = {2011 Sep},
	pages = {3139-51},
	abstract = {The assembly of microtubules during mitosis requires many identified components, such as γ-tubulin ring complex (γ-TuRC), components of the Ran pathway (e.g., TPX2, HuRP, and Rae1), and XMAP215/chTOG. However, it is far from clear how these factors function together or whether more factors exist. In this study, we used biochemistry to attempt to identify active microtubule nucleation protein complexes from Xenopus meiotic egg extracts. Unexpectedly, we found both microtubule assembly and bipolar spindle assembly required glycogen, which acted both as a crowding agent and as metabolic source glucose. By also reconstituting microtubule assembly in clarified extracts, we showed microtubule assembly does not require ribosomes, mitochondria, or membranes. Our clarified extracts will provide a powerful tool for activity-based biochemical fractionations for microtubule assembly.},
	keywords = {Animals, Cell Extracts, Cell-Free System, Centrosome, Glycogen, Meiosis, Microtubules, Mitosis, Mutation, Missense, Oocytes, Phosphates, Potassium Compounds, ran GTP-Binding Protein, Xenopus laevis},
	issn = {1939-4586},
	doi = {10.1091/mbc.E11-02-0158},
	author = {Groen, Aaron C and Coughlin, Margaret and Mitchison, Timothy J.}
}
@article {572491,
	title = {Rapid induction of apoptosis during Kinesin-5 inhibitor-induced mitotic arrest in HL60 cells.},
	journal = {Cancer Lett},
	volume = {310},
	number = {1},
	year = {2011},
	month = {2011 Nov 1},
	pages = {15-24},
	abstract = {Small molecule inhibitors of Kinesin-5 (K5Is) that arrest cells in mitosis with monopolar spindles are promising anti-cancer drug candidates. Clinical trials of K5Is revealed dose-limiting neutropenia, or loss of neutrophils, for which the molecular mechanism is unclear. We investigated the effects of a K5I on HL60 cells, a human promyelocytic leukemia cell line that is often used to model dividing neutrophil progenitors in cell culture. We found K5I treatment caused unusually rapid death of HL60 cells exclusively during mitotic arrest. This mitotic death occurred via the intrinsic apoptosis pathway with molecular events that include cytochrome c leakage into the cytoplasm, caspase activation, and Parp1 cleavage. Bcl-2 overexpression protected from death. We probed mitochondrial physiology to find candidate triggers of cytochrome c release, and observed a decrease of membrane potential (ΔΨm) before mitochondrial outer membrane permeabilization (MOMP). Interestingly, this loss of ΔΨm was not blocked by overexpressing Bcl-2, suggesting it might be a cause of Bax/Bak activation, not a consequence. Taken together, these results show that K5I induces intrinsic apoptosis during mitotic arrest in HL60 with loss of ΔΨm as an upstream event of MOMP.},
	keywords = {Adenosine Triphosphatases, Apoptosis, Apoptosis Regulatory Proteins, BH3 Interacting Domain Death Agonist Protein, Blotting, Western, Cell Cycle, Cell Line, Cell Line, Tumor, Cell Survival, Enzyme Inhibitors, HeLa Cells, HL-60 Cells, Humans, Kinesin, Membrane Potential, Mitochondrial, Membrane Proteins, Microscopy, Fluorescence, Mitosis, Proto-Oncogene Proteins, Quinolines, RNA Interference},
	issn = {1872-7980},
	doi = {10.1016/j.canlet.2011.05.024},
	author = {Tang, Yangzhong and Orth, James D and Xie, Tiao and Mitchison, Timothy J.}
}
@article {572586,
	title = {Binding partner switching on microtubules and aurora-B in the mitosis to cytokinesis transition.},
	journal = {Mol Cell Proteomics},
	volume = {9},
	number = {2},
	year = {2010},
	month = {2010 Feb},
	pages = {336-50},
	abstract = {The cytoskeleton globally reorganizes between mitosis (M phase) and cytokinesis (C phase), which presumably requires extensive regulatory changes. To reveal these changes, we undertook a comparative proteomics analysis of cells tightly drug-synchronized in each phase. We identified 25 proteins that bind selectively to microtubules in C phase and identified several novel binding partners including nucleolar and spindle-associated protein. C phase-selective microtubule binding of many of these proteins depended on activity of Aurora kinases as assayed by treatment with the drug VX680. Aurora-B binding partners switched dramatically between M phase to C phase, and we identified several novel C phase-selective Aurora-B binding partners including PRC1, KIF4, and anaphase-promoting complex/cyclosome. Our approach can be extended to other cellular compartments and cell states, and our data provide the first broad biochemical framework for understanding C phase. Concretely, we report a central role for Aurora-B in regulating the C phase cytoskeleton.},
	keywords = {Aurora Kinase B, Aurora Kinases, Cell Cycle Proteins, Cysteine, Cytokinesis, HeLa Cells, Humans, Interphase, Isotope Labeling, Microtubules, Mitosis, Models, Biological, Phosphorylation, Protein Binding, Protein-Serine-Threonine Kinases},
	issn = {1535-9484},
	doi = {10.1074/mcp.M900308-MCP200},
	author = {Ozl{\"u}, Nurhan and Monigatti, Flavio and Renard, Bernhard Y and Field, Christine M and Steen, Hanno and Mitchison, Timothy J. and Steen, Judith J}
}
@article {572566,
	title = {Cell biology: How cilia beat.},
	journal = {Nature},
	volume = {463},
	number = {7279},
	year = {2010},
	month = {2010 Jan 21},
	pages = {308-9},
	keywords = {Axoneme, Cilia, Ciliary Motility Disorders, Dyneins, Flagella, Humans, Models, Biological, Movement, Periodicity},
	issn = {1476-4687},
	doi = {10.1038/463308a},
	author = {Mitchison, T. J. and Mitchison, H M}
}
@article {572551,
	title = {Cell death when the SAC is out of commission.},
	journal = {Cell Cycle},
	volume = {9},
	number = {11},
	year = {2010},
	month = {2010 Jun 1},
	pages = {2049-50},
	keywords = {Antineoplastic Agents, Cdc20 Proteins, Cell Cycle Proteins, Cell Death, Cell Line, Tumor, Humans, Mitosis, Neoplasms, RNA Interference, RNA, Small Interfering, Spindle Apparatus},
	issn = {1551-4005},
	author = {Huang, Hsiao-Chun and Shi, Jue and Orth, James D and Mitchison, Timothy J.}
}
@article {572561,
	title = {Directly probing the mechanical properties of the spindle and its matrix.},
	journal = {J Cell Biol},
	volume = {188},
	number = {4},
	year = {2010},
	month = {2010 Feb 22},
	pages = {481-9},
	abstract = {Several recent models for spindle length regulation propose an elastic pole to pole spindle matrix that is sufficiently strong to bear or antagonize forces generated by microtubules and microtubule motors. We tested this hypothesis using microneedles to skewer metaphase spindles in Xenopus laevis egg extracts. Microneedle tips inserted into a spindle just outside the metaphase plate resulted in spindle movement along the interpolar axis at a velocity slightly slower than microtubule poleward flux, bringing the nearest pole toward the needle. Spindle velocity decreased near the pole, which often split apart slowly, eventually letting the spindle move completely off the needle. When two needles were inserted on either side of the metaphase plate and rapidly moved apart, there was minimal spindle deformation until they reached the poles. In contrast, needle separation in the equatorial direction rapidly increased spindle width as constant length spindle fibers pulled the poles together. These observations indicate that an isotropic spindle matrix does not make a significant mechanical contribution to metaphase spindle length determination.},
	keywords = {Animals, Biomechanical Phenomena, Cross-Linking Reagents, Microtubules, Needles, Spindle Apparatus, Xenopus},
	issn = {1540-8140},
	doi = {10.1083/jcb.200907110},
	author = {Gatlin, Jesse C and Matov, Alexandre and Danuser, Gaudenz and Mitchison, Timothy J. and Salmon, Edward D}
}
@article {572556,
	title = {FRET measurements of kinesin neck orientation reveal a structural basis for processivity and asymmetry.},
	journal = {Proc Natl Acad Sci U S A},
	volume = {107},
	number = {12},
	year = {2010},
	month = {2010 Mar 23},
	pages = {5453-8},
	abstract = {As the smallest and simplest motor enzymes, kinesins have served as the prototype for understanding the relationship between protein structure and mechanochemical function of enzymes in this class. Conventional kinesin (kinesin-1) is a motor enzyme that transports cargo toward the plus end of microtubules by a processive, asymmetric hand-over-hand mechanism. The coiled-coil neck domain, which connects the two kinesin motor domains, contributes to kinesin processivity (the ability to take many steps in a row) and is proposed to be a key determinant of the asymmetry in the kinesin mechanism. While previous studies have defined the orientation and position of microtubule-bound kinesin motor domains, the disposition of the neck coiled-coil remains uncertain. We determined the neck coiled-coil orientation using a multidonor fluorescence resonance energy transfer (FRET) technique to measure distances between microtubules and bound kinesin molecules. Microtubules were labeled with a new fluorescent taxol donor, TAMRA-X-taxol, and kinesin derivatives with an acceptor fluorophore attached at positions on the motor and neck coiled-coil domains were used to reconstruct the positions and orientations of the domains. FRET measurements to positions on the motor domain were largely consistent with the domain orientation determined in previous studies, validating the technique. Measurements to positions on the neck coiled-coil were inconsistent with a radial orientation and instead demonstrated that the neck coiled-coil is parallel to the microtubule surface. The measured orientation provides a structural explanation for how neck surface residues enhance processivity and suggests a simple hypothesis for the origin of kinesin step asymmetry and "limping."},
	keywords = {Animals, Binding Sites, Biophysical Phenomena, Drosophila Proteins, Fluorescence Resonance Energy Transfer, Fluorescent Dyes, Kinesin, Microtubules, Models, Molecular, Molecular Motor Proteins, Paclitaxel, Protein Binding, Protein Multimerization, Protein Structure, Quaternary, Rhodamines},
	issn = {1091-6490},
	doi = {10.1073/pnas.0914924107},
	author = {Martin, Douglas S and Fathi, Reza and Mitchison, Timothy J. and Gelles, Jeff}
}
@article {572541,
	title = {A model for cleavage plane determination in early amphibian and fish embryos.},
	journal = {Curr Biol},
	volume = {20},
	number = {22},
	year = {2010},
	month = {2010 Nov 23},
	pages = {2040-5},
	abstract = {Current models for cleavage plane determination propose that metaphase spindles are positioned and oriented by interactions of their astral microtubules with the cellular cortex, followed by cleavage in the plane of the metaphase plate [1, 2]. We show that in early frog and fish embryos, where cells are unusually large, astral microtubules in metaphase are too short to position and orient the spindle. Rather, the preceding interphase aster centers and orients a pair of centrosomes prior to nuclear envelope breakdown, and the spindle assembles between these prepositioned centrosomes. Interphase asters center and orient centrosomes with dynein-mediated pulling forces. These forces act before astral microtubules contact the cortex; thus, dynein must pull from sites in the cytoplasm, not the cell cortex as is usually proposed for smaller cells. Aster shape is determined by interactions of the expanding periphery with the cell cortex or with an interaction zone that forms between sister-asters in telophase. We propose a model to explain cleavage plane geometry in which the length of astral microtubules is limited by interaction with these boundaries, causing length asymmetries. Dynein anchored in the cytoplasm then generates length-dependent pulling forces, which move and orient centrosomes.},
	keywords = {Animals, Cell Division, Centrosome, Embryo, Nonmammalian, Embryonic Development, Female, Fertilization, Male, Metaphase, Microtubules, Models, Biological, Spermatozoa, Spindle Apparatus, Xenopus laevis, Zebrafish},
	issn = {1879-0445},
	doi = {10.1016/j.cub.2010.10.024},
	author = {W{\"u}hr, Martin and Tan, Edwin S and Parker, Sandra K and Detrich, H William and Mitchison, Timothy J.}
}
@article {572546,
	title = {Quantitative analysis of actin turnover in Listeria comet tails: evidence for catastrophic filament turnover.},
	journal = {Biophys J},
	volume = {99},
	number = {7},
	year = {2010},
	month = {2010 Oct 6},
	pages = {2153-62},
	abstract = {Rapid assembly and disassembly (turnover) of actin filaments in cytoplasm drives cell motility and shape remodeling. While many biochemical processes that facilitate filament turnover are understood in isolation, it remains unclear how they work together to promote filament turnover in cells. Here, we studied cellular mechanisms of actin filament turnover by combining quantitative microscopy with mathematical modeling. Using live cell imaging, we found that actin polymer mass decay in Listeria comet tails is very well fit by a simple exponential. By analyzing candidate filament turnover pathways using stochastic modeling, we found that exponential polymer mass decay is consistent with either slow treadmilling, slow Arp2/3-dissociation, or catastrophic bursts of disassembly, but is inconsistent with acceleration of filament turnover by severing. Imaging of single filaments in Xenopus egg extract provided evidence that disassembly by bursting dominates isolated filament turnover in a cytoplasmic context. Taken together, our results point to a pathway where filaments grow transiently from barbed ends, rapidly terminate growth to enter a long-lived stable state, and then undergo a catastrophic burst of disassembly. By keeping filament lengths largely constant over time, such catastrophic filament turnover may enable cellular actin assemblies to maintain their mechanical integrity as they are turning over.},
	keywords = {Actin Cytoskeleton, Actin-Related Protein 2-3 Complex, Actins, Animals, Cell Extracts, Comet Assay, Listeria, Models, Biological, Molecular Weight, Ovum, Xenopus laevis},
	issn = {1542-0086},
	doi = {10.1016/j.bpj.2010.07.038},
	author = {Kueh, Hao Yuan and Brieher, William M and Mitchison, Timothy J.}
}
@article {572536,
	title = {Remaining mysteries of the cytoplasm.},
	journal = {Mol Biol Cell},
	volume = {21},
	number = {22},
	year = {2010},
	month = {2010 Nov 15},
	pages = {3811-2},
	keywords = {Animals, Biochemistry, Cell Biology, Cytoplasm, Eukaryotic Cells, Prokaryotic Cells},
	issn = {1939-4586},
	doi = {10.1091/mbc.E10-02-0084},
	author = {Mitchison, Timothy J.}
}
@article {572531,
	title = {Stochastic competition between mechanistically independent slippage and death pathways determines cell fate during mitotic arrest.},
	journal = {PLoS One},
	volume = {5},
	number = {12},
	year = {2010},
	month = {2010},
	pages = {e15724},
	abstract = {Variability in cell-to-cell behavior within clonal populations can be attributed to the inherent stochasticity of biochemical reactions. Most single-cell studies have examined variation in behavior due to randomness in gene transcription. Here we investigate the mechanism of cell fate choice and the origin of cell-to-cell variation during mitotic arrest, when transcription is silenced. Prolonged mitotic arrest is commonly observed in cells treated with anti-mitotic drugs. Cell fate during mitotic arrest is determined by two alternative pathways, one promoting cell death, the other promoting cyclin B1 degradation, which leads to mitotic slippage and survival. It has been unclear whether these pathways are mechanistically coupled or independent. In this study we experimentally uncoupled these two pathways using zVAD-fmk to block cell death or Cdc20 knockdown to block slippage. We then used time-lapse imaging to score the kinetics of single cells adopting the remaining fate. We also used kinetic simulation to test whether the behaviors of death versus slippage in cell populations where both pathways are active can be quantitatively recapitulated by a model that assumes stochastic competition between the pathways. Our data are well fit by a model where the two pathways are mechanistically independent, and cell fate is determined by a stochastic kinetic competition between them that results in cell-to-cell variation.},
	keywords = {Amino Acid Chloromethyl Ketones, Antineoplastic Agents, Cdc20 Proteins, Cell Cycle Proteins, Cell Death, Cell Line, Tumor, Cell Lineage, Cyclin B1, Cysteine Proteinase Inhibitors, HeLa Cells, Humans, Kinetics, Mitosis, RNA, Small Interfering, Stochastic Processes, Transcription, Genetic},
	issn = {1932-6203},
	doi = {10.1371/journal.pone.0015724},
	author = {Huang, Hsiao-Chun and Mitchison, Timothy J. and Shi, Jue}
}
@article {572636,
	title = {Functional overlap of microtubule assembly factors in chromatin-promoted spindle assembly.},
	journal = {Mol Biol Cell},
	volume = {20},
	number = {11},
	year = {2009},
	month = {2009 Jun},
	pages = {2766-73},
	abstract = {Distinct pathways from centrosomes and chromatin are thought to contribute in parallel to microtubule nucleation and stabilization during animal cell mitotic spindle assembly, but their full mechanisms are not known. We investigated the function of three proposed nucleation/stabilization factors, TPX2, gamma-tubulin and XMAP215, in chromatin-promoted assembly of anastral spindles in Xenopus laevis egg extract. In addition to conventional depletion-add back experiments, we tested whether factors could substitute for each other, indicative of functional redundancy. All three factors were required for microtubule polymerization and bipolar spindle assembly around chromatin beads. Depletion of TPX2 was partially rescued by the addition of excess XMAP215 or EB1, or inhibiting MCAK (a Kinesin-13). Depletion of either gamma-tubulin or XMAP215 was partially rescued by adding back XMAP215, but not by adding any of the other factors. These data reveal functional redundancy between specific assembly factors in the chromatin pathway, suggesting individual proteins or pathways commonly viewed to be essential may not have entirely unique functions.},
	keywords = {Animals, Cell Cycle Proteins, Chromatin, Female, Green Fluorescent Proteins, Humans, Kinesin, Kinetics, Microscopy, Fluorescence, Microtubule-Associated Proteins, Microtubules, Nuclear Proteins, Oocytes, Phosphoproteins, Spindle Apparatus, Time Factors, Tubulin, Xenopus laevis, Xenopus Proteins},
	issn = {1939-4586},
	doi = {10.1091/mbc.E09-01-0043},
	author = {Groen, Aaron C and Maresca, Thomas J and Gatlin, Jesse C and Salmon, Edward D and Mitchison, Timothy J.}
}
@article {572641,
	title = {How does a millimeter-sized cell find its center?},
	journal = {Cell Cycle},
	volume = {8},
	number = {8},
	year = {2009},
	month = {2009 Apr 15},
	pages = {1115-21},
	abstract = {Microtubules play a central role in centering the nucleus or mitotic spindle in eukaryotic cells. However, despite common use of microtubules for centering, physical mechanisms can vary greatly, and depend on cell size and cell type. In the small fission yeast cells, the nucleus can be centered by pushing forces that are generated when growing microtubules hit the cell boundary. This mechanism may not be possible in larger cells, because the compressive force that microtubules can sustain are limited by buckling, so maximal force decreases with microtubule length. In a well-studied intermediate sized cell, the C. elegans fertilized egg, centrosomes are centered by cortex-attached motors that pull on microtubules. This mechanism is widely assumed to be general for larger cells. However, re-evaluation of classic experiments in a very large cell, the fertilized amphibian egg, argues against such generality. In these large eggs, movement of asters away from a part of the cell boundary that they are touching cannot be mediated by cortical pulling, because the astral microtubules are too short to reach the opposite cell boundary. Additionally, Herlant and Brachet discovered a century ago that multiple asters within a single egg center relative to the cell boundary, but also relative to each other. Here, we summarize current understanding of microtubule organization during the first cell cycle in a fertilized Xenopus egg, discuss how microtubule asters move towards the center of this very large cell, and how multiple asters shape and position themselves relative to each other.},
	keywords = {Animals, Cell Cycle, Microtubules, Spindle Apparatus, Xenopus, Zygote},
	issn = {1551-4005},
	author = {W{\"u}hr, Martin and Dumont, Sophie and Groen, Aaron C and Needleman, Daniel J and Mitchison, Timothy J.}
}
@article {572621,
	title = {Pin-hole array correlation imaging: highly parallel fluorescence correlation spectroscopy.},
	journal = {Biophys J},
	volume = {96},
	number = {12},
	year = {2009},
	month = {2009 Jun 17},
	pages = {5050-9},
	abstract = {In this work, we describe pin-hole array correlation imaging, a multipoint version of fluorescence correlation spectroscopy, based upon a stationary Nipkow disk and a high-speed electron multiplying charged coupled detector. We characterize the system and test its performance on a variety of samples, including 40 nm colloids, a fluorescent protein complex, a membrane dye, and a fluorescence fusion protein. Our results demonstrate that pin-hole array correlation imaging is capable of simultaneously performing tens or hundreds of fluorescence correlation spectroscopy-style measurements in cells, with sufficient sensitivity and temporal resolution to study the behaviors of membrane-bound and soluble molecules labeled with conventional chemical dyes or fluorescent proteins.},
	keywords = {Colloids, Phycoerythrin, Spectrometry, Fluorescence},
	issn = {1542-0086},
	doi = {10.1016/j.bpj.2009.03.023},
	author = {Needleman, Daniel J and Xu, Yangqing and Mitchison, Timothy J.}
}
@article {572631,
	title = {Size and speed go hand in hand in cytokinesis.},
	journal = {Cell},
	volume = {137},
	number = {5},
	year = {2009},
	month = {2009 May 29},
	pages = {798-800},
	abstract = {In animal cells, cytokinesis is mediated by the constriction of a cortical ring. In this issue, Carvalho et al. (2009) show in embryos of the worm Caenorhabditis elegans that the rate of ring constriction during cytokinesis is proportional to the initial cell perimeter, ensuring that the duration of cytokinesis is cell-size independent.},
	keywords = {Animals, Caenorhabditis elegans, Cell Size, Cytokinesis, Embryo, Nonmammalian},
	issn = {1097-4172},
	doi = {10.1016/j.cell.2009.05.012},
	author = {W{\"u}hr, Martin and Mitchison, Timothy J. and Field, Christine M}
}
@article {572646,
	title = {Spindle fusion requires dynein-mediated sliding of oppositely oriented microtubules.},
	journal = {Curr Biol},
	volume = {19},
	number = {4},
	year = {2009},
	month = {2009 Feb 24},
	pages = {287-96},
	abstract = {BACKGROUND: Bipolar spindle assembly is critical for achieving accurate segregation of chromosomes. In the absence of centrosomes, meiotic spindles achieve bipolarity by a combination of chromosome-initiated microtubule nucleation and stabilization and motor-driven organization of microtubules. Once assembled, the spindle structure is maintained on a relatively long time scale despite the high turnover of the microtubules that comprise it. To study the underlying mechanisms responsible for spindle assembly and steady-state maintenance, we used microneedle manipulation of preassembled spindles in Xenopus egg extracts.

RESULTS: When two meiotic spindles were brought close enough together, they interacted, creating an interconnected microtubule structure with supernumerary poles. Without exception, the perturbed system eventually re-established bipolarity, forming a single spindle of normal shape and size. Bipolar spindle fusion was blocked when cytoplasmic dynein function was perturbed, suggesting a critical role for the motor in this process. The fusion of Eg5-inhibited monopoles also required dynein function but only occurred if the initial interpolar separation was less than twice the microtubule radius of a typical monopole.

CONCLUSIONS: Our experiments uniquely illustrate the architectural plasticity of the spindle and reveal a robust ability of the system to attain a bipolar morphology. We hypothesize that a major mechanism driving spindle fusion is dynein-mediated sliding of oppositely oriented microtubules, a novel function for the motor, and posit that this same mechanism might also be involved in normal spindle assembly and homeostasis.},
	keywords = {Animals, Cell Polarity, Chromosome Segregation, Dyneins, Kinesin, Microtubule-Associated Proteins, Microtubules, Oocytes, Spindle Apparatus, Xenopus laevis, Xenopus Proteins},
	issn = {1879-0445},
	doi = {10.1016/j.cub.2009.01.055},
	author = {Gatlin, Jesse C and Matov, Alexandre and Groen, Aaron C and Needleman, Daniel J and Maresca, Thomas J and Danuser, Gaudenz and Mitchison, Timothy J. and Salmon, E D}
}
@article {572626,
	title = {A tissue-scale gradient of hydrogen peroxide mediates rapid wound detection in zebrafish.},
	journal = {Nature},
	volume = {459},
	number = {7249},
	year = {2009},
	month = {2009 Jun 18},
	pages = {996-9},
	abstract = {Barrier structures (for example, epithelia around tissues and plasma membranes around cells) are required for internal homeostasis and protection from pathogens. Wound detection and healing represent a dormant morphogenetic program that can be rapidly executed to restore barrier integrity and tissue homeostasis. In animals, initial steps include recruitment of leukocytes to the site of injury across distances of hundreds of micrometres within minutes of wounding. The spatial signals that direct this immediate tissue response are unknown. Owing to their fast diffusion and versatile biological activities, reactive oxygen species, including hydrogen peroxide (H(2)O(2)), are interesting candidates for wound-to-leukocyte signalling. Here we probe the role of H(2)O(2) during the early events of wound responses in zebrafish larvae expressing a genetically encoded H(2)O(2) sensor. This reporter revealed a sustained rise in H(2)O(2) concentration at the wound margin, starting approximately 3 min after wounding and peaking at approximately 20 min, which extended approximately 100-200 microm into the tail-fin epithelium as a decreasing concentration gradient. Using pharmacological and genetic inhibition, we show that this gradient is created by dual oxidase (Duox), and that it is required for rapid recruitment of leukocytes to the wound. This is the first observation, to our knowledge, of a tissue-scale H(2)O(2) pattern, and the first evidence that H(2)O(2) signals to leukocytes in tissues, in addition to its known antiseptic role.},
	keywords = {Animals, Diffusion, Hydrogen Peroxide, Larva, Leukocytes, NADPH Oxidase, Wound Healing, Wounds and Injuries, Zebrafish, Zebrafish Proteins},
	issn = {1476-4687},
	doi = {10.1038/nature08119},
	author = {Niethammer, Philipp and Grabher, Clemens and Look, A Thomas and Mitchison, Timothy J.}
}
@article {572601,
	title = {Animal cell hydraulics.},
	journal = {J Cell Sci},
	volume = {122},
	number = {Pt 18},
	year = {2009},
	month = {2009 Sep 15},
	pages = {3233-41},
	abstract = {Water is the dominant ingredient of cells and its dynamics are crucial to life. We and others have suggested a physical picture of the cell as a soft, fluid-infiltrated sponge, surrounded by a water-permeable barrier. To understand water movements in an animal cell, we imposed an external, inhomogeneous osmotic stress on cultured cancer cells. This forced water through the membrane on one side, and out on the other. Inside the cell, it created a gradient in hydration, that we visualized by tracking cellular responses using natural organelles and artificially introduced quantum dots. The dynamics of these markers at short times were the same for normal and metabolically poisoned cells, indicating that the cellular responses are primarily physical rather than chemical. Our finding of an internal gradient in hydration is inconsistent with a continuum model for cytoplasm, but consistent with the sponge model, and implies that the effective pore size of the sponge is small enough to retard water flow significantly on time scales ( approximately 10-100 seconds) relevant to cell physiology. We interpret these data in terms of a theoretical framework that combines mechanics and hydraulics in a multiphase poroelastic description of the cytoplasm and explains the experimentally observed dynamics quantitatively in terms of a few coarse-grained parameters that are based on microscopically measurable structural, hydraulic and mechanical properties. Our fluid-filled sponge model could provide a unified framework to understand a number of disparate observations in cell morphology and motility.},
	keywords = {Animals, Biomechanical Phenomena, Cell Membrane, Cells, Cytoplasm, Diffusion, Elasticity, HeLa Cells, Humans, Keratins, Metaphase, Nocodazole, Quantum Dots, Rheology, Tubulin, Vimentin, Water},
	issn = {1477-9137},
	doi = {10.1242/jcs.049262},
	author = {Charras, Guillaume T and Mitchison, Timothy J. and Mahadevan, L}
}
@article {572616,
	title = {Compression regulates mitotic spindle length by a mechanochemical switch at the poles.},
	journal = {Curr Biol},
	volume = {19},
	number = {13},
	year = {2009},
	month = {2009 Jul 14},
	pages = {1086-95},
	abstract = {BACKGROUND: Although the molecules involved in mitosis are becoming better characterized, we still lack an understanding of the emergent mechanical properties of the mitotic spindle. For example, we cannot explain how spindle length is determined. To gain insight into how forces are generated and responded to in the spindle, we developed a method to apply controlled mechanical compression to metaphase mitotic spindles in living mammalian cells while monitoring microtubules and kinetochores by fluorescence microscopy.

RESULTS: Compression caused reversible spindle widening and lengthening to a new steady state. Widening was a passive mechanical response, and lengthening was an active mechanochemical process requiring microtubule polymerization but not kinesin-5 activity. Spindle morphology during lengthening and drug perturbations suggested that kinetochore fibers are pushed outward by pole-directed forces generated within the spindle. Lengthening of kinetochore fibers occurred by inhibition of microtubule depolymerization at poles, with no change in sliding velocity, interkinetochore stretching, or kinetochore dynamics.

CONCLUSIONS: We propose that spindle length is controlled by a mechanochemical switch at the poles that regulates the depolymerization rate of kinetochore fibers in response to compression and discuss models for how this switch is controlled. Poleward force appears to be exerted along kinetochore fibers by some mechanism other than kinesin-5 activity, and we speculate that it may arise from polymerization pressure from growing plus ends of interpolar microtubules whose minus ends are anchored in the fiber. These insights provide a framework for conceptualizing mechanical integration within the spindle.},
	keywords = {Animals, Cell Division, Centrosome, Kinetochores, Mechanotransduction, Cellular, Microtubules, Potoroidae, Recombinant Fusion Proteins, Spindle Apparatus, Stress, Mechanical, Tubulin},
	issn = {1879-0445},
	doi = {10.1016/j.cub.2009.05.056},
	author = {Dumont, Sophie and Mitchison, Timothy J.}
}
@article {572581,
	title = {Evidence that mitotic exit is a better cancer therapeutic target than spindle assembly.},
	journal = {Cancer Cell},
	volume = {16},
	number = {4},
	year = {2009},
	month = {2009 Oct 6},
	pages = {347-58},
	abstract = {Current antimitotics work by perturbing spindle assembly, which activates the spindle assembly checkpoint, causes mitotic arrest, and triggers apoptosis. Cancer cells can resist such killing by premature exit, before cells initiate apoptosis, due to a weak checkpoint or rapid slippage. We reasoned blocking mitotic exit downstream of the checkpoint might circumvent this resistance. Using single-cell approaches, we showed that blocking mitotic exit by Cdc20 knockdown slowed cyclin B1 proteolysis, thus allowed more time for death initiation. Killing by Cdc20 knockdown did not require checkpoint activity and can occur by intrinsic apoptosis or an alternative death pathway when Bcl2 was overexpressed. We conclude targeting Cdc20, or otherwise blocking mitotic exit, may be a better cancer therapeutic strategy than perturbing spindle assembly.},
	keywords = {Animals, Antimitotic Agents, Apoptosis, Cdc20 Proteins, Cell Cycle Proteins, Cyclin B, Cyclin B1, Drug Resistance, Neoplasm, Enzyme Inhibitors, Genetic Therapy, HeLa Cells, Humans, Kinesin, Mice, Mitochondrial Membranes, Mitosis, Mutation, Neoplasms, Paclitaxel, Permeability, Protein Processing, Post-Translational, Proto-Oncogene Proteins c-bcl-2, RNA Interference, Spindle Apparatus, Time Factors, Transfection},
	issn = {1878-3686},
	doi = {10.1016/j.ccr.2009.08.020},
	author = {Huang, Hsiao-Chun and Shi, Jue and Orth, James D and Mitchison, Timothy J.}
}
@article {572571,
	title = {Force and length in the mitotic spindle.},
	journal = {Curr Biol},
	volume = {19},
	number = {17},
	year = {2009},
	month = {2009 Sep 15},
	pages = {R749-61},
	abstract = {The mitotic spindle assembles to a steady-state length at metaphase through the integrated action of molecular mechanisms that generate and respond to mechanical forces. While molecular mechanisms that produce force have been described, our understanding of how they integrate with each other, and with the assembly/disassembly mechanisms that regulate length, is poor. We review current understanding of the basic architecture and dynamics of the metaphase spindle, and some of the elementary force-producing mechanisms. We then discuss models for force integration and spindle length determination. We also emphasize key missing data that notably include absolute values of forces and how they vary as a function of position within the spindle.},
	keywords = {Biomechanical Phenomena, Cell Polarity, Cell Size, Elasticity, Friction, Kinetochores, Metaphase, Microtubules, Models, Biological, Spindle Apparatus},
	issn = {1879-0445},
	doi = {10.1016/j.cub.2009.07.028},
	author = {Dumont, Sophie and Mitchison, Timothy J.}
}
@article {572591,
	title = {Interaction between Poly(ADP-ribose) and NuMA contributes to mitotic spindle pole assembly.},
	journal = {Mol Biol Cell},
	volume = {20},
	number = {21},
	year = {2009},
	month = {2009 Nov},
	pages = {4575-85},
	abstract = {Poly(ADP-ribose) (pADPr), made by PARP-5a/tankyrase-1, localizes to the poles of mitotic spindles and is required for bipolar spindle assembly, but its molecular function in the spindle is poorly understood. To investigate this, we localized pADPr at spindle poles by immuno-EM. We then developed a concentrated mitotic lysate system from HeLa cells to probe spindle pole assembly in vitro. Microtubule asters assembled in response to centrosomes and Ran-GTP in this system. Magnetic beads coated with pADPr, extended from PARP-5a, also triggered aster assembly, suggesting a functional role of the pADPr in spindle pole assembly. We found that PARP-5a is much more active in mitosis than interphase. We used mitotic PARP-5a, self-modified with pADPr chains, to capture mitosis-specific pADPr-binding proteins. Candidate binding proteins included the spindle pole protein NuMA previously shown to bind to PARP-5a directly. The rod domain of NuMA, expressed in bacteria, bound directly to pADPr. We propose that pADPr provides a dynamic cross-linking function at spindle poles by extending from covalent modification sites on PARP-5a and NuMA and binding noncovalently to NuMA and that this function helps promote assembly of exactly two poles.},
	keywords = {Animals, Antigens, Nuclear, Cell Cycle, HeLa Cells, Humans, Nuclear Matrix-Associated Proteins, Poly Adenosine Diphosphate Ribose, Protein Binding, Recombinant Fusion Proteins, Spindle Apparatus, Tankyrases},
	issn = {1939-4586},
	doi = {10.1091/mbc.E09-06-0477},
	author = {Chang, Paul and Coughlin, Margaret and Mitchison, Timothy J.}
}
@article {572576,
	title = {An intermittent live cell imaging screen for siRNA enhancers and suppressors of a kinesin-5 inhibitor.},
	journal = {PLoS One},
	volume = {4},
	number = {10},
	year = {2009},
	month = {2009},
	pages = {e7339},
	abstract = {Kinesin-5 (also known as Eg5, KSP and Kif11) is required for assembly of a bipolar mitotic spindle. Small molecule inhibitors of Kinesin-5, developed as potential anti-cancer drugs, arrest cell in mitosis and promote apoptosis of cancer cells. We performed a genome-wide siRNA screen for enhancers and suppressors of a Kinesin-5 inhibitor in human cells to elucidate cellular responses, and thus identify factors that might predict drug sensitivity in cancers. Because the drug{\textquoteright}s actions play out over several days, we developed an intermittent imaging screen. Live HeLa cells expressing GFP-tagged histone H2B were imaged at 0, 24 and 48 hours after drug addition, and images were analyzed using open-source software that incorporates machine learning. This screen effectively identified siRNAs that caused increased mitotic arrest at low drug concentrations (enhancers), and vice versa (suppressors), and we report siRNAs that caused both effects. We then classified the effect of siRNAs for 15 genes where 3 or 4 out of 4 siRNA oligos tested were suppressors as assessed by time lapse imaging, and by testing for suppression of mitotic arrest in taxol and nocodazole. This identified 4 phenotypic classes of drug suppressors, which included known and novel genes. Our methodology should be applicable to other screens, and the suppressor and enhancer genes we identified may open new lines of research into mitosis and checkpoint biology.},
	keywords = {Genome, Human, Green Fluorescent Proteins, HeLa Cells, Humans, Image Processing, Computer-Assisted, Kinesin, Models, Biological, Models, Statistical, Neoplasms, Nocodazole, Paclitaxel, RNA, Small Interfering, Spindle Apparatus, Time Factors, Tubulin Modulators},
	issn = {1932-6203},
	doi = {10.1371/journal.pone.0007339},
	author = {Tsui, Melody and Xie, Tiao and Orth, James D and Carpenter, Anne E and Rudnicki, Stewart and Kim, Suejong and Shamu, Caroline E and Mitchison, Timothy J.}
}
@article {572606,
	title = {Multi-parameter phenotypic profiling: using cellular effects to characterize small-molecule compounds.},
	journal = {Nat Rev Drug Discov},
	volume = {8},
	number = {7},
	year = {2009},
	month = {2009 Jul},
	pages = {567-78},
	abstract = {Multi-parameter phenotypic profiling of small molecules provides important insights into their mechanisms of action, as well as a systems level understanding of biological pathways and their responses to small molecule treatments. It therefore deserves more attention at an early step in the drug discovery pipeline. Here, we summarize the technologies that are currently in use for phenotypic profiling--including mRNA-, protein- and imaging-based multi-parameter profiling--in the drug discovery context. We think that an earlier integration of phenotypic profiling technologies, combined with effective experimental and in silico target identification approaches, can improve success rates of lead selection and optimization in the drug discovery process.},
	keywords = {Animals, Drug Discovery, Flow Cytometry, Gene Expression Profiling, Humans, Microscopy, Phenotype, Phosphorylation, Proteomics, Statistics as Topic},
	issn = {1474-1784},
	doi = {10.1038/nrd2876},
	author = {Feng, Yan and Mitchison, Timothy J. and Bender, Andreas and Young, Daniel W and Tallarico, John A}
}
@article {572611,
	title = {Spindle assembly in the absence of a RanGTP gradient requires localized CPC activity.},
	journal = {Curr Biol},
	volume = {19},
	number = {14},
	year = {2009},
	month = {2009 Jul 28},
	pages = {1210-5},
	abstract = {During animal cell division, a gradient of GTP-bound Ran is generated around mitotic chromatin. It is generally accepted that this RanGTP gradient is essential for organizing the spindle, because it locally activates critical spindle assembly factors. Here, we show in Xenopus laevis egg extract, where the gradient is best characterized, that spindles can assemble in the absence of a RanGTP gradient. Gradient-free spindle assembly occurred around sperm nuclei but not around chromatin-coated beads and required the chromosomal passenger complex (CPC). Artificial enrichment of CPC activity within hybrid bead arrays containing both immobilized chromatin and the CPC supported local microtubule assembly even in the absence of a RanGTP gradient. We conclude that RanGTP and the CPC constitute the two major molecular signals that spatially promote microtubule polymerization around chromatin. Furthermore, we hypothesize that the two signals mainly originate from discreet physical sites on the chromosomes to localize microtubule assembly around chromatin: a RanGTP signal from any chromatin and a CPC-dependent signal predominantly generated from centromeric chromatin.},
	keywords = {Animals, Aurora Kinases, Cell Division, Centrosome, Chromatin, Kinetics, Kinetochores, Microscopy, Fluorescence, Microspheres, Microtubules, Multiprotein Complexes, Protein-Serine-Threonine Kinases, ran GTP-Binding Protein, Spindle Apparatus, Xenopus laevis},
	issn = {1879-0445},
	doi = {10.1016/j.cub.2009.05.061},
	author = {Maresca, Thomas J and Groen, Aaron C and Gatlin, Jesse C and Ohi, Ryoma and Mitchison, Timothy J. and Salmon, Edward D}
}
@article {572596,
	title = {Structural plasticity in actin and tubulin polymer dynamics.},
	journal = {Science},
	volume = {325},
	number = {5943},
	year = {2009},
	month = {2009 Aug 21},
	pages = {960-3},
	abstract = {Actin filaments and microtubules polymerize and depolymerize by adding and removing subunits at polymer ends, and these dynamics drive cytoplasmic organization, cell division, and cell motility. Since Wegner proposed the treadmilling theory for actin in 1976, it has largely been assumed that the chemical state of the bound nucleotide determines the rates of subunit addition and removal. This chemical kinetics view is difficult to reconcile with observations revealing multiple structural states of the polymer that influence polymerization dynamics but that are not strictly coupled to the bound nucleotide state. We refer to these phenomena as "structural plasticity" and discuss emerging evidence that they play a central role in polymer dynamics and function.},
	keywords = {Actin Cytoskeleton, Actin Depolymerizing Factors, Actins, Adenosine Triphosphate, Guanosine Diphosphate, Guanosine Triphosphate, Kinetics, Microfilament Proteins, Microtubules, Models, Biological, Tubulin},
	issn = {1095-9203},
	doi = {10.1126/science.1168823},
	author = {Kueh, Hao Yuan and Mitchison, Timothy J.}
}
@article {572676,
	title = {Actin disassembly by cofilin, coronin, and Aip1 occurs in bursts and is inhibited by barbed-end cappers.},
	journal = {J Cell Biol},
	volume = {182},
	number = {2},
	year = {2008},
	month = {2008 Jul 28},
	pages = {341-53},
	abstract = {Turnover of actin filaments in cells requires rapid actin disassembly in a cytoplasmic environment that thermodynamically favors assembly because of high concentrations of polymerizable monomers. We here image the disassembly of single actin filaments by cofilin, coronin, and actin-interacting protein 1, a purified protein system that reconstitutes rapid, monomer-insensitive disassembly (Brieher, W.M., H.Y. Kueh, B.A. Ballif, and T.J. Mitchison. 2006. J. Cell Biol. 175:315-324). In this three-component system, filaments disassemble in abrupt bursts that initiate preferentially, but not exclusively, from both filament ends. Bursting disassembly generates unstable reaction intermediates with lowered affinity for CapZ at barbed ends. CapZ and cytochalasin D (CytoD), a barbed-end capping drug, strongly inhibit bursting disassembly. CytoD also inhibits actin disassembly in mammalian cells, whereas latrunculin B, a monomer sequestering drug, does not. We propose that bursts of disassembly arise from cooperative separation of the two filament strands near an end. The differential effects of drugs in cells argue for physiological relevance of this new disassembly pathway and potentially explain discordant results previously found with these drugs.},
	keywords = {4-Butyrolactone, Actin Cytoskeleton, Actins, Animals, Bicyclo Compounds, Heterocyclic, CapZ Actin Capping Protein, Carrier Proteins, Cell Line, Cercopithecus aethiops, Cofilin 1, Cytochalasin D, Cytoskeleton, Haplorhini, HeLa Cells, Humans, Microfilament Proteins, Nucleic Acid Synthesis Inhibitors, Proteins, Rabbits, Signal Transduction, Thiazolidines},
	issn = {1540-8140},
	doi = {10.1083/jcb.200801027},
	author = {Kueh, Hao Yuan and Charras, Guillaume T and Mitchison, Timothy J. and Brieher, William M}
}
@article {572686,
	title = {Cell polarization during monopolar cytokinesis.},
	journal = {J Cell Biol},
	volume = {181},
	number = {2},
	year = {2008},
	month = {2008 Apr 21},
	pages = {195-202},
	abstract = {During cytokinesis, a specialized set of proteins is recruited to the equatorial region between spindle poles by microtubules and actin filaments, enabling furrow assembly and ingression before cell division. We investigate the mechanisms underlying regional specialization of the cytoskeleton in HeLa cells undergoing drug-synchronized monopolar cytokinesis. After forced mitotic exit, the cytoskeleton of monopolar mitotic cells is initially radially symmetric but undergoes a symmetry-breaking reaction that simultaneously polarizes microtubules and the cell cortex, with a concentration of cortical furrow markers into a cap at one side of the cell. Polarization requires microtubules, F-actin, RhoA, Myosin II activity, and Aurora B kinase activity. Aurora B localizes to actin cables in a gap between the monopolar midzone and the furrow-like cortex, suggesting a communication between them. We propose that feedback loops between cortical furrow components and microtubules promote symmetry breaking during monopolar cytokinesis and regional specialization of the cytoskeleton during normal bipolar cytokinesis.},
	keywords = {Aurora Kinase B, Aurora Kinases, Cell Division, Cell Polarity, Cytoskeleton, HeLa Cells, Humans, Microscopy, Electron, Microtubules, Mitosis, Protein-Serine-Threonine Kinases, Purines},
	issn = {1540-8140},
	doi = {10.1083/jcb.200711105},
	author = {Hu, Chi-Kuo and Coughlin, Margaret and Field, Christine M and Mitchison, Timothy J.}
}
@article {572701,
	title = {A chemical method for fast and sensitive detection of DNA synthesis in vivo.},
	journal = {Proc Natl Acad Sci U S A},
	volume = {105},
	number = {7},
	year = {2008},
	month = {2008 Feb 19},
	pages = {2415-20},
	abstract = {We have developed a method to detect DNA synthesis in proliferating cells, based on the incorporation of 5-ethynyl-2{\textquoteright}-deoxyuridine (EdU) and its subsequent detection by a fluorescent azide through a Cu(I)-catalyzed [3 + 2] cycloaddition reaction ("click" chemistry). Detection of the EdU label is highly sensitive and can be accomplished in minutes. The small size of the fluorescent azides used for detection results in a high degree of specimen penetration, allowing the staining of whole-mount preparations of large tissue and organ explants. In contrast to BrdU, the method does not require sample fixation or DNA denaturation and permits good structural preservation. We demonstrate the use of the method in cultured cells and in the intestine and brain of whole animals.},
	keywords = {Animals, Deoxyuridine, DNA, DNA Replication, Mice, Molecular Structure, NIH 3T3 Cells, Reproducibility of Results, Sensitivity and Specificity, Time Factors},
	issn = {1091-6490},
	doi = {10.1073/pnas.0712168105},
	author = {Salic, Adrian and Mitchison, Timothy J.}
}
@article {572706,
	title = {Coronin-1A stabilizes F-actin by bridging adjacent actin protomers and stapling opposite strands of the actin filament.},
	journal = {J Mol Biol},
	volume = {376},
	number = {3},
	year = {2008},
	month = {2008 Feb 22},
	pages = {607-13},
	abstract = {Coronins are F-actin-binding proteins that are involved, in concert with Arp2/3, Aip1, and ADF/cofilin, in rearrangements of the actin cytoskeleton. An understanding of coronin function has been hampered by the absence of any structural data on its interaction with actin. Using electron microscopy and three-dimensional reconstruction, we show that coronin-1A binds to three protomers in F-actin simultaneously: it bridges subdomain 1 and subdomain 2 of two adjacent actin subunits along the same long-pitch strand, and it staples subdomain 1 and subdomain 4 of two actin protomers on different strands. Such a mode of binding explains how coronin can stabilize actin filaments in vitro. In addition, we show which residues of F-actin may participate in the interaction with coronin-1A. Human nebulin and Xin, as well as Salmonella invasion protein A, use a similar mechanism to stabilize actin filaments. We suggest that the stapling of subdomain 1 and subdomain 4 of two actin protomers on different strands is a common mechanism for F-actin stabilization utilized by many actin-binding proteins that have no homology.},
	keywords = {Actins, Animals, Humans, Microfilament Proteins, Models, Molecular, Muscle Proteins, Protein Interaction Mapping},
	issn = {1089-8638},
	doi = {10.1016/j.jmb.2007.12.007},
	author = {Galkin, Vitold E and Orlova, Albina and Brieher, William and Kueh, Hao Yuan and Mitchison, Timothy J. and Egelman, Edward H}
}
@article {572671,
	title = {Evidence for an upper limit to mitotic spindle length.},
	journal = {Curr Biol},
	volume = {18},
	number = {16},
	year = {2008},
	month = {2008 Aug 26},
	pages = {1256-61},
	abstract = {Size specification of macromolecular assemblies in the cytoplasm is poorly understood [1]. In principle, assemblies could scale with cell size or use intrinsic mechanisms. For the mitotic spindle, scaling with cell size is expected, because the function of this assembly is to physically move sister chromatids into the center of nascent daughter cells. Eggs of Xenopus laevis are among the largest cells known that cleave completely during cell division. Cell length in this organism changes by two orders of magnitude ( approximately 1200 microm to approximately 12 microm) while it develops from a fertilized egg into a tadpole [2]. We wondered whether, and how, mitotic spindle length and morphology adapt to function at these different length scales. Here, we show that spindle length increases with cell length in small cells, but in very large cells spindle length approaches an upper limit of approximately 60 microm. Further evidence for an upper limit to spindle length comes from an embryonic extract system that recapitulates mitotic spindle assembly in a test tube. We conclude that early mitotic spindle length in Xenopus laevis is uncoupled from cell length, reaching an upper bound determined by mechanisms that are intrinsic to the spindle.},
	keywords = {Animals, Cell Size, Mitosis, Spindle Apparatus, Xenopus laevis},
	issn = {0960-9822},
	doi = {10.1016/j.cub.2008.07.092},
	author = {W{\"u}hr, Martin and Chen, Yao and Dumont, Sophie and Groen, Aaron C and Needleman, Daniel J and Salic, Adrian and Mitchison, Timothy J.}
}
@article {572696,
	title = {Implications of a poroelastic cytoplasm for the dynamics of animal cell shape.},
	journal = {Semin Cell Dev Biol},
	volume = {19},
	number = {3},
	year = {2008},
	month = {2008 Jun},
	pages = {215-23},
	abstract = {Two views have dominated recent discussions of the physical basis of cell shape change during migration and division of animal cells: the cytoplasm can be modeled as a viscoelastic continuum, and the forces that change its shape are generated only by actin polymerization and actomyosin contractility in the cell cortex. Here, we question both views: we suggest that the cytoplasm is better described as poroelastic, and that hydrodynamic forces may be generally important for its shape dynamics. In the poroelastic view, the cytoplasm consists of a porous, elastic solid (cytoskeleton, organelles, ribosomes) penetrated by an interstitial fluid (cytosol) that moves through the pores in response to pressure gradients. If the pore size is small (30-60nm), as has been observed in some cells, pressure does not globally equilibrate on time and length scales relevant to cell motility. Pressure differences across the plasma membrane drive blebbing, and potentially other type of protrusive motility. In the poroelastic view, these pressures can be higher in one part of a cell than another, and can thus cause local shape change. Local pressure transients could be generated by actomyosin contractility, or by local activation of osmogenic ion transporters in the plasma membrane. We propose that local activation of Na(+)/H(+) antiporters (NHE1) at the front of migrating cells promotes local swelling there to help drive protrusive motility, acting in combination with actin polymerization. Local shrinking at the equator of dividing cells may similarly help drive invagination during cytokinesis, acting in combination with actomyosin contractility. Testing these hypotheses is not easy, as water is a difficult analyte to track, and will require a joint effort of the cytoskeleton and ion physiology communities.},
	keywords = {Animals, Cell Movement, Cell Shape, Cytoplasm, Elasticity, Osmosis, Viscosity},
	issn = {1084-9521},
	doi = {10.1016/j.semcdb.2008.01.008},
	author = {Mitchison, T. J. and Charras, G T and Mahadevan, L}
}
@article {572711,
	title = {Integrating high-content screening and ligand-target prediction to identify mechanism of action.},
	journal = {Nat Chem Biol},
	volume = {4},
	number = {1},
	year = {2008},
	month = {2008 Jan},
	pages = {59-68},
	abstract = {High-content screening is transforming drug discovery by enabling simultaneous measurement of multiple features of cellular phenotype that are relevant to therapeutic and toxic activities of compounds. High-content screening studies typically generate immense datasets of image-based phenotypic information, and how best to mine relevant phenotypic data is an unsolved challenge. Here, we introduce factor analysis as a data-driven tool for defining cell phenotypes and profiling compound activities. This method allows a large data reduction while retaining relevant information, and the data-derived factors used to quantify phenotype have discernable biological meaning. We used factor analysis of cells stained with fluorescent markers of cell cycle state to profile a compound library and cluster the hits into seven phenotypic categories. We then compared phenotypic profiles, chemical similarity and predicted protein binding activities of active compounds. By integrating these different descriptors of measured and potential biological activity, we can effectively draw mechanism-of-action inferences.},
	keywords = {Antineoplastic Agents, Cell Cycle, Cell Nucleus, Cell Proliferation, Cluster Analysis, Computational Biology, DNA Replication, Dose-Response Relationship, Drug, Drug Design, HeLa Cells, Humans, Ligands, Models, Statistical, Molecular Structure, Predictive Value of Tests, Protein Binding, Small Molecule Libraries, Structure-Activity Relationship},
	issn = {1552-4469},
	doi = {10.1038/nchembio.2007.53},
	author = {Young, Daniel W and Bender, Andreas and Hoyt, Jonathan and McWhinnie, Elizabeth and Chirn, Gung-Wei and Tao, Charles Y and Tallarico, John A and Labow, Mark and Jenkins, Jeremy L and Mitchison, Timothy J. and Feng, Yan}
}
@article {572716,
	title = {Life and times of a cellular bleb.},
	journal = {Biophys J},
	volume = {94},
	number = {5},
	year = {2008},
	month = {2008 Mar 1},
	pages = {1836-53},
	abstract = {Blebs are spherical cellular protrusions that occur in many physiological situations. Two distinct phases make up the life of a bleb, each of which have their own biology and physics: expansion, which lasts approximately 30 s, and retraction, which lasts approximately 2 min. We investigate these phases using optical microscopy and simple theoretical concepts, seeking information on blebbing itself, and on cytomechanics in general. We show that bleb nucleation depends on pressure, membrane-cortex adhesion energy, and membrane tension, and test this experimentally. Bleb growth occurs through a combination of bulk flow of lipids and delamination from the cell cortex via the formation and propagation of tears. In extreme cases, this can give rise to a traveling wave around the cell periphery, known as "circus movement." When growth stalls, an actin cortex reforms under the bleb membrane, and retraction starts, driven by myosin-II. Using flicker spectroscopy, we find that retracting blebs are fivefold more rigid than expanding blebs, an increase entirely explained by the properties of the newly formed cortical actin mesh. Finally, using artificially nucleated blebs as pressure sensors, we show that cells rounded up in mitosis possess a substantial intracellular pressure.},
	keywords = {Actins, Animals, Blister, Cell Membrane, Cell Physiological Phenomena, Cells, Cultured, Contractile Proteins, Cytoskeleton, Humans, Intracellular Fluid, Microfilament Proteins, Microscopy, Myosin Type II},
	issn = {1542-0086},
	doi = {10.1529/biophysj.107.113605},
	author = {Charras, Guillaume T and Coughlin, Margaret and Mitchison, Timothy J. and Mahadevan, L}
}
@article {572681,
	title = {A novel small-molecule inhibitor reveals a possible role of kinesin-5 in anastral spindle-pole assembly.},
	journal = {J Cell Sci},
	volume = {121},
	number = {Pt 14},
	year = {2008},
	month = {2008 Jul 15},
	pages = {2293-300},
	abstract = {The tetrameric plus-end-directed motor, kinesin-5, is essential for bipolar spindle assembly. Small-molecule inhibitors of kinesin-5 have been important tools for investigating its function, and some are currently under evaluation as anti-cancer drugs. Most inhibitors reported to date are ;non-competitive{\textquoteright} and bind to a specific site on the motor head, trapping the motor in an ADP-bound state in which it has a weak but non-zero affinity for microtubules. Here, we used a novel ATP-competitive inhibitor, FCPT, developed at Merck (USA). We found that it induced tight binding of kinesin-5 onto microtubules in vitro. Using Xenopus egg-extract spindles, we found that FCPT not only blocked poleward microtubule sliding but also selectively induced loss of microtubules at the poles of bipolar spindles (and not asters or monoasters). We also found that the spindle-pole proteins TPX2 and gamma-tubulin became redistributed to the spindle equator, suggesting that proper kinesin-5 function is required for pole assembly.},
	keywords = {Animals, Cell Cycle Proteins, Cell Polarity, Cyclopropanes, Kinesin, Microtubule-Associated Proteins, Microtubules, Neoplasm Proteins, Nuclear Proteins, Phosphoproteins, Protein Binding, Pyridines, Small Molecule Libraries, Spindle Apparatus, Thiazoles, Tubulin, Xenopus, Xenopus Proteins},
	issn = {0021-9533},
	doi = {10.1242/jcs.024018},
	author = {Groen, Aaron C and Needleman, Daniel and Brangwynne, Clifford and Gradinaru, Christain and Fowler, Brandon and Mazitschek, Ralph and Mitchison, Timothy J.}
}
@article {572691,
	title = {Spatial patterning of metabolism by mitochondria, oxygen, and energy sinks in a model cytoplasm.},
	journal = {Curr Biol},
	volume = {18},
	number = {8},
	year = {2008},
	month = {2008 Apr 22},
	pages = {586-91},
	abstract = {Metabolite gradients might guide mitochondrial localization in cells and angiogenesis in tissues. It is unclear whether they can exist in single cells, because the length scale of most cells is small compared to the expected diffusion times of metabolites. For investigation of metabolic gradients, we need experimental systems in which spatial patterns of metabolism can be systematically measured and manipulated. We used concentrated cytoplasmic extracts from Xenopus eggs as a model cytoplasm, and visualized metabolic gradients formed in response to spatial stimuli. Restriction of oxygen supply to the edge of a drop mimicked distance to the surface of a single cell, or distance from a blood vessel in tissue. We imaged a step-like increase of Nicotinamide adenine dinucleotide (NAD) reduction approximately 600 microm distant from the oxygen source. This oxic-anoxic switch was preceded on the oxic side by a gradual rise of mitochondrial transmembrane potential (Deltapsi) and reactive oxygen species (ROS) production, extending over approximately 600 microm and approximately 300 microm, respectively. Addition of Adenosine triphosphate (ATP)-consuming beads mimicked local energy sinks in the cell. We imaged Deltapsi gradients with a decay length of approximately 50-300 microm around these beads, in the first visualization of an energy demand signaling gradient. Our study demonstrates that mitochondria can pattern the cytoplasm over length scales that are suited to convey morphogenetic information in large cells and tissues and provides a versatile model system for probing of the formation and function of metabolic gradients.},
	keywords = {Animals, Cytoplasm, Energy Metabolism, Mitochondria, NAD, Oxygen, Xenopus laevis},
	issn = {0960-9822},
	doi = {10.1016/j.cub.2008.03.038},
	author = {Niethammer, Philipp and Kueh, Hao Yuan and Mitchison, Timothy J.}
}
@article {572656,
	title = {Actin-aggregating cucurbitacins from Physocarpus capitatus.},
	journal = {J Nat Prod},
	volume = {71},
	number = {11},
	year = {2008},
	month = {2008 Nov},
	pages = {1927-9},
	abstract = {Bioassay-guided fractionation of Physocarpus capitatus yielded two new cucurbitacins (3 and 4) along with the known cucurbitacin F (1) and dihydrocucurbitacin F (2). Preliminary mechanism of action studies indicate that the cucurbitacins cause actin aggregates and inhibit cell division.},
	keywords = {Actins, Animals, Antineoplastic Agents, Phytogenic, Cell Division, Cucurbitacins, Cytokinesis, Drosophila, Rosaceae, Triterpenes},
	issn = {1520-6025},
	doi = {10.1021/np8005259},
	author = {Maloney, Katherine N and Fujita, Masaki and Eggert, Ulrike S and Schroeder, Frank C and Field, Christine M and Mitchison, Timothy J. and Clardy, Jon}
}
@article {572666,
	title = {Dynamic stabilization of actin filaments.},
	journal = {Proc Natl Acad Sci U S A},
	volume = {105},
	number = {43},
	year = {2008},
	month = {2008 Oct 28},
	pages = {16531-6},
	abstract = {We report here that actin filaments in vitro exist in two populations with significantly different shrinkage rates. Newly polymerized filaments shrink rapidly, primarily from barbed ends, at 1.8/s, but as they age they switch to a stable state that shrinks slowly, primarily from pointed ends, at approximately 0.1/s. This dynamic filament stabilization runs opposite to the classical prediction that actin filaments become more unstable with age as they hydrolyze their bound ATP and release phosphate. Upon cofilin treatment, aged filaments revert to a dynamic state that shows accelerated shrinkage from both ends at a combined rate of 5.9/s. In light of recent electron microscopy studies [Orlova A, et al. (2004) Actin-destabilizing factors disrupt filaments by means of a time reversal of polymerization. Proc Natl Acad Sci USA 101:17664-17668], we propose that dynamic stabilization arises from rearrangement of the filament structure from a relatively disordered state immediately after polymerization to the canonical Holmes helix, a change that is reversed by cofilin binding. Our results suggest that plasticity in the internal structure of the actin filament may play a fundamental role in regulating actin dynamics and may help cells build actin assemblies with vastly different turnover rates.},
	keywords = {Actin Cytoskeleton, Actin Depolymerizing Factors, Adenosine Triphosphate, Animals, Hydrolysis, Kinetics, Muscle, Skeletal, Protein Conformation, Rabbits},
	issn = {1091-6490},
	doi = {10.1073/pnas.0807394105},
	author = {Kueh, Hao Yuan and Brieher, William M and Mitchison, Timothy J.}
}
@article {572661,
	title = {Mitosis: new roles for myosin-X and actin at the spindle.},
	journal = {Curr Biol},
	volume = {18},
	number = {19},
	year = {2008},
	month = {2008 Oct 14},
	pages = {R912-4},
	abstract = {Roles for actin and myosin in positioning mitotic spindles in the cell are well established. A recent study of myosin-X function in early Xenopus embryo mitosis now reports that this unconventional myosin is required for pole integrity and normal spindle length by localizing to poles and exerting pulling forces on actin filaments within the spindle.},
	keywords = {Actins, Animals, Embryo, Nonmammalian, Mitosis, Myosins, Spindle Apparatus, Xenopus laevis, Xenopus Proteins},
	issn = {0960-9822},
	doi = {10.1016/j.cub.2008.08.043},
	author = {W{\"u}hr, Martin and Mitchison, Timothy J. and Field, Christine M}
}
@article {572651,
	title = {Quantitative live imaging of cancer and normal cells treated with Kinesin-5 inhibitors indicates significant differences in phenotypic responses and cell fate.},
	journal = {Mol Cancer Ther},
	volume = {7},
	number = {11},
	year = {2008},
	month = {2008 Nov},
	pages = {3480-9},
	abstract = {Kinesin-5 inhibitors (K5I) are promising antimitotic cancer drug candidates. They cause prolonged mitotic arrest and death of cancer cells, but their full range of phenotypic effects in different cell types has been unclear. Using time-lapse microscopy of cancer and normal cell lines, we find that a novel K5I causes several different cancer and noncancer cell types to undergo prolonged arrest in monopolar mitosis. Subsequent events, however, differed greatly between cell types. Normal diploid cells mostly slipped from mitosis and arrested in tetraploid G(1), with little cell death. Several cancer cell lines died either during mitotic arrest or following slippage. Contrary to prevailing views, mitotic slippage was not required for death, and the duration of mitotic arrest correlated poorly with the probability of death in most cell lines. We also assayed drug reversibility and long-term responses after transient drug exposure in MCF7 breast cancer cells. Although many cells divided after drug washout during mitosis, this treatment resulted in lower survival compared with washout after spontaneous slippage likely due to chromosome segregation errors in the cells that divided. Our analysis shows that K5Is cause cancer-selective cell killing, provides important kinetic information for understanding clinical responses, and elucidates mechanisms of drug sensitivity versus resistance at the level of phenotype.},
	keywords = {Antimitotic Agents, Cell Line, Tumor, Cell Proliferation, Chromosome Segregation, Humans, Image Interpretation, Computer-Assisted, Kinesin, Microscopy, Fluorescence, Mitosis, Neoplasms, Phenotype},
	issn = {1535-7163},
	doi = {10.1158/1535-7163.MCT-08-0684},
	author = {Orth, James D and Tang, Yangzhong and Shi, Jade and Loy, Clement T and Amendt, Christiane and Wilm, Claudia and Zenke, Frank T and Mitchison, Timothy J.}
}
@article {572731,
	title = {Nonredundant functions of Kinesin-13s during meiotic spindle assembly.},
	journal = {Curr Biol},
	volume = {17},
	number = {11},
	year = {2007},
	month = {2007 Jun 5},
	pages = {953-9},
	abstract = {Spatiotemporal control of microtubule depolymerization during cell division underlies the construction and dynamics of mitotic and meiotic spindles. Owing to their potent ability to disassemble microtubules, Kinesin-13s constitute an important class of microtubule destabilizing factors. Unfertilized Xenopus eggs, similar to other metazoan cells, contain the prototypical Kinesin-13 MCAK as well as a second family member, XKIF2. Here, we compare the roles of MCAK and XKIF2 during spindle assembly in Xenopus extracts. We find that although MCAK and XKIF2 have similar localization and biochemical properties, XKIF2 is not required for spindle assembly and, further, cannot substitute for MCAK. Altering dosage of the two kinesins demonstrates that spindle length is exquisitely sensitive to MCAK concentration but not XKIF2 concentration. Finally, we demonstrate that the rate of poleward microtubule flux in Xenopus-extract spindles is unaffected by XKIF2 depletion and is only modestly sensitive to reduction of MCAK action. We suggest that, in contrast to models proposed for mammalian somatic cell and embryonic Drosophila spindles, Kinesin-13s do not play a central role in poleward flux by depolymerizing minus ends. Rather, MCAK, but not XKIF2, plays a central role in regulating dynamic instability of plus ends and controls spindle length by that mechanism.},
	keywords = {Animals, Cell Extracts, Kinesin, Models, Biological, Spindle Apparatus, Xenopus, Xenopus Proteins},
	issn = {0960-9822},
	doi = {10.1016/j.cub.2007.04.057},
	author = {Ohi, Ryoma and Burbank, Kendra and Liu, Qing and Mitchison, Timothy J.}
}
@article {572721,
	title = {Slide-and-cluster models for spindle assembly.},
	journal = {Curr Biol},
	volume = {17},
	number = {16},
	year = {2007},
	month = {2007 Aug 21},
	pages = {1373-83},
	abstract = {BACKGROUND: Mitotic and meiotic spindles are assemblies of microtubules (MTs) that form during cell division to physically separate sister chromosomes. How the various components of spindles act together to establish and maintain the dynamic bipolar structure of spindles is not understood. Interactions between MTs and motors have been studied both experimentally and theoretically in many contexts, including the self-organization of arrays of MTs by motors and the competition between different classes of motors to move a single load. This work demonstrates how the interplay between two types of motors together with continual nucleation of MTs by chromosomes could organize the MTs into spindles.

RESULTS: We propose a slide-and-cluster model based on four known molecular activities: MT nucleation near chromosomes, the sliding of MTs by a plus-end-directed motor, the clustering of their minus ends by a minus-end-directed motor, and the loss of MTs by dynamic instability. Our model applies to overlapping, nonkinetochore MTs in anastral spindles, and perhaps also to interpolar MTs in astral spindles. We show mathematically that the slide-and-cluster mechanism robustly forms bipolar spindles with sharp poles and a stable steady-state length. This model accounts for several experimental observations that were difficult to explain with existing models. Three new predictions of the model were tested and verified in Xenopus egg extracts.

CONCLUSIONS: We show that a simple two-motor model could create stable, bipolar spindles under a wide range of physical parameters. Our model is the first self-contained model for anastral spindle assembly and MT sliding (known as poleward flux). Our experimental results support the slide-and-cluster scenario; most significantly, we find that MT sliding slows near spindle poles, confirming the model{\textquoteright}s primary prediction.},
	keywords = {Animals, Dyneins, Kinesin, Microtubules, Molecular Motor Proteins, Spindle Apparatus, Xenopus, Xenopus Proteins},
	issn = {0960-9822},
	doi = {10.1016/j.cub.2007.07.058},
	author = {Burbank, Kendra S and Mitchison, Timothy J. and Fisher, Daniel S}
}
@article {572726,
	title = {Symmetry of septin hourglass and ring structures.},
	journal = {J Mol Biol},
	volume = {372},
	number = {1},
	year = {2007},
	month = {2007 Sep 7},
	pages = {37-49},
	abstract = {Septin filaments form ordered hourglass and ring-shaped structures in close apposition to the yeast bud-neck membrane. The septin hourglass scaffolds the asymmetric localization of many essential cell division proteins. However, it is unknown whether the septin structures have an overall polarity along the mother-daughter axis that determines the asymmetric protein localization. Here we engineered rigid septin- green fluorescent protein (GFP) fusions with various fluorescence dipole directions by changing the position of the GFP beta-barrel relative to the septin filament axis. We then used polarized fluorescence microscopy to detect potential asymmetries in the filament organization. We found that both the hourglass and ring filament assemblies have sub-resolution C(2) symmetry and lack net polarity along the mother-daughter axis. The hourglass filaments have an additional degree of symmetry relative to the ring filaments, most likely due to a twist in their higher-order structure. We previously reported that during the hourglass to rings transition septin filaments change their direction. Here we show that the filaments also undergo a change in their lateral organization, consistent with filament untwisting. The lack of net septin polarity along the mother-daughter axis suggests that there are no septin-based structural reasons for the observed asymmetry of other proteins. We discuss possible anisotropic processes that could break the septin symmetry and establish the essential bud-neck asymmetry.},
	keywords = {Amino Acid Sequence, Cell Cycle Proteins, Cell Division, Cytoskeletal Proteins, Green Fluorescent Proteins, Models, Biological, Molecular Sequence Data, Profilins, Protein Structure, Quaternary, Recombinant Fusion Proteins, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, Sequence Homology, Amino Acid},
	issn = {0022-2836},
	doi = {10.1016/j.jmb.2007.05.100},
	author = {Vrabioiu, Alina M and Mitchison, Timothy J.}
}
@article {572771,
	title = {Animal cytokinesis: from parts list to mechanisms.},
	journal = {Annu Rev Biochem},
	volume = {75},
	year = {2006},
	month = {2006},
	pages = {543-66},
	abstract = {The mechanism underlying cytokinesis, the final step in cell division, remains one of the major unsolved questions in basic cell biology. Thanks to advances in functional genomics and proteomics, we are now able to assemble a "parts list" of proteins involved in cytokinesis. In this review, we discuss how to relate this parts list to biological mechanism. For easier analysis, we split cytokinesis into discrete steps: cleavage plane specification, rearrangement of microtubule structures, contractile ring assembly, ring ingression, and completion. We report on the advances that have been made to understand these steps and how they can be integrated into a global understanding of cytokinesis. We also discuss the extent to which classic questions have been answered and identify major outstanding questions.},
	keywords = {Animals, Cell Cycle Proteins, Cytokinesis, Cytoskeletal Proteins, Humans, Microtubules, Neoplasms, Spindle Apparatus},
	issn = {0066-4154},
	doi = {10.1146/annurev.biochem.74.082803.133425},
	author = {Eggert, Ulrike S and Mitchison, Timothy J. and Field, Christine M}
}
@article {572786,
	title = {Noble metals strip peptides from class II MHC proteins.},
	journal = {Nat Chem Biol},
	volume = {2},
	number = {4},
	year = {2006},
	month = {2006 Apr},
	pages = {197-201},
	abstract = {Class II major histocompatibility complex (MHC) proteins are essential for normal immune system function but also drive many autoimmune responses. They bind peptide antigens in endosomes and present them on the cell surface for recognition by CD4(+) T cells. A small molecule could potentially block an autoimmune response by disrupting MHC-peptide interactions, but this has proven difficult because peptides bind tightly and dissociate slowly from MHC proteins. Using a high-throughput screening assay we discovered a class of noble metal complexes that strip peptides from human class II MHC proteins by an allosteric mechanism. Biochemical experiments indicate the metal-bound MHC protein adopts a {\textquoteright}peptide-empty{\textquoteright} conformation that resembles the transition state of peptide loading. Furthermore, these metal inhibitors block the ability of antigen-presenting cells to activate T cells. This previously unknown allosteric mechanism may help resolve how gold(I) drugs affect the progress of rheumatoid arthritis and may provide a basis for developing a new class of anti-autoimmune drugs.},
	keywords = {Allosteric Site, Animals, Antigen Presentation, Autoimmune Diseases, CD4-Positive T-Lymphocytes, Chromatography, Gel, cisplatin, Dose-Response Relationship, Drug, Drosophila melanogaster, Enzyme-Linked Immunosorbent Assay, Gold Sodium Thiomalate, Histocompatibility Antigens Class II, Humans, Kinetics, Major Histocompatibility Complex, Models, Statistical, Molecular Conformation, Peptides, Protein Binding, Sodium Hypochlorite, Time Factors},
	issn = {1552-4450},
	doi = {10.1038/nchembio773},
	author = {De Wall, Stephen L and Painter, Corrie and Stone, Jennifer D and Bandaranayake, Rajintha and Wiley, Don C and Mitchison, Timothy J. and Stern, Lawrence J and DeDecker, Brian S}
}
@article {572781,
	title = {NuSAP, a mitotic RanGTP target that stabilizes and cross-links microtubules.},
	journal = {Mol Biol Cell},
	volume = {17},
	number = {6},
	year = {2006},
	month = {2006 Jun},
	pages = {2646-60},
	abstract = {Nucleolar and spindle-associated protein (NuSAP) was recently identified as a microtubule- and chromatin-binding protein in vertebrates that is nuclear during interphase. Small interfering RNA-mediated depletion of NuSAP resulted in aberrant spindle formation, missegregation of chromosomes, and ultimately blocked cell proliferation. We show here that NuSAP is enriched on chromatin-proximal microtubules at meiotic spindles in Xenopus oocytes. When added at higher than physiological levels to Xenopus egg extract, NuSAP induces extensive bundling of spindle microtubules and causes bundled microtubules within spindle-like structures to become longer. In vitro reconstitution experiments reveal two direct effects of NuSAP on microtubules: first, it can efficiently stabilize microtubules against depolymerization, and second, it can cross-link large numbers of microtubules into aster-like structures, thick fibers, and networks. With defined components we show that the activity of NuSAP is differentially regulated by Importin (Imp) alpha, Impbeta, and Imp7. While Impalpha and Imp7 appear to block the microtubule-stabilizing activity of NuSAP, Impbeta specifically suppresses aspects of the cross-linking activity of NuSAP. We propose that to achieve full NuSAP functionality at the spindle, all three importins must be dissociated by RanGTP. Once activated, NuSAP may aid to maintain spindle integrity by stabilizing and cross-linking microtubules around chromatin.},
	keywords = {Animals, Female, Gene Expression Regulation, Meiosis, Microtubules, Oocytes, Protein Transport, ran GTP-Binding Protein, RNA Interference, Spindle Apparatus, Tubulin, Xenopus laevis, Xenopus Proteins},
	issn = {1059-1524},
	doi = {10.1091/mbc.E05-12-1178},
	author = {Ribbeck, Katharina and Groen, Aaron C and Santarella, Rachel and Bohnsack, Markus T and Raemaekers, Tim and K{\"o}cher, Thomas and Gentzel, Marc and G{\"o}rlich, Dirk and Wilm, Matthias and Carmeliet, Geert and Mitchison, Timothy J. and Ellenberg, Jan and Hoenger, Andreas and Mattaj, Iain W}
}
@article {572776,
	title = {Small molecule screening by imaging.},
	journal = {Curr Opin Chem Biol},
	volume = {10},
	number = {3},
	year = {2006},
	month = {2006 Jun},
	pages = {232-7},
	abstract = {Useful small molecule tools can be discovered in imaging screens that measure phenotype in single cells or small organisms. Recent examples include identification of small molecule inhibitors of processes such as cell migration, cytokinesis, mitotic spindle length determination, melanogenesis, aggresome formation, membrane transport and nuclear export. Imaging screens are currently limited by challenges in the areas of image analysis and target identification. We discuss the use of model organisms such as zebrafish in screens and review different methods of target identification. The emerging field of automated image analysis is also introduced.},
	keywords = {Animals, Drug Evaluation, Preclinical, Humans, Image Processing, Computer-Assisted},
	issn = {1367-5931},
	doi = {10.1016/j.cbpa.2006.04.010},
	author = {Eggert, Ulrike S and Mitchison, Timothy J.}
}
@article {572791,
	title = {Small-molecule and mutational analysis of allosteric Eg5 inhibition by monastrol.},
	journal = {BMC Chem Biol},
	volume = {6},
	year = {2006},
	month = {2006},
	pages = {2},
	abstract = {BACKGROUND: A recent crystal structure of monastrol in a ternary complex with the kinesin Eg5 motor domain highlights a novel, induced-fit drug binding site at atomic resolution. Mutational obliteration of the monastrol binding site results in a monastrol-resistant, but otherwise catalytically active Eg5 motor domain. However, considering the conformational changes at this site, it is unclear what specific interactions stabilize the interaction between monastrol and the Eg5 motor domain.

RESULTS: To study the molecular complementarity of the monastrol-Eg5 interaction, we used a combination of synthetic chemistry and targeted mutations in Eg5 to measure the contribution of specific contacts to inhibition of Eg5 in vitro and in cultured cells. Structure-activity data on chemical derivatives, sequence analysis of Eg5 homologs from different species, and the effect of mutations near the drug binding site were consistent with the crystal structure.

CONCLUSION: The mechanism of monastrol revealed by our data rationalizes its specificity for Eg5 over other kinesins and highlights a potential mechanism of drug resistance for anti-cancer therapy targeting this site in Eg5.},
	issn = {1472-6769},
	doi = {10.1186/1472-6769-6-2},
	author = {Maliga, Zoltan and Mitchison, Timothy J.}
}
@article {572746,
	title = {Meiotic spindle: sculpted by severing.},
	journal = {Curr Biol},
	volume = {16},
	number = {21},
	year = {2006},
	month = {2006 Nov 7},
	pages = {R923-5},
	abstract = {Katanin is a conserved AAA ATPase with the ability to sever microtubules, but its biological function in animal cells has been obscure. A recent study using electron tomography has found that katanin stimulates the production of microtubules in the meiotic spindles of Caenorhabditis elegans oocytes.},
	keywords = {Adenosine Triphosphatases, Animals, Caenorhabditis elegans, Caenorhabditis elegans Proteins, Meiosis, Microtubules, Oocytes},
	issn = {0960-9822},
	doi = {10.1016/j.cub.2006.09.048},
	author = {Ribbeck, Katharina and Mitchison, Timothy J.}
}
@article {572766,
	title = {Microtubule dynamic instability.},
	journal = {Curr Biol},
	volume = {16},
	number = {14},
	year = {2006},
	month = {2006 Jul 25},
	pages = {R516-7},
	keywords = {Microtubules, Models, Biological},
	issn = {0960-9822},
	doi = {10.1016/j.cub.2006.06.044},
	author = {Burbank, Kendra S and Mitchison, Timothy J.}
}
@article {572741,
	title = {A new method reveals microtubule minus ends throughout the meiotic spindle.},
	journal = {J Cell Biol},
	volume = {175},
	number = {3},
	year = {2006},
	month = {2006 Nov 6},
	pages = {369-75},
	abstract = {Anastral meiotic spindles are thought to be organized differently from astral mitotic spindles, but the field lacks the basic structural information required to describe and model them, including the location of microtubule-nucleating sites and minus ends. We measured the distributions of oriented microtubules in metaphase anastral spindles in Xenopus laevis extracts by fluorescence speckle microscopy and cross-correlation analysis. We localized plus ends by tubulin incorporation and combined this with the orientation data to infer the localization of minus ends. We found that minus ends are localized throughout the spindle, sparsely at the equator and at higher concentrations near the poles. Based on these data, we propose a model for maintenance of the metaphase steady-state that depends on continuous nucleation of microtubules near chromatin, followed by sorting and outward transport of stabilized minus ends, and, eventually, their loss near poles.},
	keywords = {Animals, Densitometry, In Vitro Techniques, Meiosis, Metaphase, Microscopy, Fluorescence, Microtubules, Models, Biological, Spindle Apparatus, Tubulin, Xenopus laevis},
	issn = {0021-9525},
	doi = {10.1083/jcb.200511112},
	author = {Burbank, Kendra S and Groen, Aaron C and Perlman, Zachary E and Fisher, Daniel S and Mitchison, Timothy J.}
}
@article {572751,
	title = {Rapid actin monomer-insensitive depolymerization of Listeria actin comet tails by cofilin, coronin, and Aip1.},
	journal = {J Cell Biol},
	volume = {175},
	number = {2},
	year = {2006},
	month = {2006 Oct 23},
	pages = {315-24},
	abstract = {Actin filaments in cells depolymerize rapidly despite the presence of high concentrations of polymerizable G actin. Cofilin is recognized as a key regulator that promotes actin depolymerization. In this study, we show that although pure cofilin can disassemble Listeria monocytogenes actin comet tails, it cannot efficiently disassemble comet tails in the presence of polymerizable actin. Thymus extracts also rapidly disassemble comet tails, and this reaction is more efficient than pure cofilin when normalized to cofilin concentration. By biochemical fractionation, we identify Aip1 and coronin as two proteins present in thymus extract that facilitate the cofilin-mediated disassembly of Listeria comet tails. Together, coronin and Aip1 lower the amount of cofilin required to disassemble the comet tail and permit even low concentrations of cofilin to depolymerize actin in the presence of polymerizable G actin. The cooperative activities of cofilin, coronin, and Aip1 should provide a biochemical basis for understanding how actin filaments can grow in some places in the cell while shrinking in others.},
	keywords = {Actin Cytoskeleton, Actin Depolymerizing Factors, Actin-Related Protein 2-3 Complex, Actins, Animals, Carrier Proteins, Cattle, HeLa Cells, Humans, Listeria monocytogenes, Mass Spectrometry, Microfilament Proteins, Muscle, Skeletal, Profilins, Proteins, Rabbits, Thymus Gland},
	issn = {0021-9525},
	doi = {10.1083/jcb.200603149},
	author = {Brieher, William M and Kueh, Hao Yuan and Ballif, Bryan A and Mitchison, Timothy J.}
}
@article {572736,
	title = {Reassembly of contractile actin cortex in cell blebs.},
	journal = {J Cell Biol},
	volume = {175},
	number = {3},
	year = {2006},
	month = {2006 Nov 6},
	pages = {477-90},
	abstract = {Contractile actin cortex is involved in cell morphogenesis, movement, and cytokinesis, but its organization and assembly are poorly understood. During blebbing, the membrane detaches from the cortex and inflates. As expansion ceases, contractile cortex re-assembles under the membrane and drives bleb retraction. This cycle enabled us to measure the temporal sequence of protein recruitment to the membrane during cortex reassembly and to explore dependency relationships. Expanding blebs were devoid of actin, but proteins of the erythrocytic submembranous cytoskeleton were present. When expansion ceased, ezrin was recruited to the membrane first, followed by actin, actin-bundling proteins, and, finally, contractile proteins. Complete assembly of the contractile cortex, which was organized into a cagelike mesh of filaments, took approximately 30 s. Cytochalasin D blocked recruitment of actin and alpha-actinin, but had no effect on membrane association of ankyrin B and ezrin. Ezrin played no role in actin nucleation, but was essential for tethering the membrane to the cortex. The Rho pathway was important for cortex assembly in blebs.},
	keywords = {Actins, Contractile Proteins, Cytoskeletal Proteins, Cytoskeleton, Erythrocyte Membrane, Fluorescent Dyes, Green Fluorescent Proteins, HeLa Cells, Humans, Kinetics, Luminescent Proteins, Membrane Proteins, Microfilament Proteins, Microinjections, Microscopy, Confocal, Microscopy, Electron, Scanning, Microscopy, Electron, Transmission, Microscopy, Video, Mutation, Myosin Type II, Protein Transport, Recombinant Fusion Proteins, rhoA GTP-Binding Protein, Transfection},
	issn = {0021-9525},
	doi = {10.1083/jcb.200602085},
	author = {Charras, Guillaume T and Hu, Chi-Kuo and Coughlin, Margaret and Mitchison, Timothy J.}
}
@article {572761,
	title = {Small molecules in an RNAi world.},
	journal = {Mol Biosyst},
	volume = {2},
	number = {2},
	year = {2006},
	month = {2006 Feb},
	pages = {93-6},
	abstract = {In this Opinion, we compare and contrast small molecules and RNAi. We discuss the advantages and disadvantages of both technologies focusing on timing, specificity, dose and therapeutic use.},
	keywords = {Animals, Dose-Response Relationship, Drug, Humans, Models, Biological, RNA Interference, RNA, Small Interfering, Sensitivity and Specificity, Time Factors},
	issn = {1742-206X},
	doi = {10.1039/b515335b},
	author = {Eggert, Ulrike S and Field, Christine M and Mitchison, Timothy J.}
}
@article {572756,
	title = {Structural insights into yeast septin organization from polarized fluorescence microscopy.},
	journal = {Nature},
	volume = {443},
	number = {7110},
	year = {2006},
	month = {2006 Sep 28},
	pages = {466-9},
	abstract = {Septins are polymerizing GTPases that function in cortical organization and cell division. In Saccharomyces cerevisiae they localize at the isthmus between the mother and the daughter cells, where they undergo a transition from a non-dynamic hourglass-shaped assembly to two separate rings, at the onset of cytokinesis. Septins form filaments as pure protein and in vivo, but the filament organization within the hourglass and ring structures is controversial. Here, we use polarized fluorescence microscopy of orientationally constrained green fluorescent protein to determine septin filament organization and dynamics in living yeast. We found that the hourglass is made of filaments aligned along the yeast bud neck. During the transition from hourglass to rings the filaments rotate through 90 degrees in the membrane plane and become circumferential. These data resolve a long-standing controversy in the field and provide strong evidence that septins have a mechanical function in cell division.},
	keywords = {Cell Cycle Proteins, Cytoskeletal Proteins, Fluorescence, Fluorescence Polarization, Microscopy, Fluorescence, Profilins, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins},
	issn = {1476-4687},
	doi = {10.1038/nature05109},
	author = {Vrabioiu, Alina M and Mitchison, Timothy J.}
}
@article {572891,
	title = {Anillin binds nonmuscle myosin II and regulates the contractile ring.},
	journal = {Mol Biol Cell},
	volume = {16},
	number = {1},
	year = {2005},
	month = {2005 Jan},
	pages = {193-201},
	abstract = {We demonstrate that the contractile ring protein anillin interacts directly with nonmuscle myosin II and that this interaction is regulated by myosin light chain phosphorylation. We show that despite their interaction, anillin and myosin II are independently targeted to the contractile ring. Depletion of anillin in Drosophila or human cultured cells results in cytokinesis failure. Human cells depleted for anillin fail to properly regulate contraction by myosin II late in cytokinesis and fail in abscission. We propose a role for anillin in spatially regulating the contractile activity of myosin II during cytokinesis.},
	keywords = {Amino Acid Sequence, Animals, Blotting, Western, Cell Line, Chromatography, Chromatography, Affinity, Cloning, Molecular, Contractile Proteins, Cytokinesis, Cytoskeleton, DNA, Drosophila, HeLa Cells, Humans, Immunoprecipitation, Interphase, Metaphase, Microscopy, Fluorescence, Molecular Sequence Data, Muscle Contraction, Myosin Type II, Myosins, Phosphorylation, Protein Binding, RNA, RNA Interference, RNA, Double-Stranded, Sequence Homology, Amino Acid, Xenopus laevis},
	issn = {1059-1524},
	doi = {10.1091/mbc.E04-08-0758},
	author = {Straight, Aaron F and Field, Christine M and Mitchison, Timothy J.}
}
@article {572881,
	title = {Mechanical properties of Xenopus egg cytoplasmic extracts.},
	journal = {Biophys J},
	volume = {88},
	number = {1},
	year = {2005},
	month = {2005 Jan},
	pages = {680-9},
	abstract = {Cytoplasmic extracts prepared from Xenopus laevis eggs are used for the reconstitution of a wide range of processes in cell biology, and offer a unique environment in which to investigate the role of cytoplasmic mechanics without the complication of preorganized cellular structures. As a step toward understanding the mechanical properties of this system, we have characterized the rheology of crude interphase extracts. At macroscopic length scales, the extract forms a soft viscoelastic solid. Using a conventional mechanical rheometer, we measure the elastic modulus to be in the range of 2-10 Pa, and loss modulus in the range of 0.5-5 Pa. Using pharmacological and immunological disruption methods, we establish that actin filaments and microtubules cooperate to give mechanical strength, whereas the intermediate filament cytokeratin does not contribute to viscoelasticity. At microscopic length scales smaller than the average network mesh size, the response is predominantly viscous. We use multiple particle tracking methods to measure the thermal fluctuations of 1 microm embedded tracer particles, and measure the viscosity to be approximately 20 mPa-s. We explore the impact of rheology on actin-dependent cytoplasmic contraction, and find that although microtubules modulate contractile forces in vitro, their interactions are not purely mechanical.},
	keywords = {Actins, Animals, Cell Size, Cytoplasm, Female, Keratins, Microtubules, Mitosis, Oocytes, Polyethylene Glycols, Rheology, Time Factors, Xenopus},
	issn = {0006-3495},
	doi = {10.1529/biophysj.104.048025},
	author = {Valentine, M. T. and Perlman, Z. E. and Mitchison, T. J. and Weitz, D. A.}
}
@article {572851,
	title = {High-content screening and profiling of drug activity in an automated centrosome-duplication assay.},
	journal = {Chembiochem},
	volume = {6},
	number = {1},
	year = {2005},
	month = {2005 Jan},
	pages = {145-51},
	abstract = {Maintenance of centrosome number is essential for cell-cycle progression and genomic stability, but investigation of this regulation has been limited by assay difficulty. We present a fully automated image-based centrosome-duplication assay that is accurate and robust enough for both careful cell-biology studies and high-throughput screening, and employ this assay in a series of chemical-genetic studies. We observe that a simple cytometric profiling strategy, which is based on organelle size, groups compounds with similar mechanisms of action; this suggests a simple strategy for excluding compounds that undesirably target such activities as protein synthesis and microtubule dynamics. Screening a library of compounds of known activity, we found unexpected effects on centrosome duplication by a number of drugs, most notably isoform-specific protein kinase C inhibitors and retinoic acid receptor agonists. From a 16 320-member library of uncharacterized small molecules, we identified five potent centrosome-duplication inhibitors that do not target microtubule dynamics or protein synthesis. The analysis methodology reported here is directly relevant to studies of centrosome regulation in a variety of systems and is adaptable to a wide range of other biological problems.},
	keywords = {Animals, Biological Assay, Cells, Cultured, Centrosome, Computational Biology, Drug Evaluation, Preclinical},
	issn = {1439-4227},
	doi = {10.1002/cbic.200400266},
	author = {Perlman, Zachary E and Mitchison, Timothy J. and Mayer, Thomas U}
}
@article {572826,
	title = {Mechanism and function of poleward flux in Xenopus extract meiotic spindles.},
	journal = {Philos Trans R Soc Lond B Biol Sci},
	volume = {360},
	number = {1455},
	year = {2005},
	month = {2005 Mar 29},
	pages = {623-9},
	abstract = {In Xenopus extract meiotic spindles, microtubules slide continuously towards their minus ends, a process called poleward flux. This article discusses recent progress in determining the mechanism of poleward flux, and its functions in spindle organization and generating force on chromosomes. Bipolar organization is required for flux and inhibition of the mitotic kinesin Eg5 inhibits flux, suggesting the sliding force for flux is generated by Eg5 pushing anti-parallel microtubules apart. An important function of flux in spindle organization may be to transport minus ends nucleated at chromatin towards the pole. By pulling microtubules through attachment sites at kinetochores, flux may generate poleward force on metaphase chromosomes.},
	keywords = {Animals, Biological Transport, Chromosome Segregation, Kinesin, Kinetochores, Meiosis, Microtubules, Models, Biological, Spindle Apparatus, Xenopus, Xenopus Proteins},
	issn = {0962-8436},
	doi = {10.1098/rstb.2004.1616},
	author = {Mitchison, T. J.}
}
@article {572821,
	title = {Non-equilibration of hydrostatic pressure in blebbing cells.},
	journal = {Nature},
	volume = {435},
	number = {7040},
	year = {2005},
	month = {2005 May 19},
	pages = {365-9},
	abstract = {Current models for protrusive motility in animal cells focus on cytoskeleton-based mechanisms, where localized protrusion is driven by local regulation of actin biochemistry. In plants and fungi, protrusion is driven primarily by hydrostatic pressure. For hydrostatic pressure to drive localized protrusion in animal cells, it would have to be locally regulated, but current models treating cytoplasm as an incompressible viscoelastic continuum or viscous liquid require that hydrostatic pressure equilibrates essentially instantaneously over the whole cell. Here, we use cell blebs as reporters of local pressure in the cytoplasm. When we locally perfuse blebbing cells with cortex-relaxing drugs to dissipate pressure on one side, blebbing continues on the untreated side, implying non-equilibration of pressure on scales of approximately 10 microm and 10 s. We can account for localization of pressure by considering the cytoplasm as a contractile, elastic network infiltrated by cytosol. Motion of the fluid relative to the network generates spatially heterogeneous transients in the pressure field, and can be described in the framework of poroelasticity.},
	keywords = {Actins, Animals, Bicyclo Compounds, Heterocyclic, Cell Line, Cell Surface Extensions, Cytoplasm, Cytoskeleton, Elasticity, Hydrostatic Pressure, Microscopy, Video, Models, Biological, Movement, Perfusion, Staurosporine, Thiazoles, Thiazolidines, Time Factors},
	issn = {1476-4687},
	doi = {10.1038/nature03550},
	author = {Charras, Guillaume T and Yarrow, Justin C and Horton, Mike A and Mahadevan, L and Mitchison, T. J.}
}
@article {572836,
	title = {Roles of polymerization dynamics, opposed motors, and a tensile element in governing the length of Xenopus extract meiotic spindles.},
	journal = {Mol Biol Cell},
	volume = {16},
	number = {6},
	year = {2005},
	month = {2005 Jun},
	pages = {3064-76},
	abstract = {Metaphase spindles assemble to a steady state in length by mechanisms that involve microtubule dynamics and motor proteins, but they are incompletely understood. We found that Xenopus extract spindles recapitulate the length of egg meiosis II spindles, by using mechanisms intrinsic to the spindle. To probe these mechanisms, we perturbed microtubule polymerization dynamics and opposed motor proteins and measured effects on spindle morphology and dynamics. Microtubules were stabilized by hexylene glycol and inhibition of the catastrophe factor mitotic centromere-associated kinesin (MCAK) (a kinesin 13, previously called XKCM) and destabilized by depolymerizing drugs. The opposed motors Eg5 and dynein were inhibited separately and together. Our results are consistent with important roles for polymerization dynamics in regulating spindle length, and for opposed motors in regulating the relative stability of bipolar versus monopolar organization. The response to microtubule destabilization suggests that an unidentified tensile element acts in parallel with these conventional factors, generating spindle shortening force.},
	keywords = {Adenylyl Imidodiphosphate, Animals, Blotting, Western, Cell Extracts, Dyneins, Female, Glycols, Kinesin, Kinetics, Meiosis, Microscopy, Polarization, Microscopy, Video, Microtubules, Models, Biological, Oocytes, Spindle Apparatus, Xenopus, Xenopus Proteins},
	issn = {1059-1524},
	doi = {10.1091/mbc.E05-02-0174},
	author = {Mitchison, T. J. and Maddox, P and Gaetz, J and Groen, A and Shirasu, M and Desai, A and Salmon, E D and Kapoor, T M}
}
@article {572831,
	title = {Screening for cell migration inhibitors via automated microscopy reveals a Rho-kinase inhibitor.},
	journal = {Chem Biol},
	volume = {12},
	number = {3},
	year = {2005},
	month = {2005 Mar},
	pages = {385-95},
	abstract = {Small-molecule kinase inhibitors are predominantly discovered in pure protein assays. We have discovered an inhibitor of Rho-kinase (ROCK) through an image-based, high-throughput screen of cell monolayer wound healing. Using automated microscopy, we screened a library of approximately 16,000 compounds finding many that affected cell migration or cell morphology as well as compounds that blocked mitotic progression. We tested approximately 200 compounds in a series of subassays and chose one, 3-(4-pyridyl)indole (Rockout), for more detailed characterization. Rockout inhibits blebbing and causes dissolution of actin stress fibers, phenocopying Rho-kinase inhibitors. Testing Rho-kinase activity in vitro, Rockout inhibits with an IC50 of 25 microM ( approximately 5-fold less potent than Y-27632) but has a similar specificity profile. We also profile the wound healing assay with a library of compounds with known bioactivities, revealing multiple pathways involved in the biology.},
	keywords = {Animals, BALB 3T3 Cells, Cell Movement, Dose-Response Relationship, Drug, Intracellular Signaling Peptides and Proteins, Mice, Microscopy, Confocal, Protein Kinase Inhibitors, Protein-Serine-Threonine Kinases, rho-Associated Kinases},
	issn = {1074-5521},
	doi = {10.1016/j.chembiol.2005.01.015},
	author = {Yarrow, Justin C and Totsukawa, Go and Charras, Guillaume T and Mitchison, Timothy J.}
}
@article {572856,
	title = {Small-molecule screening and profiling by using automated microscopy.},
	journal = {Chembiochem},
	volume = {6},
	number = {1},
	year = {2005},
	month = {2005 Jan},
	pages = {33-9},
	abstract = {Automated fluorescence microscopy provides a powerful tool for analyzing the physiological state of single cells with high throughput and high information content. Here I discuss two types of experiments in which this technology was used to discover and characterize bioactive small molecules. In phenotypic-screening experiments, the goal is to find "hits" with specific effects on cells by screening large libraries of small molecules. An example is screening for small molecules that perturb mitosis by novel mechanisms. In cytological-profiling experiments, the goal is to characterize the bioactivity of a limited number of small molecules in considerable depth, and thus understand their mechanism and toxicities at the cellular level. I discuss an example in which 100 small molecules with known bioactivity were profiled by using multiple fluorescent probes, and clustered into mechanistic classes by automated statistical analysis.},
	keywords = {Algorithms, Automation, Cells, Cultured, Dose-Response Relationship, Drug, Drug Evaluation, Preclinical, Humans, Microscopy, Fluorescence},
	issn = {1439-4227},
	doi = {10.1002/cbic.200400272},
	author = {Mitchison, Timothy J.}
}
@article {572811,
	title = {Cell biology: powerful curves.},
	journal = {Nature},
	volume = {435},
	number = {7044},
	year = {2005},
	month = {2005 Jun 16},
	pages = {895-7},
	keywords = {Guanosine Diphosphate, Guanosine Triphosphate, Hydrolysis, Microtubules, Pliability, Protein Conformation, Tubulin},
	issn = {1476-4687},
	doi = {10.1038/435895a},
	author = {Mahadevan, L and Mitchison, T. J.}
}
@article {572796,
	title = {Chemical inhibitor of nonapoptotic cell death with therapeutic potential for ischemic brain injury.},
	journal = {Nat Chem Biol},
	volume = {1},
	number = {2},
	year = {2005},
	month = {2005 Jul},
	pages = {112-9},
	abstract = {The mechanism of apoptosis has been extensively characterized over the past decade, but little is known about alternative forms of regulated cell death. Although stimulation of the Fas/TNFR receptor family triggers a canonical {\textquoteright}extrinsic{\textquoteright} apoptosis pathway, we demonstrated that in the absence of intracellular apoptotic signaling it is capable of activating a common nonapoptotic death pathway, which we term necroptosis. We showed that necroptosis is characterized by necrotic cell death morphology and activation of autophagy. We identified a specific and potent small-molecule inhibitor of necroptosis, necrostatin-1, which blocks a critical step in necroptosis. We demonstrated that necroptosis contributes to delayed mouse ischemic brain injury in vivo through a mechanism distinct from that of apoptosis and offers a new therapeutic target for stroke with an extended window for neuroprotection. Our study identifies a previously undescribed basic cell-death pathway with potentially broad relevance to human pathologies.},
	keywords = {Animals, Brain Ischemia, Cell Death, Cell Line, Tumor, Humans, Imidazoles, Indoles, Mice, Molecular Structure, Protein Kinase Inhibitors, Protein-Serine-Threonine Kinases, Receptor-Interacting Protein Serine-Threonine Kinases, Signal Transduction, Tumor Necrosis Factor Receptor-Associated Peptides and Proteins},
	issn = {1552-4450},
	doi = {10.1038/nchembio711},
	author = {Degterev, Alexei and Huang, Zhihong and Boyce, Michael and Li, Yaqiao and Jagtap, Prakash and Mizushima, Noboru and Cuny, Gregory D and Mitchison, Timothy J. and Moskowitz, Michael A and Yuan, Junying}
}
@article {572816,
	title = {A small-molecule inhibitor of Mps1 blocks the spindle-checkpoint response to a lack of tension on mitotic chromosomes.},
	journal = {Curr Biol},
	volume = {15},
	number = {11},
	year = {2005},
	month = {2005 Jun 7},
	pages = {1070-6},
	abstract = {The spindle checkpoint prevents chromosome loss by preventing chromosome segregation in cells with improperly attached chromosomes [1, 2 and 3]. The checkpoint senses defects in the attachment of chromosomes to the mitotic spindle [4] and the tension exerted on chromosomes by spindle forces in mitosis [5, 6 and 7]. Because many cancers have defects in chromosome segregation, this checkpoint may be required for survival of tumor cells and may be a target for chemotherapy. We performed a phenotype-based chemical-genetic screen in budding yeast and identified an inhibitor of the spindle checkpoint, called cincreasin. We used a genome-wide collection of yeast gene-deletion strains and traditional genetic and biochemical analysis to show that the target of cincreasin is Mps1, a protein kinase required for checkpoint function [8]. Despite the requirement for Mps1 for sensing both the lack of microtubule attachment and tension at kinetochores, we find concentrations of cincreasin that selectively inhibit the tension-sensitive branch of the spindle checkpoint. At these concentrations, cincreasin causes lethal chromosome missegregation in mutants that display chromosomal instability. Our results demonstrate that Mps1 can be exploited as a target and that inhibiting the tension-sensitive branch of the spindle checkpoint may be a way of selectively killing cancer cells that display chromosomal instability.},
	keywords = {Bromobenzenes, Chromosome Segregation, Dimethyl Sulfoxide, DNA Primers, Dose-Response Relationship, Drug, Kinetochores, Microarray Analysis, Microtubules, Mitosis, Phenotype, Protein-Serine-Threonine Kinases, Protein-Tyrosine Kinases, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, Spindle Apparatus},
	issn = {0960-9822},
	doi = {10.1016/j.cub.2005.05.020},
	author = {Dorer, Russell K and Zhong, Sheng and Tallarico, John A and Wong, Wing Hung and Mitchison, Timothy J. and Murray, Andrew W}
}
@article {572801,
	title = {Tankyrase-1 polymerization of poly(ADP-ribose) is required for spindle structure and function.},
	journal = {Nat Cell Biol},
	volume = {7},
	number = {11},
	year = {2005},
	month = {2005 Nov},
	pages = {1133-9},
	abstract = {Poly(ADP-ribose) (PAR) is a large, negatively charged post-translational modification that is produced by polymerization of NAD+ by PAR polymerases (PARPs). There are at least 18 PARPs in the human genome, several of which have functions that are unknown. PAR modifications are dynamic; PAR structure depends on the balance between synthesis and hydrolysis by PAR glycohydrolase2. We previously found that PAR is enriched in vertebrate somatic-cell mitotic spindles and demonstrated a requirement for PAR in the assembly of Xenopus egg extract spindles. Here, we knockdown all characterized PARPs using RNA interference (RNAi), and identify tankyrase-1 as the PARP that is required for mitosis. Tankyrase-1 localizes to mitotic spindle poles, to telomeres and to the Golgi apparatus. Tankyrase-1 RNAi was recently shown to result in mitotic arrest, with abnormal chromosome distributions and spindle morphology observed--data that is interpreted as evidence of post-anaphase arrest induced by failure of telomere separation6. We show that tankyrase-1 RNAi results in pre-anaphase arrest, with intact sister-chromatid cohesion. We also demonstrate a requirement for tankyrase-1 in the assembly of bipolar spindles, and identify the spindle-pole protein NuMA as a substrate for covalent modification by tankyrase-1.},
	keywords = {Anaphase, Antigens, Nuclear, HeLa Cells, Humans, Mitosis, Nuclear Matrix-Associated Proteins, Nuclear Proteins, Poly Adenosine Diphosphate Ribose, RNA Interference, Spindle Apparatus, Tankyrases},
	issn = {1465-7392},
	doi = {10.1038/ncb1322},
	author = {Chang, Paul and Coughlin, Margaret and Mitchison, Timothy J.}
}
@article {572926,
	title = {Colloid surface chemistry critically affects multiple particle tracking measurements of biomaterials.},
	journal = {Biophys J},
	volume = {86},
	number = {6},
	year = {2004},
	month = {2004 Jun},
	pages = {4004-14},
	abstract = {Characterization of the properties of complex biomaterials using microrheological techniques has the promise of providing fundamental insights into their biomechanical functions; however, precise interpretations of such measurements are hindered by inadequate characterization of the interactions between tracers and the networks they probe. We here show that colloid surface chemistry can profoundly affect multiple particle tracking measurements of networks of fibrin, entangled F-actin solutions, and networks of cross-linked F-actin. We present a simple protocol to render the surface of colloidal probe particles protein-resistant by grafting short amine-terminated methoxy-poly(ethylene glycol) to the surface of carboxylated microspheres. We demonstrate that these poly(ethylene glycol)-coated tracers adsorb significantly less protein than particles coated with bovine serum albumin or unmodified probe particles. We establish that varying particle surface chemistry selectively tunes the sensitivity of the particles to different physical properties of their microenvironments. Specifically, particles that are weakly bound to a heterogeneous network are sensitive to changes in network stiffness, whereas protein-resistant tracers measure changes in the viscosity of the fluid and in the network microstructure. We demonstrate experimentally that two-particle microrheology analysis significantly reduces differences arising from tracer surface chemistry, indicating that modifications of network properties near the particle do not introduce large-scale heterogeneities. Our results establish that controlling colloid-protein interactions is crucial to the successful application of multiple particle tracking techniques to reconstituted protein networks, cytoplasm, and cells.},
	keywords = {Actins, Adsorption, Animals, Biocompatible Materials, Cattle, Fibrin, Gels, Microspheres, Polyethylene Glycols, Serum Albumin, Bovine},
	issn = {0006-3495},
	doi = {10.1529/biophysj.103.037812},
	author = {Valentine, M. T. and Perlman, Z. E. and Gardel, M. L. and Shin, J. H. and Matsudaira, P. and Mitchison, T. J. and Weitz, D. A.}
}
@article {572941,
	title = {Differentiation of cytoplasmic and meiotic spindle assembly MCAK functions by Aurora B-dependent phosphorylation.},
	journal = {Mol Biol Cell},
	volume = {15},
	number = {6},
	year = {2004},
	month = {2004 Jun},
	pages = {2895-906},
	abstract = {The KinI kinesin MCAK is a microtubule depolymerase important for governing spindle microtubule dynamics during chromosome segregation. The dynamic nature of spindle assembly and chromosome-microtubule interactions suggest that mechanisms must exist that modulate the activity of MCAK, both spatially and temporally. In Xenopus extracts, MCAK associates with and is stimulated by the inner centromere protein ICIS. The inner centromere kinase Aurora B also interacts with ICIS and MCAK raising the possibility that Aurora B may regulate MCAK activity as well. Herein, we demonstrate that recombinant Aurora B-INCENP inhibits Xenopus MCAK activity in vitro in a phosphorylation-dependent manner. Substituting endogenous MCAK in Xenopus extracts with the alanine mutant XMCAK-4A, which is resistant to inhibition by Aurora B-INCENP, led to assembly of mono-astral and monopolar structures instead of bipolar spindles. The size of these structures and extent of tubulin polymerization in XMCAK-4A extracts indicate that XM-CAK-4A is not defective for microtubule dynamics regulation throughout the cytoplasm. We further demonstrate that the ability of XMCAK-4A to localize to inner centromeres is abolished. Our results show that MCAK regulation of cytoplasmic and spindle-associated microtubules can be differentiated by Aurora B-dependent phosphorylation, and they further demonstrate that this regulation is required for bipolar meiotic spindle assembly.},
	keywords = {Amino Acid Sequence, Animals, Aurora Kinases, Cell Extracts, Centromere, Chromosomal Proteins, Non-Histone, Chromosome Segregation, Cytoplasm, Kinesin, Meiosis, Microtubules, Molecular Sequence Data, Mutation, Ovum, Phosphopeptides, Phosphorylation, Protein-Serine-Threonine Kinases, Recombinant Proteins, Spindle Apparatus, Substrate Specificity, Tubulin, Xenopus laevis},
	issn = {1059-1524},
	doi = {10.1091/mbc.E04-02-0082},
	author = {Ohi, Ryoma and Sapra, Tanuj and Howard, Jonathan and Mitchison, Timothy J.}
}
@article {572936,
	title = {Fascin-mediated propulsion of Listeria monocytogenes independent of frequent nucleation by the Arp2/3 complex.},
	journal = {J Cell Biol},
	volume = {165},
	number = {2},
	year = {2004},
	month = {2004 Apr 26},
	pages = {233-42},
	abstract = {Actin-dependent propulsion of Listeria monocytogenes is thought to require frequent nucleation of actin polymerization by the Arp2/3 complex. We demonstrate that L. monocytogenes motility can be separated into an Arp2/3-dependent nucleation phase and an Arp2/3-independent elongation phase. Elongation-based propulsion requires a unique set of biochemical factors in addition to those required for Arp2/3-dependent motility. We isolated fascin from brain extracts as the only soluble factor required in addition to actin during the elongation phase for this type of movement. The nucleation reaction assembles a comet tail of branched actin filaments directly behind the bacterium. The elongation-based reaction generates a hollow cylinder of parallel bundles that attach along the sides of the bacterium. Bacteria move faster in the elongation reaction than in the presence of Arp2/3, and the rate is limited by the concentration of G-actin. The biochemical and structural differences between the two motility reactions imply that each operates through distinct biochemical and biophysical mechanisms.},
	keywords = {Actins, Animals, Brain Chemistry, Carrier Proteins, Cattle, Cell Adhesion Molecules, Cell Line, Cell Movement, Listeria monocytogenes, Listeriosis, Microfilament Proteins, Models, Biological, Phosphoproteins, Polymers, Rabbits},
	issn = {0021-9525},
	doi = {10.1083/jcb.200311040},
	author = {Brieher, William M and Coughlin, Margaret and Mitchison, Timothy J.}
}
@article {572961,
	title = {The majority of the Saccharomyces cerevisiae septin complexes do not exchange guanine nucleotides.},
	journal = {J Biol Chem},
	volume = {279},
	number = {4},
	year = {2004},
	month = {2004 Jan 23},
	pages = {3111-8},
	abstract = {We show here that affinity-purified Saccharomyces cerevisiae septin complexes contain stoichiometric amounts of guanine nucleotides, specifically GTP and GDP. Using a (15)N-dilution assay read-out by liquid chromatography-tandem mass spectrometry, we determined that the majority of the bound guanine nucleotides do not turn over in vivo during one cell cycle period. In vitro, the isolated S. cerevisiae septin complexes have similar GTP binding and hydrolytic properties to the Drosophila septin complexes (Field, C. M., al-Awar, O., Rosenblatt, J., Wong, M. L., Alberts, B., and Mitchison, T. J. (1996) J. Cell Biol. 133, 605-616). In particular, the GTP turnover of septins is very slow when compared with the GTP turnover for Ras-like GTPases. We conclude that bound GTP and GDP play a structural, rather then regulatory, role for the majority of septins in proliferating cells as GTP does for alpha-tubulin.},
	keywords = {GTP Phosphohydrolases, Guanine Nucleotides, Guanosine Diphosphate, Guanosine Triphosphate, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins},
	issn = {0021-9258},
	doi = {10.1074/jbc.M310941200},
	author = {Vrabioiu, Alina M and Gerber, Scott A and Gygi, Steven P and Field, Christine M and Mitchison, Timothy J.}
}
@article {572951,
	title = {Microtubule plus-end dynamics in Xenopus egg extract spindles.},
	journal = {Mol Biol Cell},
	volume = {15},
	number = {4},
	year = {2004},
	month = {2004 Apr},
	pages = {1776-84},
	abstract = {Microtubule dynamics underlie spindle assembly, yet we do not know how the spindle environment affects these dynamics. We developed methods for measuring two key parameters of microtubule plus-end dynamic instability in Xenopus egg extract spindles. To measure plus-end polymerization rates and localize growing plus ends, we used fluorescence confocal imaging of EB1. This revealed plus-end polymerization throughout the spindle at approximately 11 microm/min, similar to astral microtubules, suggesting polymerization velocity is not regionally regulated by the spindle. The ratio of EB1 to microtubule fluorescence revealed an enrichment of polymerizing ends near the spindle middle, indicating enhanced nucleation or rescue there. We measured depolymerization rates by creating a front of synchronized depolymerization in spindles severed with microneedles. This front could be tracked by polarization and fluorescence microscopy as it advanced from each cut edge toward the associated pole. Both imaging modalities revealed rapid depolymerization ( approximately 30 microm/min) superimposed on a subset of microtubules stable to depolymerization. Larger spindle fragments contained a higher percentage of stable microtubules, which we believe were oriented with their minus ends facing the cut. Depolymerization was blocked by the potent microtubule stabilizing agent hexylene glycol, but was unaffected by alpha-MCAK antibody and AMPPNP, which block catastrophe and kinesin motility, respectively. These measurements move us closer to understanding the complete life history of a spindle microtubule.},
	keywords = {Adenylyl Imidodiphosphate, Animals, Glycols, Image Processing, Computer-Assisted, Kinesin, Metaphase, Microscopy, Confocal, Microscopy, Fluorescence, Microtubules, Ovum, Spindle Apparatus, Xenopus laevis},
	issn = {1059-1524},
	doi = {10.1091/mbc.E03-11-0824},
	author = {Tirnauer, Jennifer S and Salmon, E D and Mitchison, Timothy J.}
}
@article {572946,
	title = {A novel action of histone deacetylase inhibitors in a protein aggresome disease model.},
	journal = {Curr Biol},
	volume = {14},
	number = {6},
	year = {2004},
	month = {2004 Mar 23},
	pages = {488-92},
	abstract = {Protein inclusions are associated with a number of neurodegenerative diseases including amyotrophic lateral sclerosis (ALS). Whether protein aggregates are toxic or beneficial to cells is not known. In ALS animal models, mutant SOD1 forms aggresome-like structures in motor neurons and astrocytes. To better understand the role of protein aggregation in the progression of disease etiology, we performed a screen for small molecules that disrupt aggresome formation in cultured cells. After screening 20,000 compounds, we obtained two groups of compounds that specifically prevented aggresome formation. One group consists mainly of cardiac glycosides and will be the subject of another study. The second group contains two compounds: one is a known histone deacetylase (HDAC) inhibitor, Scriptaid, and the other is a Flavin analog, DPD. Cells treated with these molecules still contained microaggregates, but these microaggregates were not transported to microtubule organizing centers (MTOCs). The defect in transport was linked to modulation of the dynein/dynactin machinery as treatment with Scriptaid or DPD reversed mSOD-induced insolubilization of the dynactin subunits P50 dynamitin and P150(glued). Our findings suggest a connection between HDAC activity and aggresome formation and also lay the groundwork for a direct test of the role of aggresome formation in ALS etiology.},
	keywords = {Amyotrophic Lateral Sclerosis, Animals, Cell Fractionation, Cercopithecus aethiops, COS Cells, Disease Models, Animal, DNA Primers, Green Fluorescent Proteins, Histone Deacetylase Inhibitors, Hydroxylamines, Immunoblotting, Inclusion Bodies, Luminescent Proteins, Microtubule-Associated Proteins, Microtubule-Organizing Center, Quinolines, Superoxide Dismutase},
	issn = {0960-9822},
	doi = {10.1016/j.cub.2004.03.003},
	author = {Corcoran, Lisa J and Mitchison, Timothy J. and Liu, Qing}
}
@article {572956,
	title = {Rate-limiting guanosine 5{\textquoteright}-triphosphate hydrolysis during nucleotide turnover by FtsZ, a prokaryotic tubulin homologue involved in bacterial cell division.},
	journal = {Biochemistry},
	volume = {43},
	number = {1},
	year = {2004},
	month = {2004 Jan 13},
	pages = {282-8},
	abstract = {FtsZ is a prokaryotic tubulin homologue that polymerizes into a dynamic ring during cell division. GTP binding and hydrolysis provide the energy for FtsZ dynamics. However, the precise role of hydrolysis in polymer assembly and turnover is not understood, limiting our understanding of how FtsZ functions in the cell. Here we investigate GTP hydrolysis during the FtsZ polymerization cycle using several complementary approaches that avoid technical caveats of previous studies. We find that at steady state approximately 80\% of FtsZ polymer subunits are bound to GTP. In addition, we use pre-steady-state, single turnover assays to directly measure the rate of hydrolysis. Hydrolysis was found to occur at approximately 8/min and to be a rate-limiting step in GTP turnover; phosphate release rapidly followed. These results clarify previously conflicting results in the literature and suggest that pure FtsZ polymers, unlike microtubules, may not be able to undergo dynamic instability or to store energy in the polymer for force production.},
	keywords = {Bacterial Proteins, Cell Division, Cytoskeletal Proteins, Guanine Nucleotides, Guanosine Diphosphate, Guanosine Triphosphate, Hydrolysis, Polymers, Protein Binding, Protein Subunits, Sugar Phosphates, Tubulin},
	issn = {0006-2960},
	doi = {10.1021/bi035465r},
	author = {Romberg, Laura and Mitchison, Timothy J.}
}
@article {572931,
	title = {A small-molecule approach to studying invasive mechanisms of Toxoplasma gondii.},
	journal = {Proc Natl Acad Sci U S A},
	volume = {101},
	number = {19},
	year = {2004},
	month = {2004 May 11},
	pages = {7433-8},
	abstract = {Toxoplasma gondii is the most common protozoan parasite of humans. Infection with T. gondii can lead to life-threatening disease as a result of repeated cycles of host cell invasion, parasite replication, and host cell lysis. Relatively little is known about the invasive mechanisms of T. gondii and related parasites within the Phylum Apicomplexa (including Plasmodium spp., the causative agents of malaria), due to difficulties associated with studying genes essential to invasion in haploid obligate intracellular organisms. To circumvent this problem, we have developed a high-throughput microscope-based assay, which we have used to screen a collection of 12,160 structurally diverse small molecules for inhibitors of T. gondii invasion. A total of 24 noncytotoxic invasion inhibitors were identified. Secondary assays demonstrated that different inhibitors perturb different aspects of invasion, including gliding motility, secretion of host cell adhesins from apical organelles (the micronemes), and extension of a unique tubulin-based structure at the anterior of the parasite (the conoid). Unexpectedly, the screen also identified six small molecules that dramatically enhance invasion, gliding motility, and microneme secretion. The small molecules identified here reveal a previously unrecognized complexity in the control of parasite motility and microneme secretion, and they constitute a set of useful probes for dissecting the invasive mechanisms of T. gondii and related parasites. Small-molecule-based approaches provide a powerful means to address experimentally challenging problems in host-pathogen interaction, while simultaneously identifying new potential targets for drug development.},
	keywords = {Animals, Antiprotozoal Agents, Organelles, Toxoplasma},
	issn = {0027-8424},
	doi = {10.1073/pnas.0307769101},
	author = {Carey, Kimberly L and Westwood, Nicholas J and Mitchison, Timothy J. and Ward, Gary E}
}
@article {572921,
	title = {Synthesis and phenotypic screening of a Guanine-mimetic library.},
	journal = {Chembiochem},
	volume = {5},
	number = {7},
	year = {2004},
	month = {2004 Jul 5},
	pages = {1010-2},
	keywords = {Actins, Biomimetics, Cytoskeleton, Exocytosis, Guanine, HeLa Cells, Humans, Ligands, Mass Spectrometry, Peptide Library, Phenotype},
	issn = {1439-4227},
	doi = {10.1002/cbic.200400039},
	author = {Miller, Stephen C and Mitchison, Timothy J.}
}
@article {572901,
	title = {Bipolarization and poleward flux correlate during Xenopus extract spindle assembly.},
	journal = {Mol Biol Cell},
	volume = {15},
	number = {12},
	year = {2004},
	month = {2004 Dec},
	pages = {5603-15},
	abstract = {We investigated the mechanism by which meiotic spindles become bipolar and the correlation between bipolarity and poleward flux, using Xenopus egg extracts. By speckle microscopy and computational alignment, we find that monopolar sperm asters do not show evidence for flux, partially contradicting previous work. We account for the discrepancy by describing spontaneous bipolarization of sperm asters that was missed previously. During spontaneous bipolarization, onset of flux correlated with onset of bipolarity, implying that antiparallel microtubule organization may be required for flux. Using a probe for TPX2 in addition to tubulin, we describe two pathways that lead to spontaneous bipolarization, new pole assembly near chromatin, and pole splitting. By inhibiting the Ran pathway with excess importin-alpha, we establish a role for chromatin-derived, antiparallel overlap bundles in generating the sliding force for flux, and we examine these bundles by electron microscopy. Our results highlight the importance of two processes, chromatin-initiated microtubule nucleation, and sliding forces generated between antiparallel microtubules, in self-organization of spindle bipolarity and poleward flux.},
	keywords = {Animals, Cell Cycle Proteins, Cell Extracts, Cell Polarity, Chromatin, Female, Male, Meiosis, Microscopy, Electron, Microtubule-Associated Proteins, Microtubules, Neoplasm Proteins, Nuclear Proteins, Oocytes, Phosphoproteins, ran GTP-Binding Protein, Signal Transduction, Spermatozoa, Spindle Apparatus, Xenopus laevis, Xenopus Proteins},
	issn = {1059-1524},
	doi = {10.1091/mbc.E04-05-0440},
	author = {Mitchison, T. J. and Maddox, P and Groen, A and Cameron, L and Perlman, Z. and Ohi, R and Desai, A and Salmon, E D and Kapoor, T M}
}
@article {572876,
	title = {Eg5 causes elongation of meiotic spindles when flux-associated microtubule depolymerization is blocked.},
	journal = {Curr Biol},
	volume = {14},
	number = {21},
	year = {2004},
	month = {2004 Nov 9},
	pages = {1941-5},
	abstract = {In higher eukaryotes, microtubules (MT) in both halves of the mitotic spindle translocate continuously away from the midzone in a phenomenon called poleward microtubule flux. Because the spindle maintains constant length and microtubule density, this microtubule translocation must somehow be coupled to net MT depolymerization at spindle poles. The molecular mechanisms underlying both flux-associated translocation and flux-associated depolymerization are not well understood, but it can be predicted that blocking pole-based destabilization will increase spindle length, an idea that has not been tested in meiotic spindles. Here, we show that simultaneous addition of two pole-disrupting reagents p50/dynamitin and a truncated version of Xklp2 results in continuous spindle elongation in Xenopus egg extracts, and we quantitatively correlate this elongation rate with the poleward translocation of stabilized microtubules. We further use this system to demonstrate that this poleward translocation requires the activity of the kinesin-related protein Eg5. These results suggest that Eg5 is responsible for flux-associated MT translocation and that dynein and Xklp2 regulate flux-associated microtubule depolymerization at spindle poles.},
	keywords = {Animals, Cell Cycle Proteins, Cell Extracts, Fluorescence, Kinesin, Microspheres, Microtubule-Associated Proteins, Microtubules, Ovum, Spindle Apparatus, Tubulin, Xenopus, Xenopus Proteins},
	issn = {0960-9822},
	doi = {10.1016/j.cub.2004.10.029},
	author = {Shirasu-Hiza, Mimi and Perlman, Zachary E and Wittmann, Torsten and Karsenti, Eric and Mitchison, Timothy J.}
}
@article {572906,
	title = {A high-throughput cell migration assay using scratch wound healing, a comparison of image-based readout methods.},
	journal = {BMC Biotechnol},
	volume = {4},
	year = {2004},
	month = {2004 Sep 9},
	pages = {21},
	abstract = {BACKGROUND: Cell migration is a complex phenomenon that requires the coordination of numerous cellular processes. Investigation of cell migration and its underlying biology is of interest to basic scientists and those in search of therapeutics. Current migration assays for screening small molecules, siRNAs, or other perturbations are difficult to perform in parallel at the scale required to screen large libraries.

RESULTS: We have adapted the commonly used scratch wound healing assay of tissue-culture cell monolayers to a 384 well plate format. By mechanically scratching the cell substrate with a pin array, we are able to create characteristically sized wounds in all wells of a 384 well plate. Imaging of the healing wounds with an automated fluorescence microscope allows us to distinguish perturbations that affect cell migration, morphology, and division. Readout requires ~1 hr per plate but is high in information content i.e. high content. We compare readouts using different imaging technologies, automated microscopy, scanners and a fluorescence macroscope, and evaluate the trade-off between information content and data acquisition rate.

CONCLUSIONS: The adaptation of a wound healing assay to a 384 well format facilitates the study of aspects of cell migration, tissue reorganization, cell division, and other processes that underlie wound healing. This assay allows greater than 10,000 perturbations to be screened per day with a quantitative, high-content readout, and can also be used to characterize small numbers of perturbations in detail.},
	keywords = {Actins, Animals, Biological Assay, Cell Movement, Cercopithecus aethiops, DNA, Image Interpretation, Computer-Assisted, Kidney, Microscopy, Fluorescence, Models, Biological, Staining and Labeling, Wound Healing},
	issn = {1472-6750},
	doi = {10.1186/1472-6750-4-21},
	author = {Yarrow, Justin C and Perlman, Zachary E and Westwood, Nicholas J and Mitchison, Timothy J.}
}
@article {572871,
	title = {Multidimensional drug profiling by automated microscopy.},
	journal = {Science},
	volume = {306},
	number = {5699},
	year = {2004},
	month = {2004 Nov 12},
	pages = {1194-8},
	abstract = {We present a method for high-throughput cytological profiling by microscopy. Our system provides quantitative multidimensional measures of individual cell states over wide ranges of perturbations. We profile dose-dependent phenotypic effects of drugs in human cell culture with a titration-invariant similarity score (TISS). This method successfully categorized blinded drugs and suggested targets for drugs of uncertain mechanism. Multivariate single-cell analysis is a starting point for identifying relationships among drug effects at a systems level and a step toward phenotypic profiling at the single-cell level. Our methods will be useful for discovering the mechanism and predicting the toxicity of new drugs.},
	keywords = {Automation, Cell Cycle, Cluster Analysis, DNA, Dose-Response Relationship, Drug, Drug Evaluation, Preclinical, Fluorescent Dyes, HeLa Cells, Humans, Image Processing, Computer-Assisted, Microscopy, Fluorescence, Pharmacology, Phenotype, Statistics as Topic, Toxicity Tests},
	issn = {1095-9203},
	doi = {10.1126/science.1100709},
	author = {Perlman, Zachary E and Slack, Michael D and Feng, Yan and Mitchison, Timothy J. and Wu, Lani F and Altschuler, Steven J}
}
@article {572896,
	title = {A standardized kinesin nomenclature.},
	journal = {J Cell Biol},
	volume = {167},
	number = {1},
	year = {2004},
	month = {2004 Oct 11},
	pages = {19-22},
	abstract = {In recent years the kinesin superfamily has become so large that several different naming schemes have emerged, leading to confusion and miscommunication. Here, we set forth a standardized kinesin nomenclature based on 14 family designations. The scheme unifies all previous phylogenies and nomenclature proposals, while allowing individual sequence names to remain the same, and for expansion to occur as new sequences are discovered.},
	keywords = {Animals, Humans, Kinesin, Multigene Family, Terminology as Topic},
	issn = {0021-9525},
	doi = {10.1083/jcb.200408113},
	author = {Lawrence, Carolyn J and Dawe, R Kelly and Christie, Karen R and Cleveland, Don W and Dawson, Scott C and Endow, Sharyn A and Goldstein, Lawrence S B and Goodson, Holly V and Hirokawa, Nobutaka and Howard, Jonathon and Malmberg, Russell L and McIntosh, J Richard and Miki, Harukata and Mitchison, Timothy J. and Okada, Yasushi and Reddy, Anireddy S N and Saxton, William M and Schliwa, Manfred and Scholey, Jonathan M and Vale, Ronald D and Walczak, Claire E and Wordeman, Linda}
}
@article {572916,
	title = {Targeting cell division: small-molecule inhibitors of FtsZ GTPase perturb cytokinetic ring assembly and induce bacterial lethality.},
	journal = {Proc Natl Acad Sci U S A},
	volume = {101},
	number = {32},
	year = {2004},
	month = {2004 Aug 10},
	pages = {11821-6},
	abstract = {FtsZ, the ancestral homolog of eukaryotic tubulins, is a GTPase that assembles into a cytokinetic ring structure essential for cell division in prokaryotic cells. Similar to tubulin, purified FtsZ polymerizes into dynamic protofilaments in the presence of GTP; polymer assembly is accompanied by GTP hydrolysis. We used a high-throughput protein-based chemical screen to identify small molecules that target assembly-dependent GTPase activity of FtsZ. Here, we report the identification of five structurally diverse compounds, named Zantrins, which inhibit FtsZ GTPase either by destabilizing the FtsZ protofilaments or by inducing filament hyperstability through increased lateral association. These two classes of FtsZ inhibitors are reminiscent of the antitubulin drugs colchicine and Taxol, respectively. We also show that Zantrins perturb FtsZ ring assembly in Escherichia coli cells and cause lethality to a variety of bacteria in broth cultures, indicating that FtsZ antagonists may serve as chemical leads for the development of new broad-spectrum antibacterial agents. Our results illustrate the utility of small-molecule chemical probes to study FtsZ polymerization dynamics and the feasibility of FtsZ as a novel therapeutic target.},
	keywords = {Anti-Bacterial Agents, Bacterial Proteins, Biopolymers, Cytoskeletal Proteins, Drug Evaluation, Preclinical, Enzyme Inhibitors, Escherichia coli, Mycobacterium tuberculosis, Protein Conformation, Structure-Activity Relationship},
	issn = {0027-8424},
	doi = {10.1073/pnas.0404439101},
	author = {Margalit, Danielle N and Romberg, Laura and Mets, Rebecca B and Hebert, Alan M and Mitchison, Timothy J. and Kirschner, Marc W and RayChaudhuri, Debabrata}
}
@article {572911,
	title = {Vertebrate shugoshin links sister centromere cohesion and kinetochore microtubule stability in mitosis.},
	journal = {Cell},
	volume = {118},
	number = {5},
	year = {2004},
	month = {2004 Sep 3},
	pages = {567-78},
	abstract = {Drosophila MEI-S332 and fungal Sgo1 genes are essential for sister centromere cohesion in meiosis I. We demonstrate that the related vertebrate Sgo localizes to kinetochores and is required to prevent premature sister centromere separation in mitosis, thus providing an explanation for the differential cohesion observed between the arms and the centromeres of mitotic sister chromatids. Sgo is degraded by the anaphase-promoting complex, allowing the separation of sister centromeres in anaphase. Intriguingly, we show that Sgo interacts strongly with microtubules in vitro and that it regulates kinetochore microtubule stability in vivo, consistent with a direct microtubule interaction. Sgo is thus critical for mitotic progression and chromosome segregation and provides an unexpected link between sister centromere cohesion and microtubule interactions at kinetochores.},
	keywords = {Anaphase-Promoting Complex-Cyclosome, Animals, Cells, Cultured, Centromere, Chromosome Segregation, DNA, Complementary, Kinetochores, Microtubules, Mitosis, Molecular Sequence Data, Nuclear Proteins, Sequence Homology, Amino Acid, Sequence Homology, Nucleic Acid, Spindle Apparatus, Ubiquitin-Protein Ligase Complexes, Xenopus, Xenopus Proteins},
	issn = {0092-8674},
	doi = {10.1016/j.cell.2004.08.016},
	author = {Salic, Adrian and Waters, Jennifer C and Mitchison, Timothy J.}
}
@article {572886,
	title = {XRHAMM functions in ran-dependent microtubule nucleation and pole formation during anastral spindle assembly.},
	journal = {Curr Biol},
	volume = {14},
	number = {20},
	year = {2004},
	month = {2004 Oct 26},
	pages = {1801-11},
	abstract = {BACKGROUND: The regulated assembly of microtubules is essential for bipolar spindle formation. Depending on cell type, microtubules nucleate through two different pathways: centrosome-driven or chromatin-driven. The chromatin-driven pathway dominates in cells lacking centrosomes.

RESULTS: Human RHAMM (receptor for hyaluronic-acid-mediated motility) was originally implicated in hyaluronic-acid-induced motility but has since been shown to associate with centrosomes and play a role in astral spindle pole integrity in mitotic systems. We have identified the Xenopus ortholog of human RHAMM as a microtubule-associated protein that plays a role in focusing spindle poles and is essential for efficient microtubule nucleation during spindle assembly without centrosomes. XRHAMM associates both with gamma-TuRC, a complex required for microtubule nucleation and with TPX2, a protein required for microtubule nucleation and spindle pole organization.

CONCLUSIONS: XRHAMM facilitates Ran-dependent, chromatin-driven nucleation in a process that may require coordinate activation of TPX2 and gamma-TuRC.},
	keywords = {Animals, Base Sequence, Cell Cycle Proteins, Centrosome, Chromatography, Liquid, DNA Primers, Fluorescent Antibody Technique, Immunoblotting, Mass Spectrometry, Microtubule-Associated Proteins, Microtubules, Molecular Sequence Data, Neoplasm Proteins, Nuclear Proteins, Phosphoproteins, Plasmids, ran GTP-Binding Protein, Sequence Analysis, DNA, Spindle Apparatus, Xenopus, Xenopus Proteins},
	issn = {0960-9822},
	doi = {10.1016/j.cub.2004.10.002},
	author = {Groen, Aaron C and Cameron, Lisa A and Coughlin, Margaret and Miyamoto, David T and Mitchison, Timothy J. and Ohi, Ryoma}
}
@article {572841,
	title = {The kinesin Eg5 drives poleward microtubule flux in Xenopus laevis egg extract spindles.},
	journal = {J Cell Biol},
	volume = {167},
	number = {5},
	year = {2004},
	month = {2004 Dec 6},
	pages = {813-8},
	abstract = {Although mitotic and meiotic spindles maintain a steady-state length during metaphase, their antiparallel microtubules slide toward spindle poles at a constant rate. This "poleward flux" of microtubules occurs in many organisms and may provide part of the force for chromosome segregation. We use quantitative image analysis to examine the role of the kinesin Eg5 in poleward flux in metaphase Xenopus laevis egg extract spindles. Pharmacological inhibition of Eg5 results in a dose-responsive slowing of flux, and biochemical depletion of Eg5 significantly decreases the flux rate. Our results suggest that ensembles of nonprocessive Eg5 motors drive flux in metaphase Xenopus extract spindles.},
	keywords = {Animals, Cell Division, Cell Extracts, Cell Polarity, Chromosome Segregation, Cytokinesis, Dose-Response Relationship, Drug, Enzyme Inhibitors, Female, Image Cytometry, Kinesin, Metaphase, Microscopy, Fluorescence, Microtubules, Molecular Motor Proteins, Oocytes, Pyrimidines, Spindle Apparatus, Subcellular Fractions, Thiones, Xenopus laevis, Xenopus Proteins},
	issn = {0021-9525},
	doi = {10.1083/jcb.200407126},
	author = {Miyamoto, David T and Perlman, Zachary E and Burbank, Kendra S and Groen, Aaron C and Mitchison, Timothy J.}
}
@article {572866,
	title = {Kinetic mechanism of blebbistatin inhibition of nonmuscle myosin IIb.},
	journal = {Biochemistry},
	volume = {43},
	number = {46},
	year = {2004},
	month = {2004 Nov 23},
	pages = {14832-9},
	abstract = {We examined the effect of blebbistatin on the kinetic properties of nonmuscle myosin IIB subfragment 1 (NMIIB S1). Blebbistatin is a small molecule that affects cell blebbing during the process of cell division, which has been shown to decrease the myosin ATPase activity of a number of myosins [Straight et al. (2003) Science 299, 1743-1747]. The steady-state actin-activated ATPase activity of NMIIB S1 was decreased approximately 90\% at 40 microM actin in the presence of blebbistatin. Stopped-flow techniques were employed to elucidate the effect of blebbistatin on the various steps of the NMIIB S1 cross-bridge cycle. Blebbistatin did not affect ATP binding and hydrolysis. Binding to actin in the presence of ADP (0.57 +/-0.08 microM(-1) s(-1)) was reduced slightly in the presence of blebbistatin (0.38 +/- 0.03 microM(-1) s(-1)), while mantADP dissociation from acto-NMIIB S1 was reduced (approximately 30\%). P(i) release was blocked in the presence of blebbistatin. Accordingly, the apparent affinity of NMIIB S1 for actin in the presence of ATP was greatly reduced. Based on the above data, we surmise that blebbistatin inhibits the ATPase activity of NMIIB S1 primarily by blocking entry into the strong binding state; secondarily, it reduces the rate of ADP release. These effects are likely mediated by binding of blebbistatin within the myosin cleft that progressively closes in forming the acto-myosin rigor state.},
	keywords = {Actins, Adenosine Diphosphate, Adenosine Triphosphatases, Adenosine Triphosphate, Animals, Chickens, Heterocyclic Compounds with 4 or More Rings, Hydrolysis, Kinetics, Myosin Subfragments, Nonmuscle Myosin Type IIB, ortho-Aminobenzoates, Peptide Fragments, Phosphates, Photochemistry, Protein Binding, Pyrenes},
	issn = {0006-2960},
	doi = {10.1021/bi0490284},
	author = {Ramamurthy, Bhagavathi and Yengo, Christopher M and Straight, Aaron F and Mitchison, Timothy J. and Sweeney, H Lee}
}
@article {572861,
	title = {Parallel chemical genetic and genome-wide RNAi screens identify cytokinesis inhibitors and targets.},
	journal = {PLoS Biol},
	volume = {2},
	number = {12},
	year = {2004},
	month = {2004 Dec},
	pages = {e379},
	abstract = {Cytokinesis involves temporally and spatially coordinated action of the cell cycle and cytoskeletal and membrane systems to achieve separation of daughter cells. To dissect cytokinesis mechanisms it would be useful to have a complete catalog of the proteins involved, and small molecule tools for specifically inhibiting them with tight temporal control. Finding active small molecules by cell-based screening entails the difficult step of identifying their targets. We performed parallel chemical genetic and genome-wide RNA interference screens in Drosophila cells, identifying 50 small molecule inhibitors of cytokinesis and 214 genes important for cytokinesis, including a new protein in the Aurora B pathway (Borr). By comparing small molecule and RNAi phenotypes, we identified a small molecule that inhibits the Aurora B kinase pathway. Our protein list provides a starting point for systematic dissection of cytokinesis, a direction that will be greatly facilitated by also having diverse small molecule inhibitors, which we have identified. Dissection of the Aurora B pathway, where we found a new gene and a specific small molecule inhibitor, should benefit particularly. Our study shows that parallel RNA interference and small molecule screening is a generally useful approach to identifying active small molecules and their target pathways.},
	keywords = {Animals, Aurora Kinases, Caenorhabditis elegans, Cell Line, Cytokinesis, Drosophila, Formamides, Genetic Techniques, Genome, Genomics, Microscopy, Fluorescence, Phenotype, Protein-Serine-Threonine Kinases, Pyrazoles, RNA Interference, Saccharomyces cerevisiae},
	issn = {1545-7885},
	doi = {10.1371/journal.pbio.0020379},
	author = {Eggert, Ulrike S and Kiger, Amy A and Richter, Constance and Perlman, Zachary E and Perrimon, Norbert and Mitchison, Timothy J. and Field, Christine M}
}
@article {572846,
	title = {Poly(ADP-ribose) is required for spindle assembly and structure.},
	journal = {Nature},
	volume = {432},
	number = {7017},
	year = {2004},
	month = {2004 Dec 2},
	pages = {645-9},
	abstract = {The mitotic spindle is typically thought of as an array of microtubules, microtubule-associated proteins and motors that self-organizes to align and segregate chromosomes. The major spindle components consist of proteins and DNA, the primary structural elements of the spindle. Other macromolecules including RNA and lipids also associate with spindles, but their spindle function, if any, is unknown. Poly(ADP-ribose) (PAR) is a large, branched, negatively charged polymeric macromolecule whose polymerization onto acceptor proteins is catalysed by a family of poly(ADP-ribose) polymerases (PARPs). Several PARPs localize to the spindle in vertebrate cells, suggesting that PARPs and/or PAR have a role in spindle function. Here we show that PAR is enriched in the spindle and is required for spindle function--PAR hydrolysis or perturbation leads to rapid disruption of spindle structure, and hydrolysis during spindle assembly blocks the formation of bipolar spindles. PAR exhibits localization dynamics that differ from known spindle proteins and are consistent with a low rate of turnover in the spindle. Thus, PAR is a non-proteinaceous, non-chromosomal component of the spindle required for bipolar spindle assembly and function.},
	keywords = {Animals, Cattle, Cytoplasm, Glycoside Hydrolases, Hydrolysis, Kinesin, Nuclear Proteins, Oocytes, Poly Adenosine Diphosphate Ribose, Poly(ADP-ribose) Polymerases, Spindle Apparatus, Xenopus laevis, Xenopus Proteins},
	issn = {1476-4687},
	doi = {10.1038/nature03061},
	author = {Chang, Paul and Jacobson, Myron K and Mitchison, Timothy J.}
}
@article {572976,
	title = {Determining the position of the cell division plane.},
	journal = {Nature},
	volume = {424},
	number = {6952},
	year = {2003},
	month = {2003 Aug 28},
	pages = {1074-8},
	abstract = {Proper positioning of the cell division plane during mitosis is essential for determining the size and position of the two daughter cells--a critical step during development and cell differentiation. A bipolar microtubule array has been proposed to be a minimum requirement for furrow positioning in mammalian cells, with furrows forming at the site of microtubule plus-end overlap between the spindle poles. Observations in other species have suggested, however, that this may not be true. Here we show, by inducing mammalian tissue cells with monopolar spindles to enter anaphase, that furrow formation in cultured mammalian cells does not require a bipolar spindle. Unexpectedly, cytokinesis occurs at high frequency in monopolar cells. Division always occurs at a cortical position distal to the chromosomes. Analysis of microtubules during cytokinesis in cells with monopolar and bipolar spindles shows that a subpopulation of stable microtubules extends past chromosomes and binds to the cell cortex at the site of furrow formation. Our data are consistent with a model in which chromosomes supply microtubules with factors that promote microtubule stability and furrowing.},
	keywords = {Anaphase, Animals, Cell Differentiation, Cell Division, Cell Line, Cell Polarity, Chromosomes, Microtubules, Mitosis, Pyrimidines, Spindle Apparatus, Thiones},
	issn = {1476-4687},
	doi = {10.1038/nature01860},
	author = {Canman, Julie C and Cameron, Lisa A and Maddox, Paul S and Straight, Aaron and Tirnauer, Jennifer S and Mitchison, Timothy J. and Fang, Guowei and Kapoor, Tarun M and Salmon, E D}
}
@article {572981,
	title = {Direct observation of microtubule dynamics at kinetochores in Xenopus extract spindles: implications for spindle mechanics.},
	journal = {J Cell Biol},
	volume = {162},
	number = {3},
	year = {2003},
	month = {2003 Aug 4},
	pages = {377-82},
	abstract = {Microtubule plus ends dynamically attach to kinetochores on mitotic chromosomes. We directly imaged this dynamic interface using high resolution fluorescent speckle microscopy and direct labeling of kinetochores in Xenopus extract spindles. During metaphase, kinetochores were stationary and under tension while plus end polymerization and poleward microtubule flux (flux) occurred at velocities varying from 1.5-2.5 micro m/min. Because kinetochore microtubules polymerize at metaphase kinetochores, the primary source of kinetochore tension must be the spindle forces that produce flux and not a kinetochore-based mechanism. We infer that the kinetochore resists translocation of kinetochore microtubules through their attachment sites, and that the polymerization state of the kinetochore acts a "slip-clutch" mechanism that prevents detachment at high tension. At anaphase onset, kinetochores switched to depolymerization of microtubule plus ends, resulting in chromosome-to-pole rates transiently greater than flux. Kinetochores switched from persistent depolymerization to persistent polymerization and back again during anaphase, bistability exhibited by kinetochores in vertebrate tissue cells. These results provide the most complete description of spindle microtubule poleward flux to date, with important implications for the microtubule-kinetochore interface and for how flux regulates kinetochore function.},
	keywords = {Anaphase, Animals, Biomechanical Phenomena, Cell Extracts, Cell Movement, Cell Polarity, Chromosomes, Eukaryotic Cells, Female, Kinetochores, Metaphase, Microtubules, Mitosis, Oocytes, Polymers, Spindle Apparatus, Xenopus laevis},
	issn = {0021-9525},
	doi = {10.1083/jcb.200301088},
	author = {Maddox, Paul and Straight, Aaron and Coughlin, Peg and Mitchison, Timothy J. and Salmon, Edward D}
}
@article {572996,
	title = {Dissecting temporal and spatial control of cytokinesis with a myosin II Inhibitor.},
	journal = {Science},
	volume = {299},
	number = {5613},
	year = {2003},
	month = {2003 Mar 14},
	pages = {1743-7},
	abstract = {Completion of cell division during cytokinesis requires temporally and spatially regulated communication from the microtubule cytoskeleton to the actin cytoskeleton and the cell membrane. We identified a specific inhibitor of nonmuscle myosin II, blebbistatin, that inhibited contraction of the cleavage furrow without disrupting mitosis or contractile ring assembly. Using blebbistatin and other drugs, we showed that exit from the cytokinetic phase of the cell cycle depends on ubiquitin-mediated proteolysis. Continuous signals from microtubules are required to maintain the position of the cleavage furrow, and these signals control the localization of myosin II independently of other furrow components.},
	keywords = {Anaphase, Aurora Kinases, Cell Division, Cell Movement, Contractile Proteins, Cyclin-Dependent Kinases, DNA Replication, Enzyme Inhibitors, HeLa Cells, Heterocyclic Compounds with 4 or More Rings, Humans, Kinesin, Leupeptins, Microtubules, Mitosis, Myosin Type II, Nocodazole, Protein-Serine-Threonine Kinases, Staurosporine, Ubiquitin},
	issn = {1095-9203},
	doi = {10.1126/science.1081412},
	author = {Straight, Aaron F and Cheung, Amy and Limouze, John and Chen, Irene and Westwood, Nick J and Sellers, James R and Mitchison, Timothy J.}
}
@article {573001,
	title = {The force-velocity relationship for the actin-based motility of Listeria monocytogenes.},
	journal = {Curr Biol},
	volume = {13},
	number = {4},
	year = {2003},
	month = {2003 Feb 18},
	pages = {329-32},
	abstract = {The intracellular movement of the bacterial pathogen Listeria monocytogenes has helped identify key molecular constituents of actin-based motility (recent reviews ). However, biophysical as well as biochemical data are required to understand how these molecules generate the forces that extrude eukaryotic membranes. For molecular motors and for muscle, force-velocity curves have provided key biophysical data to distinguish between mechanistic theories. Here we manipulate and measure the viscoelastic properties of tissue extracts to provide the first force-velocity curve for Listeria monocytogenes. We find that the force-velocity relationship is highly curved, almost biphasic, suggesting a high cooperativity between biochemical catalysis and force generation. Using high-resolution motion tracking in low-noise extracts, we find long trajectories composed exclusively of molecular-sized steps. Robust statistics from these trajectories show a correlation between the duration of steps and macroscopic Listeria speed, but not between average step size and speed. Collectively, our data indicate how the molecular properties of the Listeria polymerization engine regulate speed, and that regulation occurs during molecular-scale pauses.},
	keywords = {Actins, Listeria monocytogenes},
	issn = {0960-9822},
	author = {McGrath, James L and Eungdamrong, Narat J and Fisher, Charles I and Peng, Fay and Mahadevan, Lakshminarayanan and Mitchison, Timothy J. and Kuo, Scot C}
}
@article {572971,
	title = {An inner centromere protein that stimulates the microtubule depolymerizing activity of a KinI kinesin.},
	journal = {Dev Cell},
	volume = {5},
	number = {2},
	year = {2003},
	month = {2003 Aug},
	pages = {309-21},
	abstract = {Mitosis requires precise control of microtubule dynamics. The KinI kinesin MCAK, a microtubule depolymerase, is critical for this regulation. In a screen to discover previously uncharacterized microtubule-associated proteins, we identified ICIS, a protein that stimulates MCAK activity in vitro. Consistent with this biochemical property, blocking ICIS function in Xenopus extracts with antibodies caused excessive microtubule growth and inhibited spindle formation. Prior to anaphase, ICIS localized in an MCAK-dependent manner to inner centromeres, the chromosomal region located in between sister kinetochores. From Xenopus extracts, ICIS coimmunoprecipitated MCAK and the inner centromere proteins INCENP and Aurora B, which are thought to promote chromosome biorientation. By immunoelectron microscopy, we found that ICIS is present on the surface of inner centromeres, placing it in an ideal location to depolymerize microtubules associated laterally with inner centromeres. At inner centromeres, MCAK-ICIS may destabilize these microtubules and provide a mechanism that prevents kinetochore-microtubule attachment errors.},
	keywords = {Amino Acid Sequence, Animals, Aurora Kinase B, Aurora Kinases, Cell Cycle Proteins, Cell Line, Centromere, Chromosomal Proteins, Non-Histone, Humans, Kinesin, Microtubule-Associated Proteins, Microtubules, Mitosis, Molecular Sequence Data, Oocytes, Protein-Serine-Threonine Kinases, S-Phase Kinase-Associated Proteins, Sequence Alignment, Spindle Apparatus, Tissue Extracts, Xenopus laevis, Xenopus Proteins},
	issn = {1534-5807},
	author = {Ohi, Ryoma and Coughlin, Margaret L and Lane, William S and Mitchison, Timothy J.}
}
@article {572991,
	title = {Phenotypic screening of small molecule libraries by high throughput cell imaging.},
	journal = {Comb Chem High Throughput Screen},
	volume = {6},
	number = {4},
	year = {2003},
	month = {2003 Jun},
	pages = {279-86},
	abstract = {We have developed high throughput fluorescence cell imaging methods to screen chemical libraries for compounds with effects on diverse aspects of cell physiology. We describe screens for compounds that arrest cells in mitosis, that block cell migration, and that block the secretory pathway. Each of these screens yielded specific inhibitors for research use, and the mitosis screen identified Eg5 as a potential target protein for cancer chemotherapy. Cell imaging provides a large amount of information from primary screening data that can be used to distinguish compounds with different effects on cells, and together with automated analysis, to quantitate compound effects.},
	keywords = {Animals, Combinatorial Chemistry Techniques, Eukaryotic Cells, Humans, Image Processing, Computer-Assisted, Microscopy, Fluorescence, Phenotype},
	issn = {1386-2073},
	author = {Yarrow, J C and Feng, Y and Perlman, Z. E. and Kirchhausen,T. and Mitchison, T. J.}
}
@article {572986,
	title = {Rapid de-localization of actin leading edge components with BDM treatment.},
	journal = {BMC Cell Biol},
	volume = {4},
	year = {2003},
	month = {2003 Jun 3},
	pages = {5},
	abstract = {BACKGROUND: 2,3-butanedione monoxime (BDM) has been widely used as a non-muscle myosin inhibitor to investigate the role of non-muscle myosinII in the process of actin retrograde flow and other actin cytoskeletal processes. Recent reports show that BDM does not inhibit any non-muscle myosins so far tested, including nm-myosinII, prompting the question, how were these process affected in BDM studies?

RESULTS: We have found that treatment of mammalian cells with BDM for only 1 min blocks actin incorporation at the leading edge in a permeabilized cell system. We show that inhibition of actin incorporation occurs through de-localization of leading edge proteins involved in actin polymerization--the Arp2/3 complex, WAVE, and VASP--that de-localize concomitantly with the leading edge actin network.

CONCLUSION: De-localization of actin leading edge components by BDM treatment is a newly described effect of this compound. It may explain many of the results previously ascribed to inhibition of non-muscle myosinII by BDM, particularly in studies of leading edge dynamics. Though this effect of BDM is intriguing, future studies probing actin dynamics at the leading edge should use more potent and specific inhibitors.},
	keywords = {Actin-Related Protein 3, Actins, Animals, Cell Adhesion Molecules, Cell Line, Cell Membrane Permeability, Cercopithecus aethiops, Cytoskeletal Proteins, Diacetyl, Kidney, Mice, Microfilament Proteins, Myosin Type II, Phosphoproteins, Swiss 3T3 Cells, Wiskott-Aldrich Syndrome Protein Family},
	issn = {1471-2121},
	doi = {10.1186/1471-2121-4-5},
	author = {Yarrow, Justin C and Lechler, Terry and Li, Rong and Mitchison, Timothy J.}
}
@article {572966,
	title = {Dynamics of the mitotic spindle--potential therapeutic targets.},
	journal = {Prog Cell Cycle Res},
	volume = {5},
	year = {2003},
	month = {2003},
	pages = {349-60},
	abstract = {Inhibition of mitosis is a useful strategy for treating diseases involving excessive cell proliferation. Antimitotic drugs currently in clinical use perturb microtubule dynamics and thereby disrupt the function of the mitotic spindle. Protein regulators of microtubule dynamics and microtubule motors are also essential for mitotic spindle function. In this chapter, we evaluate the potential of these proteins as candidate targets for antimitotic drugs. We review in depth a number of proteins of particular interest, highlighting their known functions in mitosis and the effects of their inhibition on cell cycle progression.},
	keywords = {Animals, Antineoplastic Agents, Cell Cycle Proteins, Cell Division, Humans, Microtubules, Molecular Motor Proteins, Spindle Apparatus},
	issn = {1087-2957},
	author = {Miyamoto, David T and Perlman, Zachary E and Mitchison, Timothy J. and Shirasu-Hiza, Mimi}
}
@article {573026,
	title = {Cytoskeleton: what does GTP do for septins?},
	journal = {Curr Biol},
	volume = {12},
	number = {22},
	year = {2002},
	month = {2002 Nov 19},
	pages = {R788-90},
	abstract = {The recent observation of GTP-promoted polymerization of a single septin polypeptide suggests that this protein has tubulin-like biochemical properties. This model cannot, however, explain the GTP-biochemistry of heteromeric septin complexes from cytosol.},
	keywords = {Animals, Cytoskeleton, GTP-Binding Proteins, Guanosine Triphosphate, Mammals, Protein Folding, Recombinant Proteins},
	issn = {0960-9822},
	author = {Mitchison, Timothy J. and Field, Christine M}
}
@article {573036,
	title = {EB1-microtubule interactions in Xenopus egg extracts: role of EB1 in microtubule stabilization and mechanisms of targeting to microtubules.},
	journal = {Mol Biol Cell},
	volume = {13},
	number = {10},
	year = {2002},
	month = {2002 Oct},
	pages = {3614-26},
	abstract = {EB1 targets to polymerizing microtubule ends, where it is favorably positioned to regulate microtubule polymerization and confer molecular recognition of the microtubule end. In this study, we focus on two aspects of the EB1-microtubule interaction: regulation of microtubule dynamics by EB1 and the mechanism of EB1 association with microtubules. Immunodepletion of EB1 from cytostatic factor-arrested M-phase Xenopus egg extracts dramatically reduced microtubule length; this was complemented by readdition of EB1. By time-lapse microscopy, EB1 increased the frequency of microtubule rescues and decreased catastrophes, resulting in increased polymerization and decreased depolymerization and pausing. Imaging of EB1 fluorescence revealed a novel structure: filamentous extensions on microtubule plus ends that appeared during microtubule pauses; loss of these extensions correlated with the abrupt onset of polymerization. Fluorescent EB1 localized to comets at the polymerizing plus ends of microtubules in cytostatic factor extracts and uniformly along the lengths of microtubules in interphase extracts. The temporal decay of EB1 fluorescence from polymerizing microtubule plus ends predicted a dissociation half-life of seconds. Fluorescence recovery after photobleaching also revealed dissociation and rebinding of EB1 to the microtubule wall with a similar half-life. EB1 targeting to microtubules is thus described by a combination of higher affinity binding to polymerizing ends and lower affinity binding along the wall, with continuous dissociation. The latter is likely to be attenuated in interphase. The highly conserved effect of EB1 on microtubule dynamics suggests it belongs to a core set of regulatory factors conserved in higher organisms, and the complex pattern of EB1 targeting to microtubules could be exploited by the cell for coordinating microtubule behaviors.},
	keywords = {Animals, Cell Cycle, Cell Polarity, Centrosome, Fluorescence Recovery After Photobleaching, Humans, Microtubule-Associated Proteins, Microtubules, Oocytes, Polymers, Protein Binding, Tubulin, Xenopus laevis},
	issn = {1059-1524},
	doi = {10.1091/mbc.E02-04-0210},
	author = {Tirnauer, Jennifer S and Grego, Sonia and Salmon, E D and Mitchison, Timothy J.}
}
@article {573046,
	title = {Evidence that monastrol is an allosteric inhibitor of the mitotic kinesin Eg5.},
	journal = {Chem Biol},
	volume = {9},
	number = {9},
	year = {2002},
	month = {2002 Sep},
	pages = {989-96},
	abstract = {Monastrol, a cell-permeable inhibitor of the kinesin Eg5, has been used to probe the dynamic organization of the mitotic spindle. The mechanism by which monastrol inhibits Eg5 function is unknown. We found that monastrol inhibits both the basal and the microtubule-stimulated ATPase activity of the Eg5 motor domain. Unlike many ATPase inhibitors, monastrol does not compete with ATP binding to Eg5. Monastrol appears to inhibit microtubule-stimulated ADP release from Eg5 but does not compete with microtubule binding, suggesting that monastrol binds a novel allosteric site in the motor domain. Finally, we established that (S)-monastrol, as compared to the (R)-enantiomer, is a more potent inhibitor of Eg5 activity in vitro and in vivo. Future structural studies should help in designing more potent Eg5 inhibitors for possible use as anticancer drugs and cell biological reagents.},
	keywords = {Adenosine Diphosphate, Adenosine Triphosphate, Allosteric Regulation, Animals, Cells, Cultured, Cercopithecus aethiops, Enzyme Inhibitors, Fluorescent Dyes, Humans, Hydrolysis, Kidney, Kinesin, Kinetics, Microtubules, Models, Biological, Protein Structure, Tertiary, Pyrimidines, Spectrometry, Fluorescence, Stereoisomerism, Thiones, Xenopus Proteins},
	issn = {1074-5521},
	author = {Maliga, Zoltan and Kapoor, Tarun M and Mitchison, Timothy J.}
}
@article {573021,
	title = {Mammalian septins nomenclature.},
	journal = {Mol Biol Cell},
	volume = {13},
	number = {12},
	year = {2002},
	month = {2002 Dec},
	pages = {4111-3},
	abstract = {There are 10 known mammalian septin genes, some of which produce multiple splice variants. The current nomenclature for the genes and gene products is very confusing, with several different names having been given to the same gene product and distinct names given to splice variants of the same gene. Moreover, some names are based on those of yeast or Drosophila septins that are not the closest homologues. Therefore, we suggest that the mammalian septin field adopt a common nomenclature system, based on that adopted by the Mouse Genomic Nomenclature Committee and accepted by the Human Genome Organization Gene Nomenclature Committee. The human and mouse septin genes will be named SEPT1-SEPT10 and Sept1-Sept10, respectively. Splice variants will be designated by an underscore followed by a lowercase "v" and a number, e.g., SEPT4_v1.},
	keywords = {Alternative Splicing, Animals, Cytoskeletal Proteins, Fungal Proteins, GTP Phosphohydrolases, GTP-Binding Proteins, Humans, Phylogeny, Protein Structure, Tertiary, Septins, Terminology as Topic},
	issn = {1059-1524},
	doi = {10.1091/mbc.E02-07-0438},
	author = {Macara, Ian G and Baldarelli, Richard and Field, Christine M and Glotzer, Michael and Hayashi, Yasuhide and Hsu, Shu-Chan and Kennedy, Mary B and Kinoshita, Makoto and Longtine, Mark and Low, Claudia and Maltais, Lois J and McKenzie, Louise and Mitchison, Timothy J. and Nishikawa, Toru and Noda, Makoto and Petty, Elizabeth M and Peifer, Mark and Pringle, John R and Robinson, Phillip J and Roth, Dagmar and Russell, S E Hilary and Stuhlmann, Heidi and Tanaka, Manami and Tanaka, Tomoo and Trimble, William S and Ware, Jerry and Zeleznik-Le, Nancy J and Zieger, Barbara}
}
@article {573031,
	title = {The microtubule-destabilizing kinesin XKCM1 is required for chromosome positioning during spindle assembly.},
	journal = {Curr Biol},
	volume = {12},
	number = {21},
	year = {2002},
	month = {2002 Oct 29},
	pages = {1885-9},
	abstract = {Xenopus kinesin catastrophe modulator-1 (XKCM1) is a Kin I kinesin family member that uses the energy of ATP hydrolysis to depolymerize microtubules. We demonstrated previously that XKCM1 is essential for mitotic-spindle assembly in vitro and acts by regulating microtubule dynamics as a pure protein, in extracts and in cells. A portion of the XKCM1 pool is specifically localized to centromeres during mitosis and may be important in chromosome movement. To selectively analyze the function of centromere-bound XKCM1, we generated glutathione-S-transferase (GST) fusion proteins containing the N-terminal globular domain (GST-NT), the centrally located catalytic domain (GST-CD), and the C-terminal alpha-helical tail (GST-CT) of XKCM1. The GST-NT protein targeted to centromeres during spindle assembly, suggesting that the N-terminal domain of XKCM1 is sufficient for centromere localization. Addition of GST-NT prior to or after spindle assembly replaced endogenous XKCM1, indicating that centromere targeting is a dynamic process. Loss of endogenous XKCM1 from centromeres caused a misalignment of chromosomes on the metaphase plate without affecting global spindle structure. These results suggest that centromere bound XKCM1 has an important role in chromosome positioning on the spindle.},
	keywords = {Amino Acid Sequence, Animals, Centromere, Glutathione Transferase, Kinesin, Molecular Sequence Data, Sequence Homology, Amino Acid, Spindle Apparatus, Xenopus, Xenopus Proteins},
	issn = {0960-9822},
	author = {Walczak, Claire E and Gan, Eugene C and Desai, Arshad and Mitchison, Timothy J. and Kline-Smith, Susan L}
}
@article {573041,
	title = {Poleward microtubule flux is a major component of spindle dynamics and anaphase a in mitotic Drosophila embryos.},
	journal = {Curr Biol},
	volume = {12},
	number = {19},
	year = {2002},
	month = {2002 Oct 1},
	pages = {1670-4},
	abstract = {During cell division, eukaryotic cells assemble dynamic microtubule-based spindles to segregate replicated chromosomes. Rapid spindle microtubule turnover, likely derived from dynamic instability, has been documented in yeasts, plants and vertebrates. Less studied is concerted spindle microtubule poleward translocation (flux) coupled to depolymerization at spindle poles. Microtubule flux has been observed only in vertebrates, although there is indirect evidence for it in insect spermatocytes and higher plants. Here we use fluorescent speckle microscopy (FSM) to demonstrate that mitotic spindles of syncytial Drosophila embryos exhibit poleward microtubule flux, indicating that flux is a widely conserved property of spindles. By simultaneously imaging chromosomes (or kinetochores) and flux, we provide evidence that flux is the dominant mechanism driving chromosome-to-pole movement (anaphase A) in these spindles. At 18 degrees C and 24 degrees C, separated sister chromatids moved poleward at average rates (3.6 and 6.6 microm/min, respectively) slightly greater than the mean rates of poleward flux (3.2 and 5.2 microm/min, respectively). However, at 24 degrees C the rate of kinetochore-to-pole movement varied from slower than to twice the mean rate of flux, suggesting that although flux is the dominant mechanism, kinetochore-associated microtubule depolymerization contributes to anaphase A.},
	keywords = {Anaphase, Animals, Chromosome Segregation, Drosophila, Embryo, Nonmammalian, Kinetochores, Microscopy, Fluorescence, Microtubules, Mitosis, Spindle Apparatus},
	issn = {0960-9822},
	author = {Maddox, Paul and Desai, Arshad and Oegema, Karen and Mitchison, Timothy J. and Salmon, E D}
}
@article {573051,
	title = {Single-molecule speckle analysis of actin filament turnover in lamellipodia.},
	journal = {Science},
	volume = {295},
	number = {5557},
	year = {2002},
	month = {2002 Feb 8},
	pages = {1083-6},
	abstract = {Lamellipodia are thin, veil-like extensions at the edge of cells that contain a dynamic array of actin filaments. We describe an approach for analyzing spatial regulation of actin polymerization and depolymerization in vivo in which we tracked single molecules of actin fused to the green fluorescent protein. Polymerization and the lifetime of actin filaments in lamellipodia were measured with high spatial precision. Basal polymerization and depolymerization occurred throughout lamellipodia with largely constant kinetics, and polymerization was promoted within one micron of the lamellipodium tip. Most of the actin filaments in the lamellipodium were generated by polymerization away from the tip.},
	keywords = {Actin Cytoskeleton, Actin-Related Protein 2, Actin-Related Protein 3, Actins, Animals, Biopolymers, Cell Line, Cytoskeletal Proteins, Depsipeptides, Fibroblasts, Fluorescence, Green Fluorescent Proteins, Half-Life, Luminescent Proteins, Models, Biological, Peptides, Cyclic, Pseudopodia, Recombinant Fusion Proteins, Xenopus},
	issn = {1095-9203},
	doi = {10.1126/science.1067470},
	author = {Watanabe, Naoki and Mitchison, Timothy J.}
}
@article {573056,
	title = {A small-molecule inhibitor of skeletal muscle myosin II.},
	journal = {Nat Cell Biol},
	volume = {4},
	number = {1},
	year = {2002},
	month = {2002 Jan},
	pages = {83-8},
	abstract = {We screened a small-molecule library for inhibitors of rabbit muscle myosin II subfragment 1 (S1) actin-stimulated ATPase activity. The best inhibitor, N-benzyl-p-toluene sulphonamide (BTS), an aryl sulphonamide, inhibited the Ca2+-stimulated S1 ATPase, and reversibly blocked gliding motility. Although BTS does not compete for the nucleotide-binding site of myosin, it weakens myosin{\textquoteright}s interaction with F-actin. BTS reversibly suppressed force production in skinned skeletal muscle fibres from rabbit and frog skin at micromolar concentrations. BTS suppressed twitch production of intact frog fibres with minimum alteration of Ca2+ metabolism. BTS is remarkably specific, as it was much less effective in suppressing contraction in rat myocardial or rabbit slow-twitch muscle, and did not inhibit platelet myosin II. The isolation of BTS and the recently discovered Eg5 kinesin inhibitor, monastrol, suggests that motor proteins may be potential targets for therapeutic applications.},
	keywords = {Adenosine Triphosphatases, Animals, Calcium, Enzyme Inhibitors, In Vitro Techniques, Molecular Motor Proteins, Muscle Contraction, Muscle, Skeletal, Myosin Subfragments, Peptide Library, Rabbits, Ranidae, Rats, Skeletal Muscle Myosins, Sulfonamides, Toluene},
	issn = {1465-7392},
	doi = {10.1038/ncb734},
	author = {Cheung, A and Dantzig, J A and Hollingworth, S and Baylor, S M and Goldman, Y E and Mitchison, T. J. and Straight, A F}
}
@article {573016,
	title = {EB1 targets to kinetochores with attached, polymerizing microtubules.},
	journal = {Mol Biol Cell},
	volume = {13},
	number = {12},
	year = {2002},
	month = {2002 Dec},
	pages = {4308-16},
	abstract = {Microtubule polymerization dynamics at kinetochores is coupled to chromosome movements, but its regulation there is poorly understood. The plus end tracking protein EB1 is required both for regulating microtubule dynamics and for maintaining a euploid genome. To address the role of EB1 in aneuploidy, we visualized its targeting in mitotic PtK1 cells. Fluorescent EB1, which localized to polymerizing ends of astral and spindle microtubules, was used to track their polymerization. EB1 also associated with a subset of attached kinetochores in late prometaphase and metaphase, and rarely in anaphase. Localization occurred in a narrow crescent, concave toward the centromere, consistent with targeting to the microtubule plus end-kinetochore interface. EB1 did not localize to kinetochores lacking attached kinetochore microtubules in prophase or early prometaphase, or upon nocodazole treatment. By time lapse, EB1 specifically targeted to kinetochores moving antipoleward, coupled to microtubule plus end polymerization, and not during plus end depolymerization. It localized independently of spindle bipolarity, the spindle checkpoint, and dynein/dynactin function. EB1 is the first protein whose targeting reflects kinetochore directionality, unlike other plus end tracking proteins that show enhanced kinetochore binding in the absence of microtubules. Our results suggest EB1 may modulate kinetochore microtubule polymerization and/or attachment.},
	keywords = {Cell Nucleus, Cloning, Molecular, Dyneins, Fluorescent Antibody Technique, Indirect, Humans, Kinetochores, Microscopy, Fluorescence, Microtubule-Associated Proteins, Microtubules, Protein Binding},
	issn = {1059-1524},
	doi = {10.1091/mbc.E02-04-0236},
	author = {Tirnauer, Jennifer S and Canman, Julie C and Salmon, E D and Mitchison, Timothy J.}
}
@article {573011,
	title = {Self- and actin-templated assembly of Mammalian septins.},
	journal = {Dev Cell},
	volume = {3},
	number = {6},
	year = {2002},
	month = {2002 Dec},
	pages = {791-802},
	abstract = {Septins are polymerizing GTPases required for cytokinesis and cortical organization. The principles by which they are targeted to, and assemble at, specific cell regions are unknown. We show that septins in mammalian cells switch between a linear organization along actin bundles and cytoplasmic rings, approximately 0.6 microm in diameter. A recombinant septin complex self-assembles into rings resembling those in cells. Linear organization along actin bundles was reconstituted by adding an adaptor protein, anillin. Perturbation of septin organization in cells by expression of a septin-interacting fragment of anillin or by septin depletion via siRNA causes loss of actin bundles. We conclude that septins alone self-assemble into rings, that adaptor proteins recruit septins to actin bundles, and that septins help organize these bundles.},
	keywords = {3T3 Cells, Actin Cytoskeleton, Adaptor Proteins, Signal Transducing, Animals, Binding Sites, Blood Proteins, Carrier Proteins, cdc42 GTP-Binding Protein, Cell Compartmentation, Contractile Proteins, Cytoskeletal Proteins, Eukaryotic Cells, Green Fluorescent Proteins, GTP Phosphohydrolase Activators, GTP Phosphohydrolases, GTP-Binding Protein Regulators, GTP-Binding Proteins, HeLa Cells, Humans, Luminescent Proteins, Mice, Protein Structure, Tertiary, Recombinant Fusion Proteins, Septins},
	issn = {1534-5807},
	author = {Kinoshita, Makoto and Field, Christine M and Coughlin, Margaret L and Straight, Aaron F and Mitchison, Timothy J.}
}
@article {573006,
	title = {Small molecules, big impact: a history of chemical inhibitors and the cytoskeleton.},
	journal = {Chem Biol},
	volume = {9},
	number = {12},
	year = {2002},
	month = {2002 Dec},
	pages = {1275-85},
	abstract = {Chemical inhibitors, whether natural products or synthetic, have had an enormous impact on the study of the eukaryotic cytoskeleton. Here we review the history of some of the most widely used cytoskeletal poisons and their influence on our understanding of cytoskeletal functions. We then highlight several new inhibitors and the targeted screens used to identify them and discuss why this approach has been successful.},
	keywords = {Animals, Cell Size, Cytoskeletal Proteins, Cytoskeleton, Humans, Microtubules},
	issn = {1074-5521},
	author = {Peterson, Jeffrey R and Mitchison, Timothy J.}
}
@article {572806,
	title = {Probing cell division with "chemical genetics".},
	journal = {Harvey Lect},
	volume = {98},
	year = {2002},
	month = {2002-2003},
	pages = {19-40},
	keywords = {Animals, Cell Division, Combinatorial Chemistry Techniques, Cytokinesis, Drug Evaluation, Preclinical, Heterocyclic Compounds with 4 or More Rings, Humans, Mitosis, Models, Biological, Myosin Type II, Phenotype},
	issn = {0073-0874},
	author = {Mitchison, T. J.}
}
@article {573076,
	title = {Mitosis: a history of division.},
	journal = {Nat Cell Biol},
	volume = {3},
	number = {1},
	year = {2001},
	month = {2001 Jan},
	pages = {E17-21},
	abstract = {Mitosis has been studied since the early 1880s, to the extent that we now have a detailed, but still incomplete, description of spindle dynamics and mechanics, a sense of potential mechanochemical and regulatory mechanisms at a molecular level, and a long list of mitotic proteins. Here we present a personal view of how far we have come, and where we need to go to fully understand the mechanisms involved in mitosis.},
	keywords = {Animals, Genetics, History, 19th Century, History, 20th Century, Humans, Mitosis},
	issn = {1465-7392},
	doi = {10.1038/35050656},
	author = {Mitchison, T. J. and Salmon, E D}
}
@article {573071,
	title = {Psychosine, cytokinesis, and orphan receptors. Unexpected connections.},
	journal = {J Cell Biol},
	volume = {153},
	number = {2},
	year = {2001},
	month = {2001 Apr 16},
	pages = {F1-3},
	keywords = {Animals, Cell Division, Cell Line, GTP-Binding Proteins, Humans, Leukodystrophy, Globoid Cell, Lipid Metabolism, Molecular Structure, Psychosine, Receptors, Cell Surface},
	issn = {0021-9525},
	author = {Mitchison, T. J.}
}
@article {573066,
	title = {A chemical inhibitor of N-WASP reveals a new mechanism for targeting protein interactions.},
	journal = {Proc Natl Acad Sci U S A},
	volume = {98},
	number = {19},
	year = {2001},
	month = {2001 Sep 11},
	pages = {10624-9},
	abstract = {Cell morphology and motility are governed largely by complex signaling networks that ultimately engage the actin cytoskeleton. Understanding how individual circuits contribute to the process of forming cellular structures would be aided greatly by the availability of specific chemical inhibitors. We have used a novel chemical screen in Xenopus cell-free extracts to identify compounds that inhibit signaling pathways regulating actin polymerization. Here we report the results of a high-throughput screen for compounds that inhibit phosphatidylinositol 4,5-bisphosphate (PIP(2))-induced actin assembly and the identification of the first compound, a cyclic peptide, known to block actin assembly by inhibiting an upstream signaling component. We identify the target of this compound as N-WASP, a protein that has been investigated for its role as a node interconnecting various actin signaling networks. We show that this compound prevents activation of the Arp2/3 complex by N-WASP by allosterically stabilizing the autoinhibited conformation of N-WASP.},
	keywords = {Actin-Related Protein 2, Actin-Related Protein 3, Actins, Animals, cdc42 GTP-Binding Protein, Cytoskeletal Proteins, Guanosine 5{\textquoteright}-O-(3-Thiotriphosphate), Nerve Tissue Proteins, Peptides, Cyclic, Phosphatidylinositol 4,5-Diphosphate, Polymers, Wiskott-Aldrich Syndrome Protein, Neuronal, Xenopus laevis},
	issn = {0027-8424},
	doi = {10.1073/pnas.201393198},
	author = {Peterson, J R and Lokey, R S and Mitchison, T. J. and Kirschner, M W}
}
@article {573061,
	title = {Eg5 is static in bipolar spindles relative to tubulin: evidence for a static spindle matrix.},
	journal = {J Cell Biol},
	volume = {154},
	number = {6},
	year = {2001},
	month = {2001 Sep 17},
	pages = {1125-33},
	abstract = {We used fluorescent speckle microscopy to probe the dynamics of the mitotic kinesin Eg5 in Xenopus extract spindles, and compared them to microtubule dynamics. We found significant populations of Eg5 that were static over several seconds while microtubules flux towards spindle poles. Eg5 dynamics are frozen by adenylimidodiphosphate. Bulk turnover experiments showed that Eg5 can exchange between the spindle and the extract with a half life of ,
	keywords = {Adenosine Triphosphatases, Adenylyl Imidodiphosphate, Animals, Cytoplasm, Humans, Kinesin, Kinetics, Microscopy, Fluorescence, Microtubule-Associated Proteins, Microtubules, Molecular Motor Proteins, Movement, Pyrimidines, Spindle Apparatus, Thiones, Xenopus, Xenopus Proteins},
	issn = {0021-9525},
	doi = {10.1083/jcb.200106011},
	author = {Kapoor, T M and Mitchison, T. J.}
}
@article {573096,
	title = {Actin-dependent propulsion of endosomes and lysosomes by recruitment of N-WASP.},
	journal = {J Cell Biol},
	volume = {148},
	number = {3},
	year = {2000},
	month = {2000 Feb 7},
	pages = {519-30},
	abstract = {We examined the spatial and temporal control of actin assembly in living Xenopus eggs. Within minutes of egg activation, dynamic actin-rich comet tails appeared on a subset of cytoplasmic vesicles that were enriched in protein kinase C (PKC), causing the vesicles to move through the cytoplasm. Actin comet tail formation in vivo was stimulated by the PKC activator phorbol myristate acetate (PMA), and this process could be reconstituted in a cell-free system. We used this system to define the characteristics that distinguish vesicles associated with actin comet tails from other vesicles in the extract. We found that the protein, N-WASP, was recruited to the surface of every vesicle associated with an actin comet tail, suggesting that vesicle movement results from actin assembly nucleated by the Arp2/3 complex, the immediate downstream target of N-WASP. The motile vesicles accumulated the dye acridine orange, a marker for endosomes and lysosomes. Furthermore, vesicles associated with actin comet tails had the morphological features of multivesicular endosomes as revealed by electron microscopy. Endosomes and lysosomes from mammalian cells preferentially nucleated actin assembly and moved in the Xenopus egg extract system. These results define endosomes and lysosomes as recruitment sites for the actin nucleation machinery and demonstrate that actin assembly contributes to organelle movement. Conversely, by nucleating actin assembly, intracellular membranes may contribute to the dynamic organization of the actin cytoskeleton.},
	keywords = {Actins, Animals, Cell-Free System, Endosomes, Female, HeLa Cells, Humans, Intracellular Membranes, Isoenzymes, Lysosomes, Male, Nerve Tissue Proteins, Ovum, Protein Kinase C, Protein Kinase C-alpha, Recombinant Fusion Proteins, Tetradecanoylphorbol Acetate, Wiskott-Aldrich Syndrome Protein, Neuronal, Xenopus},
	issn = {0021-9525},
	author = {Taunton, J and Rowning, B A and Coughlin, M L and M. Wu and Moon, R T and Mitchison, T. J. and Larabell, C A}
}
@article {573091,
	title = {Dissecting cellular processes using small molecules: identification of colchicine-like, taxol-like and other small molecules that perturb mitosis.},
	journal = {Chem Biol},
	volume = {7},
	number = {4},
	year = {2000},
	month = {2000 Apr},
	pages = {275-86},
	abstract = {BACKGROUND: Understanding the molecular mechanisms of complex cellular processes requires unbiased means to identify and to alter conditionally gene products that function in a pathway of interest. Although random mutagenesis and screening (forward genetics) provide a useful means to this end, the complexity of the genome, long generation time and redundancy of gene function have limited their use with mammalian systems. We sought to develop an analogous process using small molecules to modulate conditionally the function of proteins. We hoped to identify simultaneously small molecules that may serve as leads for the development of therapeutically useful agents.

RESULTS: We report the results of a high-throughput, phenotype-based screen for identifying cell-permeable small molecules that affect mitosis of mammalian cells. The predominant class of compounds that emerged directly alters the stability of microtubules in the mitotic spindle. Although many of these compounds show the colchicine-like property of destabilizing microtubules, one member shows the taxol-like property of stabilizing microtubules. Another class of compounds alters chromosome segregation by novel mechanisms that do not involve direct interactions with microtubules.

CONCLUSIONS: The identification of structurally diverse small molecules that affect the mammalian mitotic machinery from a large library of synthetic compounds illustrates the use of chemical genetics in dissecting an essential cellular pathway. This screen identified five compounds that affect mitosis without directly targeting microtubules. Understanding the mechanism of action of these compounds, along with future screening efforts, promises to help elucidate the molecular mechanisms involved in chromosome segregation during mitosis.},
	keywords = {Animals, Cell Line, Chromosomes, Colchicine, Drug Evaluation, Preclinical, Humans, Microscopy, Fluorescence, Microtubules, Mitosis, Molecular Structure, Paclitaxel, Spindle Apparatus, Tubulin},
	issn = {1074-5521},
	author = {Haggarty, S J and Mayer, T U and Miyamoto, D T and Fathi, R and R. W. King and Mitchison, T. J. and Schreiber, S L}
}
@article {573086,
	title = {Functional analysis of a human homologue of the Drosophila actin binding protein anillin suggests a role in cytokinesis.},
	journal = {J Cell Biol},
	volume = {150},
	number = {3},
	year = {2000},
	month = {2000 Aug 7},
	pages = {539-52},
	abstract = {We have characterized a human homologue of anillin, a Drosophila actin binding protein. Like Drosophila anillin, the human protein localizes to the nucleus during interphase, the cortex following nuclear envelope breakdown, and the cleavage furrow during cytokinesis. Anillin also localizes to ectopic cleavage furrows generated between two spindles in fused PtK(1) cells. Microinjection of antianillin antibodies slows cleavage, leading to furrow regression and the generation of multinucleate cells. GFP fusions that contain the COOH-terminal 197 amino acids of anillin, which includes a pleckstrin homology (PH) domain, form ectopic cortical foci during interphase. The septin Hcdc10 localizes to these ectopic foci, whereas myosin II and actin do not, suggesting that anillin interacts with the septins at the cortex. Robust cleavage furrow localization requires both this COOH-terminal domain and additional NH(2)-terminal sequences corresponding to an actin binding domain defined by in vitro cosedimentation assays. Endogenous anillin and Hcdc10 colocalize to punctate foci associated with actin cables throughout mitosis and the accumulation of both proteins at the cell equator requires filamentous actin. These results indicate that anillin is a conserved cleavage furrow component important for cytokinesis. Interactions with at least two other furrow proteins, actin and the septins, likely contribute to anillin function.},
	keywords = {Amino Acid Sequence, Animals, Binding Sites, Blood Proteins, Cell Compartmentation, Cell Cycle Proteins, Cell Division, Conserved Sequence, Contractile Proteins, Drosophila, Evolution, Molecular, Humans, Microfilament Proteins, Molecular Sequence Data, Phosphoproteins, Protein Structure, Tertiary, Recombinant Fusion Proteins, Septins, Sequence Homology, Amino Acid},
	issn = {0021-9525},
	author = {Oegema, K and Savoian, M S and Mitchison, T. J. and Field, C. M.}
}
@article {573081,
	title = {Probing spindle assembly mechanisms with monastrol, a small molecule inhibitor of the mitotic kinesin, Eg5.},
	journal = {J Cell Biol},
	volume = {150},
	number = {5},
	year = {2000},
	month = {2000 Sep 4},
	pages = {975-88},
	abstract = {Monastrol, a cell-permeable small molecule inhibitor of the mitotic kinesin, Eg5, arrests cells in mitosis with monoastral spindles. Here, we use monastrol to probe mitotic mechanisms. We find that monastrol does not inhibit progression through S and G2 phases of the cell cycle or centrosome duplication. The mitotic arrest due to monastrol is also rapidly reversible. Chromosomes in monastrol-treated cells frequently have both sister kinetochores attached to microtubules extending to the center of the monoaster (syntelic orientation). Mitotic arrest-deficient protein 2 (Mad2) localizes to a subset of kinetochores, suggesting the activation of the spindle assembly checkpoint in these cells. Mad2 localizes to some kinetochores that have attached microtubules in monastrol-treated cells, indicating that kinetochore microtubule attachment alone may not satisfy the spindle assembly checkpoint. Monastrol also inhibits bipolar spindle formation in Xenopus egg extracts. However, it does not prevent the targeting of Eg5 to the monoastral spindles that form. Imaging bipolar spindles disassembling in the presence of monastrol allowed direct observations of outward directed forces in the spindle, orthogonal to the pole-to-pole axis. Monastrol is thus a useful tool to study mitotic processes, detection and correction of chromosome malorientation, and contributions of Eg5 to spindle assembly and maintenance.},
	keywords = {Animals, Antineoplastic Agents, Calcium-Binding Proteins, Carrier Proteins, Cell Cycle, Cell Cycle Proteins, Cell Line, Cell-Free System, Centrosome, Chromosomes, Fungal Proteins, Kinesin, Microtubules, Mitosis, Nuclear Proteins, Oocytes, Pyrimidines, Spindle Apparatus, Thiones, Xenopus laevis, Xenopus Proteins},
	issn = {0021-9525},
	author = {Kapoor, T M and Mayer, T U and Coughlin, M L and Mitchison, T. J.}
}
@article {573136,
	title = {The bipolar kinesin, KLP61F, cross-links microtubules within interpolar microtubule bundles of Drosophila embryonic mitotic spindles.},
	journal = {J Cell Biol},
	volume = {144},
	number = {1},
	year = {1999},
	month = {1999 Jan 11},
	pages = {125-38},
	abstract = {Previous genetic and biochemical studies have led to the hypothesis that the essential mitotic bipolar kinesin, KLP61F, cross-links and slides microtubules (MTs) during spindle assembly and function. Here, we have tested this hypothesis by immunofluorescence and immunoelectron microscopy (immunoEM). We show that Drosophila embryonic spindles at metaphase and anaphase contain abundant bundles of MTs running between the spindle poles. These interpolar MT bundles are parallel near the poles and antiparallel in the midzone. We have observed that KLP61F motors, phosphorylated at a cdk1/cyclin B consensus domain within the BimC box (BCB), localize along the length of these interpolar MT bundles, being concentrated in the midzone region. Nonphosphorylated KLP61F motors, in contrast, are excluded from the spindle and display a cytoplasmic localization. Immunoelectron microscopy further suggested that phospho-KLP61F motors form cross-links between MTs within interpolar MT bundles. These bipolar KLP61F MT-MT cross-links should be capable of organizing parallel MTs into bundles within half spindles and sliding antiparallel MTs apart in the spindle midzone. Thus we propose that bipolar kinesin motors and MTs interact by a "sliding filament mechanism" during the formation and function of the mitotic spindle.},
	keywords = {Anaphase, Animals, Cross-Linking Reagents, Drosophila melanogaster, Drosophila Proteins, Freezing, Kinesin, Metaphase, Microscopy, Immunoelectron, Microtubule-Associated Proteins, Microtubules, Phosphorylation, Rabbits, Spindle Apparatus},
	issn = {0021-9525},
	author = {Sharp, D J and McDonald, K L and Brown, H M and Matthies, H J and Walczak, C and Vale, R D and Mitchison, T. J. and Scholey, J M}
}
@article {573121,
	title = {Characterization of two related Drosophila gamma-tubulin complexes that differ in their ability to nucleate microtubules.},
	journal = {J Cell Biol},
	volume = {144},
	number = {4},
	year = {1999},
	month = {1999 Feb 22},
	pages = {721-33},
	abstract = {gamma-tubulin exists in two related complexes in Drosophila embryo extracts (Moritz, M., Y. Zheng, B.M. Alberts, and K. Oegema. 1998. J. Cell Biol. 142:1- 12). Here, we report the purification and characterization of both complexes that we name gamma-tubulin small complex (gammaTuSC; approximately 280,000 D) and Drosophila gammaTuRC ( approximately 2,200,000 D). In addition to gamma-tubulin, the gammaTuSC contains Dgrip84 and Dgrip91, two proteins homologous to the Spc97/98p protein family. The gammaTuSC is a structural subunit of the gammaTuRC, a larger complex containing about six additional polypeptides. Like the gammaTuRC isolated from Xenopus egg extracts (Zheng, Y., M.L. Wong, B. Alberts, and T. Mitchison. 1995. Nature. 378:578-583), the Drosophila gammaTuRC can nucleate microtubules in vitro and has an open ring structure with a diameter of 25 nm. Cryo-electron microscopy reveals a modular structure with approximately 13 radially arranged structural repeats. The gammaTuSC also nucleates microtubules, but much less efficiently than the gammaTuRC, suggesting that assembly into a larger complex enhances nucleating activity. Analysis of the nucleotide content of the gammaTuSC reveals that gamma-tubulin binds preferentially to GDP over GTP, rendering gamma-tubulin an unusual member of the tubulin superfamily.},
	keywords = {Amino Acid Sequence, Animals, Cryoelectron Microscopy, Drosophila, Guanosine Diphosphate, Guanosine Triphosphate, Insect Proteins, Macromolecular Substances, Microtubules, Molecular Sequence Data, Molecular Weight, Protein Binding, Tubulin, Xenopus},
	issn = {0021-9525},
	author = {Oegema, K and Wiese, C and Martin, O C and Milligan, R A and Iwamatsu, A and Mitchison, T. J. and Zheng, Y}
}
@article {573126,
	title = {Kin I kinesins are microtubule-destabilizing enzymes.},
	journal = {Cell},
	volume = {96},
	number = {1},
	year = {1999},
	month = {1999 Jan 8},
	pages = {69-78},
	abstract = {Using in vitro assays with purified proteins, we show that XKCM1 and XKIF2, two distinct members of the internal catalytic domain (Kin I) kinesin subfamily, catalytically destabilize microtubules using a novel mechanism. Both XKCM1 and XKIF2 influence microtubule stability by targeting directly to microtubule ends where they induce a destabilizing conformational change. ATP hydrolysis recycles XKCM1/XKIF2 for multiple rounds of action by dissociating a XKCM1/ XKIF2-tubulin dimer complex released upon microtubule depolymerization. These results establish Kin I kinesins as microtubule-destabilizing enzymes, distinguish them mechanistically from kinesin superfamily members that use ATP hydrolysis to translocate along microtubules, and have important implications for the regulation of microtubule dynamics and for the intracellular functions and evolution of the kinesin superfamily.},
	keywords = {Animals, Catalytic Domain, Dimerization, Kinesin, Microtubules, Peptides, Protein Conformation, Tubulin, Xenopus, Xenopus Proteins},
	issn = {0092-8674},
	author = {Desai, A and Verma, S and Mitchison, T. J. and Walczak, C E}
}
@article {573131,
	title = {The use of Xenopus egg extracts to study mitotic spindle assembly and function in vitro.},
	journal = {Methods Cell Biol},
	volume = {61},
	year = {1999},
	month = {1999},
	pages = {385-412},
	keywords = {Anaphase, Animals, Cell Extracts, Mitosis, Ovum, Proto-Oncogene Proteins c-mos, Spindle Apparatus, Xenopus},
	issn = {0091-679X},
	author = {Desai, A and A Murray and Mitchison, T. J. and Walczak, C E}
}
@article {573116,
	title = {Allele-specific activators and inhibitors for kinesin.},
	journal = {Proc Natl Acad Sci U S A},
	volume = {96},
	number = {16},
	year = {1999},
	month = {1999 Aug 3},
	pages = {9106-11},
	abstract = {Members of the kinesin superfamily are force-generating ATPases that drive movement and influence cytoskeleton organization in cells. Often, more than one kinesin is implicated in a cellular process, and many kinesins are proposed to have overlapping functions. By using conventional kinesin as a model system, we have developed an approach to activate or inhibit a specific kinesin allele in the presence of other similar motor proteins. Modified ATP analogs are described that do not activate either conventional kinesin or another superfamily member, Eg5. However, a kinesin allele with Arg-14 in its nucleotide binding pocket mutated to alanine can use a subset of these nucleotide analogs to drive microtubule gliding. Cyclopentyl-ATP is one such analog. Cyclopentyl-adenylylimidodiphosphate, a nonhydrolyzable form of this analog, inhibits the mutant allele in microtubule-gliding assays, but not wild-type kinesin or Eg5. We anticipate that the incorporation of kinesin mutants and allele-specific activators and inhibitors in in vitro assays should clarify the role of individual motor proteins in complex cellular processes.},
	keywords = {Adenosine Diphosphate, Adenosine Triphosphate, Adenylyl Imidodiphosphate, Binding Sites, Dimerization, Humans, Kinesin, Kinetics, Macromolecular Substances, Models, Molecular, Mutagenesis, Site-Directed, Recombinant Proteins, Structure-Activity Relationship},
	issn = {0027-8424},
	author = {Kapoor, T M and Mitchison, T. J.}
}
@article {573101,
	title = {Dynamic confocal imaging of mitochondria in swimming Tetrahymena and of microtubule poleward flux in Xenopus extract spindles.},
	journal = {Biol Bull},
	volume = {197},
	number = {2},
	year = {1999},
	month = {1999 Oct},
	pages = {263-5},
	keywords = {Animals, Microscopy, Confocal, Mitochondria, Spindle Apparatus, Tetrahymena, Xenopus laevis},
	issn = {0006-3185},
	author = {Maddox, P and Desai, A and Salmon, E D and Mitchison, T. J. and Oogema, K and Kapoor, T and Matsumoto, B and Inou{\'e}, S}
}
@article {573111,
	title = {Small molecule inhibitor of mitotic spindle bipolarity identified in a phenotype-based screen.},
	journal = {Science},
	volume = {286},
	number = {5441},
	year = {1999},
	month = {1999 Oct 29},
	pages = {971-4},
	abstract = {Small molecules that perturb specific protein functions are valuable tools for dissecting complex processes in mammalian cells. A combination of two phenotype-based screens, one based on a specific posttranslational modification, the other visualizing microtubules and chromatin, was used to identify compounds that affect mitosis. One compound, here named monastrol, arrested mammalian cells in mitosis with monopolar spindles. In vitro, monastrol specifically inhibited the motility of the mitotic kinesin Eg5, a motor protein required for spindle bipolarity. All previously known small molecules that specifically affect the mitotic machinery target tubulin. Monastrol will therefore be a particularly useful tool for studying mitotic mechanisms.},
	keywords = {Actins, Animals, Cattle, Cell Line, Cytoskeleton, Golgi Apparatus, Kinesin, Microtubules, Mitosis, Molecular Motor Proteins, Phenotype, Phosphoproteins, Protein Processing, Post-Translational, Pyrimidines, RNA-Binding Proteins, Spindle Apparatus, Thiones, Tumor Cells, Cultured, Xenopus, Xenopus Proteins},
	issn = {0036-8075},
	author = {Mayer, T U and Kapoor, T M and Haggarty, S J and R. W. King and Schreiber, S L and Mitchison, T. J.}
}
@article {573106,
	title = {Spatial control of actin polymerization during neutrophil chemotaxis.},
	journal = {Nat Cell Biol},
	volume = {1},
	number = {2},
	year = {1999},
	month = {1999 Jun},
	pages = {75-81},
	abstract = {Neutrophils respond to chemotactic stimuli by increasing the nucleation and polymerization of actin filaments, but the location and regulation of these processes are not well understood. Here, using a permeabilized-cell assay, we show that chemotactic stimuli cause neutrophils to organize many discrete sites of actin polymerization, the distribution of which is biased by external chemotactic gradients. Furthermore, the Arp2/3 complex, which can nucleate actin polymerization, dynamically redistributes to the region of living neutrophils that receives maximal chemotactic stimulation, and the least-extractable pool of the Arp2/3 complex co-localizes with sites of actin polymerization. Our observations indicate that chemoattractant-stimulated neutrophils may establish discrete foci of actin polymerization that are similar to those generated at the posterior surface of the intracellular bacterium Listeria monocytogenes. We propose that asymmetrical establishment and/or maintenance of sites of actin polymerization produces directional migration of neutrophils in response to chemotactic gradients.},
	keywords = {Actin-Related Protein 2, Actins, Animals, Cell Membrane, Cell Membrane Permeability, Cell Polarity, Chemotaxis, Leukocyte, Cytoskeletal Proteins, Humans, Listeria monocytogenes, N-Formylmethionine Leucyl-Phenylalanine, Neutrophils, Rabbits, Signal Transduction},
	issn = {1465-7392},
	doi = {10.1038/10042},
	author = {Weiner, O D and Servant, G and Welch, M D and Mitchison, T. J. and Sedat, J W and Bourne, H R}
}
@article {573186,
	title = {Actin, cofilin and cognition.},
	journal = {Nature},
	volume = {393},
	number = {6687},
	year = {1998},
	month = {1998 Jun 25},
	pages = {739-40},
	keywords = {Actin Depolymerizing Factors, Actins, Cell Movement, Cognition, DNA-Binding Proteins, Humans, Lim Kinases, Microfilament Proteins, Phosphorylation, Polymers, Protein Kinases, Protein-Serine-Threonine Kinases, Williams Syndrome},
	issn = {0028-0836},
	doi = {10.1038/31597},
	author = {Rosenblatt, J and Mitchison, T. J.}
}
@article {573196,
	title = {Anaphase A chromosome movement and poleward spindle microtubule flux occur At similar rates in Xenopus extract spindles.},
	journal = {J Cell Biol},
	volume = {141},
	number = {3},
	year = {1998},
	month = {1998 May 4},
	pages = {703-13},
	abstract = {We have used local fluorescence photoactivation to mark the lattice of spindle microtubules during anaphase A in Xenopus extract spindles. We find that both poleward spindle microtubule flux and anaphase A chromosome movement occur at similar rates ( approximately 2 microm/min). This result suggests that poleward microtubule flux, coupled to microtubule depolymerization near the spindle poles, is the predominant mechanism for anaphase A in Xenopus egg extracts. In contrast, in vertebrate somatic cells a "Pacman" kinetochore mechanism, coupled to microtubule depolymerization near the kinetochore, predominates during anaphase A. Consistent with the conclusion from fluorescence photoactivation analysis, both anaphase A chromosome movement and poleward spindle microtubule flux respond similarly to pharmacological perturbations in Xenopus extracts. Furthermore, the pharmacological profile of anaphase A in Xenopus extracts differs from the previously established profile for anaphase A in vertebrate somatic cells. The difference between these profiles is consistent with poleward microtubule flux playing the predominant role in anaphase chromosome movement in Xenopus extracts, but not in vertebrate somatic cells. We discuss the possible biological implications of the existence of two distinct anaphase A mechanisms and their differential contributions to poleward chromosome movement in different cell types.},
	keywords = {Adenylyl Imidodiphosphate, Anaphase, Animals, Chromosomes, Enzyme Inhibitors, Kinetochores, Metaphase, Microtubules, Ovum, Protein Kinase Inhibitors, Protein Kinases, Spindle Apparatus, Xenopus},
	issn = {0021-9525},
	author = {Desai, A and Maddox, P S and Mitchison, T. J. and Salmon, E D}
}
@article {573181,
	title = {Caged fluorescent probes.},
	journal = {Methods Enzymol},
	volume = {291},
	year = {1998},
	month = {1998},
	pages = {63-78},
	keywords = {Animals, Fluorescent Dyes, Microscopy, Fluorescence, Molecular Structure, Photochemistry, Proteins},
	issn = {0076-6879},
	author = {Mitchison, T. J. and Sawin, K E and Theriot, J A and Gee, K and Mallavarapu, A}
}
@article {573191,
	title = {Interaction of human Arp2/3 complex and the Listeria monocytogenes ActA protein in actin filament nucleation.},
	journal = {Science},
	volume = {281},
	number = {5373},
	year = {1998},
	month = {1998 Jul 3},
	pages = {105-8},
	abstract = {Actin filament assembly at the cell surface of the pathogenic bacterium Listeria monocytogenes requires the bacterial ActA surface protein and the host cell Arp2/3 complex. Purified Arp2/3 complex accelerated the nucleation of actin polymerization in vitro, but pure ActA had no effect. However, when combined, the Arp2/3 complex and ActA synergistically stimulated the nucleation of actin filaments. This mechanism of activating the host Arp2/3 complex at the L. monocytogenes surface may be similar to the strategy used by cells to control Arp2/3 complex activity and hence the spatial and temporal distribution of actin polymerization.},
	keywords = {Actin-Related Protein 2, Actin-Related Protein 3, Actins, Bacterial Proteins, Biopolymers, Cell Membrane, Cytochalasin D, Cytoskeletal Proteins, Humans, Kinetics, Listeria monocytogenes, Membrane Proteins, Microscopy, Electron},
	issn = {0036-8075},
	author = {Welch, M D and Rosenblatt, J and Skoble, J and Portnoy, D A and Mitchison, T. J.}
}
@article {573176,
	title = {A model for the proposed roles of different microtubule-based motor proteins in establishing spindle bipolarity.},
	journal = {Curr Biol},
	volume = {8},
	number = {16},
	year = {1998},
	month = {1998 Jul 30-Aug },
	pages = {903-13},
	abstract = {BACKGROUND: In eukaryotes, assembly of the mitotic spindle requires the interaction of chromosomes with microtubules. During this process, several motor proteins that move along microtubules promote formation of a bipolar microtubule array, but the precise mechanism is unclear. In order to examine the roles of different motor proteins in building a bipolar spindle, we have used a simplified system in which spindles assemble around beads coated with plasmid DNA and incubated in extracts from Xenopus eggs. Using this system, we can study spindle assembly in the absence of paired cues, such as centrosomes and kinetochores, whose microtubule-organizing properties might mask the action of motor proteins.

RESULTS: We blocked the function of individual motor proteins in the Xenopus extracts using specific antibodies. Inhibition of Xenopus kinesin-like protein 1 (Xklp1) led either to the dissociation of chromatin beads from microtubule arrays, or to collapsed microtubule bundles on beads. Inhibition of Eg5 resulted in monopolar microtubule arrays emanating from chromatin beads. Addition of antibodies against dynein inhibited the focusing of microtubule ends into spindle poles in a dose-dependent manner. Inhibition of Xenopus carboxy-terminal kinesin 2 (XCTK2) affected both pole formation and spindle stability. Co-inhibition of XCTK2 and dynein dramatically increased the severity of spindle pole defects. Inhibition of Xklp2 caused only minor spindle pole defects.

CONCLUSIONS: Multiple microtubule-based motor activities are required for the bipolar organization of microtubules around chromatin beads, and we propose a model for the roles of the individual motor proteins in this process.},
	keywords = {Animals, Cell Polarity, Chromatin, Female, Kinesin, Meiosis, Metaphase, Microtubule-Associated Proteins, Microtubules, Models, Biological, Oocytes, Spindle Apparatus, Tissue Extracts, Xenopus laevis, Xenopus Proteins},
	issn = {0960-9822},
	author = {Walczak, C E and Vernos, I and Mitchison, T. J. and Karsenti, E and Heald, R}
}
@article {573171,
	title = {Tubulin and FtsZ structures: functional and therapeutic implications.},
	journal = {Bioessays},
	volume = {20},
	number = {7},
	year = {1998},
	month = {1998 Jul},
	pages = {523-7},
	abstract = {The microtubule cytoskeleton has lagged nearly a decade behind the actin cytoskeleton with respect to structural information on the basic polymer subunit. This structural inferiority complex has finally been lifted by two recent papers describing the structures of the alpha beta tubulin dimer and FtsZ, a protein similar to tubulin that is essential for cell division in prokaryotes.},
	keywords = {Bacterial Proteins, Binding Sites, Cell Division, Cytoskeletal Proteins, Dimerization, Guanosine Triphosphate, Models, Molecular, Paclitaxel, Prokaryotic Cells, Protein Conformation, Tubulin},
	issn = {0265-9247},
	doi = {10.1002/(SICI)1521-1878(199807)20:7<523::AID-BIES1>3.0.CO;2-L},
	author = {Desai, A and Mitchison, T. J.}
}
@article {573146,
	title = {An efficient substitution reaction for the preparation of thyroid hormone analogues.},
	journal = {Bioorg Med Chem},
	volume = {6},
	number = {8},
	year = {1998},
	month = {1998 Aug},
	pages = {1179-83},
	abstract = {The substitution of the sterically hindered carbon of the potent thyroid hormone agonist, GC-1, was effected by a reaction based on the solvolysis of the benzylic hydroxyl group. The reaction was found to proceed in high yield with a variety of nucleophiles including alcohols, thiols, allyl silanes and electron-rich aromatic compounds, providing a convenient route to the synthesis of new thyroid hormone analogues.},
	keywords = {Benzophenones, Ligands, Receptors, Thyroid Hormone, Thyroid Hormones, Triiodothyronine},
	issn = {0968-0896},
	author = {Yoshihara, H A and Chiellini, G and Mitchison, T. J. and Scanlan, T S}
}
@article {573141,
	title = {Polymerization of purified yeast septins: evidence that organized filament arrays may not be required for septin function.},
	journal = {J Cell Biol},
	volume = {143},
	number = {3},
	year = {1998},
	month = {1998 Nov 2},
	pages = {737-49},
	abstract = {The septins are a family of proteins required for cytokinesis in a number of eukaryotic cell types. In budding yeast, these proteins are thought to be the structural components of a filament system present at the mother-bud neck, called the neck filaments. In this study, we report the isolation of a protein complex containing the yeast septins Cdc3p, Cdc10p, Cdc11p, and Cdc12p that is capable of forming long filaments in vitro. To investigate the relationship between these filaments and the neck filaments, we purified septin complexes from cells deleted for CDC10 or CDC11. These complexes were not capable of the polymerization exhibited by wild-type preparations, and analysis of the neck region by electron microscopy revealed that the cdc10Delta and cdc11Delta cells did not contain detectable neck filaments. These results strengthen the hypothesis that the septins are the major structural components of the neck filaments. Surprisingly, we found that septin dependent processes like cytokinesis and the localization of Bud4p to the neck still occurred in cdc10Delta cells. This suggests that the septins may be able to function in the absence of normal polymerization and the formation of a higher order filament structure.},
	keywords = {Cell Cycle Proteins, Cytoskeletal Proteins, Fungal Proteins, GTP Phosphohydrolases, GTP-Binding Proteins, Membrane Proteins, Polymers, Profilins, Recombinant Fusion Proteins, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, Schizosaccharomyces pombe Proteins, Transcription Factors},
	issn = {0021-9525},
	author = {Frazier, J A and Wong, M L and Longtine, M S and Pringle, J R and Mann, M and Mitchison, T. J. and Field, C}
}
@article {573161,
	title = {Preparation and characterization of caged fluorescein tubulin.},
	journal = {Methods Enzymol},
	volume = {298},
	year = {1998},
	month = {1998},
	pages = {125-32},
	keywords = {Animals, Brain, Cattle, Fluoresceins, Fluorescent Dyes, Hydrogen-Ion Concentration, Microinjections, Microscopy, Fluorescence, Microtubules, Molecular Probes, Photosensitizing Agents, Polymers, Spectrophotometry, Tubulin, Ultraviolet Rays, Xenopus},
	issn = {0076-6879},
	author = {Desai, A and Mitchison, T. J.}
}
@article {573156,
	title = {Purification and assay of a septin complex from Drosophila embryos.},
	journal = {Methods Enzymol},
	volume = {298},
	year = {1998},
	month = {1998},
	pages = {279-95},
	keywords = {Ammonium Sulfate, Animals, Anion Exchange Resins, Cation Exchange Resins, Cell Cycle Proteins, Chemical Precipitation, Chromatography, Chromatography, Affinity, Chromatography, Agarose, Cross-Linking Reagents, Drosophila melanogaster, Drosophila Proteins, Durapatite, Guanosine Triphosphate, Insect Proteins, Microfilament Proteins, Polymers, Resins, Synthetic, Ultraviolet Rays},
	issn = {0076-6879},
	author = {Oegema, K and Desai, A and Wong, M L and Mitchison, T. J. and Field, C. M.}
}
@article {573166,
	title = {Purification and assay of the platelet Arp2/3 complex.},
	journal = {Methods Enzymol},
	volume = {298},
	year = {1998},
	month = {1998},
	pages = {52-61},
	keywords = {Actin-Related Protein 2, Actin-Related Protein 3, Actins, Blood Platelets, Cell Extracts, Centrifugation, Chromatography, Agarose, Chromatography, Gel, Cytoskeletal Proteins, Cytoskeleton, Indoles, Listeria, Microscopy, Fluorescence, Octoxynol, Solubility},
	issn = {0076-6879},
	author = {Welch, M D and Mitchison, T. J.}
}
@article {573151,
	title = {Purification of cytoskeletal proteins using peptide antibodies.},
	journal = {Methods Enzymol},
	volume = {298},
	year = {1998},
	month = {1998},
	pages = {525-41},
	keywords = {Animals, Antibodies, Antibody Specificity, Antigen-Antibody Complex, Cell Cycle Proteins, Chromatography, Chromatography, Affinity, Cytoskeletal Proteins, Dithionitrobenzoic Acid, Drosophila melanogaster, Epitopes, Hemocyanin, Kinesin, Ovum, Peptides, Precipitin Tests, Rabbits, Solubility, Staphylococcal Protein A, Tubulin, Xenopus laevis},
	issn = {0076-6879},
	author = {Field, C. M. and Oegema, K and Zheng, Y and Mitchison, T. J. and Walczak, C E}
}
@article {573231,
	title = {Actin dynamics in vivo.},
	journal = {Curr Opin Cell Biol},
	volume = {9},
	number = {1},
	year = {1997},
	month = {1997 Feb},
	pages = {54-61},
	abstract = {Actin dynamics in lamellipodia are driven by continuous cycles of actin polymerization, retrograde flow, and depolymerization. In the past year, advances have been made in identifying signaling pathways that regulate actin-filament uncapping and polymerization, in determining the role of myosin motor proteins in retrograde flow, and in evaluating the role of severing proteins in actin depolymerization. Both Listeria monocytogenes and Saccharomyces cerevisiae have emerged as powerful model organisms for studying actin dynamics in cells.},
	keywords = {Actins, Bacterial Proteins, Fungal Proteins},
	issn = {0955-0674},
	author = {Welch, M D and Mallavarapu, A and Rosenblatt, J and Mitchison, T. J.}
}
@article {573236,
	title = {Actin polymerization is induced by Arp2/3 protein complex at the surface of Listeria monocytogenes.},
	journal = {Nature},
	volume = {385},
	number = {6613},
	year = {1997},
	month = {1997 Jan 16},
	pages = {265-9},
	abstract = {The pathogenic bacterium Listeria monocytogenes is capable of directed movement within the cytoplasm of infected host cells. Propulsion is thought to be driven by actin polymerization at the bacterial cell surface, and moving bacteria leave in their wake a tail of actin filaments. Determining the mechanism by which L. monocytogenes polymerizes actin may aid the understanding of how actin polymerization is controlled in the cell. Actin assembly by L. monocytogenes requires the bacterial surface protein ActA and protein components present in host cell cytoplasm. We have purified an eight-polypeptide complex that possesses the properties of the host-cell actin polymerization factor. The pure complex is sufficient to initiate ActA-dependent actin polymerization at the surface of L. monocytogenes, and is required to mediate actin tail formation and motility. Two subunits of this protein complex are actin-related proteins (ARPs) belonging to the Arp2 and Arp3 subfamilies. The Arp3 subunit localizes to the surface of stationary bacteria and the tails of motile bacteria in tissue culture cells infected with L. monocytogenes; this is consistent with a role for the complex in promoting actin assembly in vivo. The activity and subunit composition of the Arp2/3 complex suggests that it forms a template that nucleates actin polymerization.},
	keywords = {Actin-Related Protein 2, Actin-Related Protein 3, Actins, Amino Acid Sequence, Animals, Bacterial Proteins, Biopolymers, Blood Platelets, Cell Membrane, Cytoskeletal Proteins, HeLa Cells, Humans, Listeria monocytogenes, Membrane Proteins, Molecular Sequence Data, Movement, Xenopus laevis},
	issn = {0028-0836},
	doi = {10.1038/385265a0},
	author = {Welch, M D and Iwamatsu, A and Mitchison, T. J.}
}
@article {573221,
	title = {Identification of novel graded polarity actin filament bundles in locomoting heart fibroblasts: implications for the generation of motile force.},
	journal = {J Cell Biol},
	volume = {136},
	number = {6},
	year = {1997},
	month = {1997 Mar 24},
	pages = {1287-305},
	abstract = {We have determined the structural organization and dynamic behavior of actin filaments in entire primary locomoting heart fibroblasts by S1 decoration, serial section EM, and photoactivation of fluorescence. As expected, actin filaments in the lamellipodium of these cells have uniform polarity with barbed ends facing forward. In the lamella, cell body, and tail there are two observable types of actin filament organization. A less abundant type is located on the inner surface of the plasma membrane and is composed of short, overlapping actin bundles (0.25-2.5 microm) that repeatedly alternate in polarity from uniform barbed ends forward to uniform pointed ends forward. This type of organization is similar to the organization we show for actin filament bundles (stress fibers) in nonlocomoting cells (PtK2 cells) and to the known organization of muscle sarcomeres. The more abundant type of actin filament organization in locomoting heart fibroblasts is mostly ventrally located and is composed of long, overlapping bundles (average 13 microm, but can reach up to about 30 microm) which span the length of the cell. This more abundant type has a novel graded polarity organization. In each actin bundle, polarity gradually changes along the length of the bundle. Actual actin filament polarity at any given point in the bundle is determined by position in the cell; the closer to the front of the cell the more barbed ends of actin filaments face forward. By photoactivation marking in locomoting heart fibroblasts, as expected in the lamellipodium, actin filaments flow rearward with respect to substrate. In the lamella, all marked and observed actin filaments remain stationary with respect to substrate as the fibroblast locomotes. In the cell body of locomoting fibroblasts there are two dynamic populations of actin filaments: one remains stationary and the other moves forward with respect to substrate at the rate of the cell body. This is the first time that the structural organization and dynamics of actin filaments have been determined in an entire locomoting cell. The organization, dynamics, and relative abundance of graded polarity actin filament bundles have important implications for the generation of motile force during primary heart fibroblast locomotion.},
	keywords = {Actin Cytoskeleton, Actins, Animals, Cell Line, Cell Movement, Cell Polarity, Chick Embryo, Fibroblasts, Kidney, Macropodidae, Microscopy, Electron, Microscopy, Fluorescence, Myocardial Contraction, Myocardium, Organ Specificity, Photochemistry},
	issn = {0021-9525},
	author = {Cramer, L P and Siebert, M and Mitchison, T. J.}
}
@article {573246,
	title = {Investigation of the mechanism of retraction of the cell margin and rearward flow of nodules during mitotic cell rounding.},
	journal = {Mol Biol Cell},
	volume = {8},
	number = {1},
	year = {1997},
	month = {1997 Jan},
	pages = {109-19},
	abstract = {We have studied two types of cell motility directed toward the cell center: retraction of the cell margin and rearward flow of small cytoplasmic nodules during mitotic cell rounding in Potoroo tridactylis kidney (PtK2) cells by time-lapse video microscopy, drug treatments, and photoactivation of fluorescence. Nodules flow rearward on thin, actin-rich fibers (retraction fibers) exposed as the cell margin retracts. Retraction of the cell margin and rearward flow of nodules require intact actin filaments, but are insensitive to an inhibitor of myosin function (butanedione monoxime). Using photoactivation of fluorescence marking, we have determined that actin filaments in the majority of retraction fibers remain stationary while the cell margin retracts and nodules flow rearward. The pointed ends of retraction fiber actin filaments face the cell center. We argue that nodule motility is driven by a novel actin-based force that perhaps also partially contributes to retraction of the cell margin during cell rounding at mitosis.},
	keywords = {Actins, Animals, Cell Movement, Cells, Cultured, Cytochalasin D, Cytoskeletal Proteins, Diacetyl, Fluorescence, Kidney, Microscopy, Video, Mitosis, Nucleic Acid Synthesis Inhibitors},
	issn = {1059-1524},
	author = {Cramer, L P and Mitchison, T. J.}
}
@article {573241,
	title = {Microtubule polymerization dynamics.},
	journal = {Annu Rev Cell Dev Biol},
	volume = {13},
	year = {1997},
	month = {1997},
	pages = {83-117},
	abstract = {The polymerization dynamics of microtubules are central to their biological functions. Polymerization dynamics allow microtubules to adopt spatial arrangements that can change rapidly in response to cellular needs and, in some cases, to perform mechanical work. Microtubules utilize the energy of GTP hydrolysis to fuel a unique polymerization mechanism termed dynamic instability. In this review, we first describe progress toward understanding the mechanism of dynamic instability of pure tubulin and then discuss the function and regulation of microtubule dynamic instability in living cells.},
	keywords = {Animals, Dimerization, Guanosine Triphosphate, Microtubule-Associated Proteins, Microtubules, Polymers, Thermodynamics, Tubulin},
	issn = {1081-0706},
	doi = {10.1146/annurev.cellbio.13.1.83},
	author = {Desai, A and Mitchison, T. J.}
}
@article {573226,
	title = {XCTK2: a kinesin-related protein that promotes mitotic spindle assembly in Xenopus laevis egg extracts.},
	journal = {J Cell Biol},
	volume = {136},
	number = {4},
	year = {1997},
	month = {1997 Feb 24},
	pages = {859-70},
	abstract = {We used a peptide antibody to a conserved sequence in the motor domain of kinesins to screen a Xenopus ovary cDNA expression library. Among the clones isolated were two that encoded a protein we named XCTK2 for Xenopus COOH-terminal kinesin 2. XCTK2 contains an NH2-terminal globular domain, a central alpha-helical stalk, and a COOH-terminal motor domain. XCTK2 is similar to CTKs in other organisms and is most homologous to CHO2. Antibodies raised against XCTK2 recognize a 75-kD protein in Xenopus egg extracts that cosediments with microtubules. In Xenopus tissue culture cells, the anti-XCTK2 antibodies stain mitotic spindles as well as a subset of interphase nuclei. To probe the function of XCTK2, we have used an in vitro assay for spindle assembly in Xenopus egg extracts. Addition of antibodies to cytostatic factor-arrested extracts causes a 70\% reduction in the percentage of bipolar spindles formed. XCTK2 is not required for maintenance of bipolar spindles, as antibody addition to preformed spindles has no effect. To further evaluate the function of XCTK2, we expressed XCTK2 in insect Sf-9 cells using the baculovirus expression system. When purified (recombinant XCTK2 is added to Xenopus egg extracts at a fivefold excess over endogenous levels) there is a stimulation in both the rate and extent of bipolar spindle formation. XCTK2 exists in a large complex in extracts and can be coimmunoprecipitated with two other proteins from extracts. XCTK2 likely plays an important role in the establishment and structural integrity of mitotic spindles.},
	keywords = {Amino Acid Sequence, Animals, Cell Extracts, Kinesin, Macromolecular Substances, Molecular Sequence Data, Ovum, Spindle Apparatus, Xenopus laevis, Xenopus Proteins},
	issn = {0021-9525},
	author = {Walczak, C E and Verma, S and Mitchison, T. J.}
}
@article {573206,
	title = {The human Arp2/3 complex is composed of evolutionarily conserved subunits and is localized to cellular regions of dynamic actin filament assembly.},
	journal = {J Cell Biol},
	volume = {138},
	number = {2},
	year = {1997},
	month = {1997 Jul 28},
	pages = {375-84},
	abstract = {The Arp2/3 protein complex has been implicated in the control of actin polymerization in cells. The human complex consists of seven subunits which include the actin related proteins Arp2 and Arp3, and five others referred to as p41-Arc, p34-Arc, p21-Arc, p20-Arc, and p16-Arc (p omplex). We have determined the predicted amino acid sequence of all seven subunits. Each has homologues in diverse eukaryotes, implying that the structure and function of the complex has been conserved through evolution. Human Arp2 and Arp3 are very similar to family members from other species. p41-Arc is a new member of the Sop2 family of WD (tryptophan and aspartate) repeat-containing proteins and may be posttranslationally modified, suggesting that it may be involved in regulating the activity and/or localization of the complex. p34-Arc, p21-Arc, p20-Arc, and p16-Arc define novel protein families. We sought to evaluate the function of the Arp2/3 complex in cells by determining its intracellular distribution. Arp3, p34-Arc, and p21-Arc were localized to the lamellipodia of stationary and locomoting fibroblasts, as well to Listeria monocytogenes assembled actin tails. They were not detected in cellular bundles of actin filaments. Taken together with the ability of the Arp2/3 complex to induce actin polymerization, these observations suggest that the complex promotes actin assembly in lamellipodia and may participate in lamellipodial protrusion.},
	keywords = {Actin Cytoskeleton, Actin-Related Protein 2, Actin-Related Protein 2-3 Complex, Actin-Related Protein 3, Actins, Amino Acid Sequence, Animals, Carrier Proteins, Cell Movement, Chick Embryo, Cytoskeletal Proteins, Fibroblasts, Humans, Listeria monocytogenes, Microfilament Proteins, Molecular Sequence Data, Pseudopodia, Sequence Homology, Amino Acid},
	issn = {0021-9525},
	author = {Welch, M D and DePace, A H and Verma, S and Iwamatsu, A and Mitchison, T. J.}
}
@article {573201,
	title = {A method that allows the assembly of kinetochore components onto chromosomes condensed in clarified Xenopus egg extracts.},
	journal = {Proc Natl Acad Sci U S A},
	volume = {94},
	number = {23},
	year = {1997},
	month = {1997 Nov 11},
	pages = {12378-83},
	abstract = {Kinetochores are complex macromolecular structures that link mitotic chromosomes to spindle microtubules. Although a small number of kinetochore components have been identified, including the kinesins CENP-E and XKCM1 as well as cytoplasmic dynein, neither how these and other proteins are organized to produce a kinetochore nor their exact functions within this structure are understood. For this reason, we have developed an assay that allows kinetochore components to assemble onto discrete foci on in vitro-condensed chromosomes. The source of the kinetochore components is a clarified cell extract from Xenopus eggs that can be fractionated or immunodepleted of individual proteins. Kinetochore assembly in these clarified extracts requires preincubating the substrate sperm nuclei in an extract under low ATP conditions. Immunodepletion of XKCM1 from the extracts prevents the localization of kinetochore-associated XKCM1 without affecting the targeting of CENP-E and cytoplasmic dynein or the binding of monomeric tubulin to the kinetochore. Extension of this assay for the analysis of other components should help to dissect the protein-protein interactions involved in kinetochore assembly and function.},
	keywords = {Animals, Chromosomes, Female, Kinetochores, Macromolecular Substances, Male, Oocytes, Spermatozoa, Xenopus},
	issn = {0027-8424},
	author = {Desai, A and Deacon, H W and Walczak, C E and Mitchison, T. J.}
}
@article {573211,
	title = {Rappaport rules: cleavage furrow induction in animal cells.},
	journal = {Proc Natl Acad Sci U S A},
	volume = {94},
	number = {10},
	year = {1997},
	month = {1997 May 13},
	pages = {4817-20},
	keywords = {Actins, Anaphase, Animals, Cell Cycle, Cell Division, Cell Line, Echinodermata, Models, Biological, Myosins, Ovum, Sea Urchins, Signal Transduction, Spindle Apparatus},
	issn = {0027-8424},
	author = {Oegema, K and Mitchison, T. J.}
}
@article {573216,
	title = {Xenopus actin depolymerizing factor/cofilin (XAC) is responsible for the turnover of actin filaments in Listeria monocytogenes tails.},
	journal = {J Cell Biol},
	volume = {136},
	number = {6},
	year = {1997},
	month = {1997 Mar 24},
	pages = {1323-32},
	abstract = {In contrast to the slow rate of depolymerization of pure actin in vitro, populations of actin filaments in vivo turn over rapidly. Therefore, the rate of actin depolymerization must be accelerated by one or more factors in the cell. Since the actin dynamics in Listeria monocytogenes tails bear many similarities to those in the lamellipodia of moving cells, we have used Listeria as a model system to isolate factors required for regulating the rapid actin filament turnover involved in cell migration. Using a cell-free Xenopus egg extract system to reproduce the Listeria movement seen in a cell, we depleted candidate depolymerizing proteins and analyzed the effect that their removal had on the morphology of Listeria tails. Immunodepletion of Xenopus actin depolymerizing factor (ADF)/cofilin (XAC) from Xenopus egg extracts resulted in Listeria tails that were approximately five times longer than the tails from undepleted extracts. Depletion of XAC did not affect the tail assembly rate, suggesting that the increased tail length was caused by an inhibition of actin filament depolymerization. Immunodepletion of Xenopus gelsolin had no effect on either tail length or assembly rate. Addition of recombinant wild-type XAC or chick ADF protein to XAC-depleted extracts restored the tail length to that of control extracts, while addition of mutant ADF S3E that mimics the phosphorylated, inactive form of ADF did not reduce the tail length. Addition of excess wild-type XAC to Xenopus egg extracts reduced the length of Listeria tails to a limited extent. These observations show that XAC but not gelsolin is essential for depolymerizing actin filaments that rapidly turn over in Xenopus extracts. We also show that while the depolymerizing activities of XAC and Xenopus extract are effective at depolymerizing normal filaments containing ADP, they are unable to completely depolymerize actin filaments containing AMPPNP, a slowly hydrolyzible ATP analog. This observation suggests that the substrate for XAC is the ADP-bound subunit of actin and that the lifetime of a filament is controlled by its nucleotide content.},
	keywords = {Actin Cytoskeleton, Actin Depolymerizing Factors, Actins, Adenosine Triphosphate, Animals, Biopolymers, Cell Movement, Cell-Free System, Cofilin 1, Cofilin 2, Destrin, Gelsolin, Kinetics, Listeria monocytogenes, Microfilament Proteins, Nerve Tissue Proteins, Oocytes, Phosphoproteins, Recombinant Fusion Proteins, Xenopus laevis, Xenopus Proteins},
	issn = {0021-9525},
	author = {Rosenblatt, J and Agnew, B J and Abe, H and Bamburg, J R and Mitchison, T. J.}
}
@article {573271,
	title = {Actin-based cell motility and cell locomotion.},
	journal = {Cell},
	volume = {84},
	number = {3},
	year = {1996},
	month = {1996 Feb 9},
	pages = {371-9},
	keywords = {Actins, Animals, Biophysical Phenomena, Biophysics, Cell Movement, Humans, Models, Biological},
	issn = {0092-8674},
	author = {Mitchison, T. J. and Cramer, L P}
}
@article {573286,
	title = {XKCM1: a Xenopus kinesin-related protein that regulates microtubule dynamics during mitotic spindle assembly.},
	journal = {Cell},
	volume = {84},
	number = {1},
	year = {1996},
	month = {1996 Jan 12},
	pages = {37-47},
	abstract = {We isolated a cDNA clone encoding a kinesin-related protein, which we named XKCM1. Antibodies to XKCM1 stain mitotic centromeres and spindle poles. Immunodepletion and antibody addition experiments in an in vitro spindle assembly assay show that XKCM1 is required for both establishment and maintenance of mitotic spindles. The structures that form in the absence of XKCM1 contain abnormally long microtubules. This long microtubule defect can be rescued by the addition of purified XKCM1 protein. Analysis of microtubule dynamics in a clarified mitotic extract reveals that loss of XKCM1 function causes a 4-fold suppression in the catastrophe frequency. XKCM1 thus exhibits a novel activity for a kinesin-related protein by promoting microtubule depolymerization during mitotic spindle assembly.},
	keywords = {Amino Acid Sequence, Animals, Antibody Specificity, Cell Extracts, Centromere, Chromosomes, Cloning, Molecular, DNA, Complementary, Gene Expression, Gene Library, Kinesin, Microtubule-Associated Proteins, Molecular Sequence Data, Ovum, Spindle Apparatus, Xenopus, Xenopus Proteins},
	issn = {0092-8674},
	author = {Walczak, C E and Mitchison, T. J. and Desai, A}
}
@article {573266,
	title = {Identification of a protein that interacts with tubulin dimers and increases the catastrophe rate of microtubules.},
	journal = {Cell},
	volume = {84},
	number = {4},
	year = {1996},
	month = {1996 Feb 23},
	pages = {623-31},
	abstract = {Using a polymerization inhibition assay, we have purified a small, heat stable protein that physically interacts with tubulin dimers and increases the catastrophe rate of microtubules. Sequence analysis identified this protein as oncoprotein 18 (Op18)/stathmin, a conserved phosphoprotein that is highly expressed in leukemia cells. Immunodepletion experiments in Xenopus egg extracts showed that Op18/stathmin is involved in physiological regulation of mitotic microtubule dynamics. Op18/stathmin is a microtubule regulator that preferentially interacts with unpolymerized subunits. It is a candidate for increasing the microtubule catastrophe rate in mitosis and might also regulate microtubule dynamics in response to external signals.},
	keywords = {Animals, Cattle, Microtubule Proteins, Microtubules, Phosphoproteins, Stathmin, Thymus Gland, Tissue Extracts, Tubulin, Xenopus, Xenopus Proteins},
	issn = {0092-8674},
	author = {Belmont, L D and Mitchison, T. J.}
}
@article {573256,
	title = {Kinesin-related proteins at mitotic spindle poles: function and regulation.},
	journal = {Cell},
	volume = {85},
	number = {7},
	year = {1996},
	month = {1996 Jun 28},
	pages = {943-6},
	keywords = {Animals, Calcium-Binding Proteins, Kinesin, Muscle Proteins, Spindle Apparatus},
	issn = {0092-8674},
	author = {Walczak, C E and Mitchison, T. J.}
}
@article {573251,
	title = {The kinetochore microtubule minus-end disassembly associated with poleward flux produces a force that can do work.},
	journal = {Mol Biol Cell},
	volume = {7},
	number = {10},
	year = {1996},
	month = {1996 Oct},
	pages = {1547-58},
	abstract = {During metaphase and anaphase in newt lung cells, tubulin subunits within the kinetochore microtubule (kMT) lattice flux slowly poleward as kMTs depolymerize at their minus-ends within in the pole. Very little is known about how and where the force that moves the tubulin subunits poleward is generated and what function it serves during mitosis. We found that treatment with the drug taxol (10 microM) caused separated centrosomes in metaphase newt lung cells to move toward one another with an average velocity of 0.89 microns/min, until the interpolar distance was reduced by 22-62\%. This taxol-induced spindle shortening occurred as kMTs between the chromosomes and the poles shortened. Photoactivation of fluorescent marks on kMTs revealed that taxol inhibited kinetochore microtubule assembly/disassembly at kinetochores, whereas minus-end MT disassembly continued at a rate typical of poleward flux in untreated metaphase cells. This poleward flux was strong enough to stretch the centromeric chromatin between sister kinetochores as much as it is stretched in control metaphase cells. In anaphase, taxol blocked kMT disassembly/assembly at the kinetochore whereas minus-end disassembly continued at a rate similar to flux in control cells (approximately 0.2 microns/min). These results reveal that the mechanism for kMT poleward flux 1) is not dependent on kMT plus-end dynamics and 2) produces pulling forces capable of generating tension across the centromeres of bioriented chromosomes.},
	keywords = {Anaphase, Animals, Biomechanical Phenomena, Cell Polarity, Cells, Cultured, Chromatin, Image Processing, Computer-Assisted, Kinetochores, Lung, Metaphase, Microscopy, Fluorescence, Microtubules, Paclitaxel, Photochemistry, Salamandridae, Spindle Apparatus, Stress, Mechanical, Tubulin},
	issn = {1059-1524},
	author = {Waters, J C and Mitchison, T. J. and Rieder, C L and Salmon, E D}
}
@article {573261,
	title = {A purified Drosophila septin complex forms filaments and exhibits GTPase activity.},
	journal = {J Cell Biol},
	volume = {133},
	number = {3},
	year = {1996},
	month = {1996 May},
	pages = {605-16},
	abstract = {Septin proteins are necessary for cytokinesis in budding yeast and Drosophila and are thought to be the subunits of the yeast neck filaments. To test whether septins actually form filaments, an immunoaffinity approach was used to isolate a septin complex from Drosophila embryos. The purified complex is comprised of the three previously identified septin polypeptides Pnut, Sep2, and Sep1. Hydrodynamic and sequence data suggest that the complex is composed of a heterotrimer of homodimers. The complex copurifies with one molecule of bound guanine nucleotide per septin polypeptide. It binds and hydrolyzes exogenously added GTP. These observations together with conserved sequence motifs identify the septins as members of the GTPase superfamily. We discuss a model of filament structure and speculate as to how the filaments are organized within cells.},
	keywords = {Actin Cytoskeleton, Amino Acid Sequence, Animals, Blotting, Western, Deoxyribonucleases, Drosophila, Drosophila Proteins, Exoribonucleases, Fungal Proteins, GTP Phosphohydrolases, Guanosine Triphosphate, Hydrolysis, Microfilament Proteins, Microscopy, Electron, Molecular Sequence Data, Proteins, Saccharomyces cerevisiae Proteins},
	issn = {0021-9525},
	author = {Field, C. M. and al-Awar, O and Rosenblatt, J and Wong, M L and Alberts, B and Mitchison, T. J.}
}
@article {573336,
	title = {The bulk of unpolymerized actin in Xenopus egg extracts is ATP-bound.},
	journal = {Mol Biol Cell},
	volume = {6},
	number = {2},
	year = {1995},
	month = {1995 Feb},
	pages = {227-36},
	abstract = {Non-muscle cells contain 15-500 microM actin, a large fraction of which is unpolymerized. Thus, the concentration of unpolymerized actin is well above the critical concentration for polymerization in vitro (0.2 microM). This fraction of actin could be prevented from polymerization by being ADP bound (therefore less favored to polymerize) or by being ATP bound and sequestered by a protein such as thymosin beta 4, or both. We isolated the unpolymerized actin from Xenopus egg extracts using immobilized DNase 1 and assayed the bound nucleotide. High-pressure liquid chromatography analysis showed that the bulk of soluble actin is ATP bound. Analysis of actin-bound nucleotide exchange rates suggested the existence of two pools of unpolymerized actin, one of which exchanges nucleotide relatively rapidly and another that apparently does not exchange. Native gel electrophoresis of Xenopus egg extracts demonstrated that most of the soluble actin exists in complexes with other proteins, one of which might be thymosin beta 4. These results are consistent with actin polymerization being controlled by the sequestration and release of ATP-bound actin, and argue against nucleotide exchange playing a major role in regulating actin polymerization.},
	keywords = {Actins, Adenosine Triphosphate, Animals, Cell-Free System, Chromatography, Gel, Chromatography, High Pressure Liquid, Deoxyribonuclease I, Electrophoresis, Polyacrylamide Gel, Female, Molecular Weight, Muscles, Oocytes, Protein Binding, Rabbits, Ultracentrifugation, Xenopus},
	issn = {1059-1524},
	author = {Rosenblatt, J and Peluso, P and Mitchison, T. J.}
}
@article {573316,
	title = {Evolution of a dynamic cytoskeleton.},
	journal = {Philos Trans R Soc Lond B Biol Sci},
	volume = {349},
	number = {1329},
	year = {1995},
	month = {1995 Sep 29},
	pages = {299-304},
	abstract = {Actin filaments and microtubules form the cytoskeleton of all eukaryotic cells, and they are responsible for organizing the cytoplasm and supporting motile processes. Both polymers are highly dynamic, and their polymerization dynamics are central to their organization. Though their evolutionary origins appear to be distinct, actin and tubulin have a similar mechanism for promoting polymerization dynamics in which the energy of nucleotide triphosphate hydrolysis during polymerization is used to weaken the bonds between subunits, thus promoting subsequent depolymerization. The evolutionary origins of actin and tubulin are unclear. It is likely that motile mechanisms driven by reversible polymerization, termed thermal ratchets, are older than those based on ATPase motor proteins. Such mechanisms are still important in modern eukaryotes, and may have powered early versions of the critical motile processes of phagocytosis and chromosome segregation in primitive cells. Thus evolution of dynamic cytoskeletal polymers may have been one of the earliest and most important steps leading to the evolution of eukaryotes. Plausible evolutionary pathways can be constructed leading from simple enzymes to dynamic cytoskeletal polymers.},
	keywords = {Actins, Adenosine Triphosphate, Animals, Cytoskeleton, Eukaryotic Cells, Evolution, Molecular, Humans, Microtubules, Models, Biological, Tubulin},
	issn = {0962-8436},
	doi = {10.1098/rstb.1995.0117},
	author = {Mitchison, T. J.}
}
@article {573341,
	title = {Identification and partial characterization of mitotic centromere-associated kinesin, a kinesin-related protein that associates with centromeres during mitosis.},
	journal = {J Cell Biol},
	volume = {128},
	number = {1-2},
	year = {1995},
	month = {1995 Jan},
	pages = {95-104},
	abstract = {Using antipeptide antibodies to conserved regions of the kinesin motor domain, we cloned a kinesin-related protein that associates with the centromere region of mitotic chromosomes. We call the protein MCAK, for mitotic centromere-associated kinesin. MCAK appears concentrated on centromeres at prophase and persists until telophase, after which time the localization disperses. It is found throughout the centromere region and between the kinetochore plates of isolated mitotic CHO chromosomes, in contrast to two other kinetochore-associated microtubule motors: cytoplasmic dynein and CENP-E (Yen et al., 1992), which are closer to the outer surface of the kinetochore plates. Sequence analysis shows MCAK to be a kinesin-related protein with the motor domain located in the center of the protein. It is 60-70\% similar to kif2, a kinesin-related protein originally cloned from mouse brain with a centrally located motor domain (Aizawa et al., 1992). MCAK protein is present in interphase and mitotic CHO cells and is transcribed as a single 3.4-kb message.},
	keywords = {Amino Acid Sequence, Animals, Base Sequence, Binding Sites, Centromere, CHO Cells, Chromosomal Proteins, Non-Histone, Cloning, Molecular, Cricetinae, Dyneins, Kinesin, Kinetochores, Mitosis, Molecular Sequence Data, Plasmids, Polymerase Chain Reaction, Protein Structure, Secondary, Reading Frames, Spindle Apparatus, Subcellular Fractions},
	issn = {0021-9525},
	author = {Wordeman, L and Mitchison, T. J.}
}
@article {573331,
	title = {Mutations in the kinesin-like protein Eg5 disrupting localization to the mitotic spindle.},
	journal = {Proc Natl Acad Sci U S A},
	volume = {92},
	number = {10},
	year = {1995},
	month = {1995 May 9},
	pages = {4289-93},
	abstract = {Eg5, a member of the bimC subfamily of kinesin-like microtubule motor proteins, localizes to spindle microtubules in mitosis but not to interphase microtubules. We investigated the molecular basis for spindle localization by transient transfection of Xenopus A6 cells with myc-tagged derivatives of Eg5. Expressed at constitutively high levels from a cytomegalovirus promoter, mycEg5 protein is cytoplasmic throughout interphase, begins to bind microtubules in early prophase, and remains localized to spindle and/or midbody microtubules through mitosis to the end of telophase. Both N- and C-terminal regions of Eg5 are required for this cell-cycle-regulated targeting. Eg5 also contains within its C-terminal domain a sequence conserved among bimC subfamily proteins that includes a potential p34cdc2 phosphorylation site. We show that mutation of a single threonine (T937) within this site to nonphosphorylatable alanine abolishes localization of the mutant protein to the spindle, whereas mutation of T937 to serine preserves spindle localization. We hypothesize that phosphorylation of Eg5 may regulate its localization to the spindle in the cell cycle.},
	keywords = {Amino Acid Sequence, Animals, Base Sequence, Cell Line, Conserved Sequence, Cytomegalovirus, DNA Primers, Kinesin, Microtubule-Associated Proteins, Molecular Sequence Data, Mutagenesis, Site-Directed, Polymerase Chain Reaction, Promoter Regions, Genetic, Recombinant Proteins, Spindle Apparatus, Transfection, Xenopus, Xenopus Proteins},
	issn = {0027-8424},
	author = {Sawin, K E and Mitchison, T. J.}
}
@article {573301,
	title = {Myosin is involved in postmitotic cell spreading.},
	journal = {J Cell Biol},
	volume = {131},
	number = {1},
	year = {1995},
	month = {1995 Oct},
	pages = {179-89},
	abstract = {We have investigated a role for myosin in postmitotic Potoroo tridactylis kidney (PtK2) cell spreading by inhibitor studies, time-lapse video microscopy, and immunofluorescence. We have also determined the spatial organization and polarity of actin filaments in postmitotic spreading cells. We show that butanedione monoxime (BDM), a known inhibitor of muscle myosin II, inhibits nonmuscle myosin II and myosin V adenosine triphosphatases. BDM reversibly inhibits PtK2 postmitotic cell spreading. Listeria motility is not affected by this drug. Electron microscopy studies show that some actin filaments in spreading edges are part of actin bundles that are also found in long, thin, structures that are connected to spreading edges and substrate (retraction fibers), and that 90\% of this actin is oriented with barbed ends in the direction of spreading. The remaining actin in spreading edges has a more random orientation and spatial arrangement. Myosin II is associated with actin polymer in spreading cell edges, but not retraction fibers. Myosin II is excluded from lamellipodia that protrude from the cell edge at the end of spreading. We suggest that spreading involves myosin, possibly myosin II.},
	keywords = {Actins, Cell Line, Cell Movement, Cholinesterase Reactivators, Diacetyl, Epithelial Cells, Epithelium, Humans, Kidney, Listeria monocytogenes, Microscopy, Electron, Mitosis, Myosins},
	issn = {0021-9525},
	author = {Cramer, L P and Mitchison, T. J.}
}
@article {573346,
	title = {A new role for motor proteins as couplers to depolymerizing microtubules.},
	journal = {J Cell Biol},
	volume = {128},
	number = {1-2},
	year = {1995},
	month = {1995 Jan},
	pages = {1-4},
	keywords = {Animals, Chromosomal Proteins, Non-Histone, Chromosomes, Kinetochores, Macromolecular Substances, Microtubule Proteins, Microtubules, Models, Biological},
	issn = {0021-9525},
	author = {Desai, A and Mitchison, T. J.}
}
@article {573326,
	title = {The SMC family: from chromosome condensation to dosage compensation.},
	journal = {Curr Opin Cell Biol},
	volume = {7},
	number = {3},
	year = {1995},
	month = {1995 Jun},
	pages = {329-36},
	abstract = {Recent genetic analyses in yeasts and biochemical studies in vertebrate cells have led to the discovery of a family of putative ATPases that play a fundamental role in chromosome condensation and segregation in mitosis. One of the members was also found to be involved in dosage compensation in Caenorhabditis elegans, providing a new link between global regulation of gene expression and chromosome structure. This unique family of proteins may control higher-order chromosome dynamics by regulated self-assembly or mechanochemical activity.},
	keywords = {Adenosine Triphosphatases, Amino Acid Sequence, Animals, Caenorhabditis elegans, Chromosomal Proteins, Non-Histone, Chromosomes, Dosage Compensation, Genetic, Female, Male, Mitosis, Molecular Sequence Data, Saccharomyces cerevisiae, Xenopus},
	issn = {0955-0674},
	author = {Hirano, T and Mitchison, T. J. and Swedlow, J R}
}
@article {573366,
	title = {Actin-dependent motile forces and cell motility.},
	journal = {Curr Opin Cell Biol},
	volume = {6},
	number = {1},
	year = {1994},
	month = {1994 Feb},
	pages = {82-6},
	abstract = {Force arising from actin polymerization and myosin activity drives a number of different actin-based cell movements. Several new reports support previous data suggesting that actin polymerization drives lamellipodial protrusion and bacterial propulsion, and one report describes a more indirect role for actin assembly in axonal elongation. The major new findings of the past year concerning possible motility roles for myosin describe myosin-driven protrusion of cell margins.},
	keywords = {Actins, Animals, Cell Movement, Humans, Macromolecular Substances, Models, Biological, Myosins},
	issn = {0955-0674},
	author = {Cramer, L P and Mitchison, T. J. and Theriot, J A}
}
@article {573361,
	title = {Involvement of profilin in the actin-based motility of L. monocytogenes in cells and in cell-free extracts.},
	journal = {Cell},
	volume = {76},
	number = {3},
	year = {1994},
	month = {1994 Feb 11},
	pages = {505-17},
	abstract = {Within hours of Listeria monocytogenes infection, host cell actin filaments form a dense cloud around the intracytoplasmic bacteria and then rearrange to form a polarized comet tail that is associated with moving bacteria. We have devised a cell-free extract system capable of faithfully reconstituting L. monocytogenes motility, and we have used this system to demonstrate that profilin, a host actin monomer-binding protein, is necessary for bacterial actin-based motility. We find that extracts from which profilin has been depleted do not support comet tail formation or bacterial motility. In extracts and host cells, profilin is localized to the back half of the surface of motile L. monocytogenes, the site of actin filament assembly in the tail. This association is not observed with L. monocytogenes mutants that do not express the ActA protein, a bacterial gene product necessary for motility and virulence. Profilin also fails to bind L. monocytogenes grown outside of host cytoplasm, suggesting that at least one other host cell factor is required for this association.},
	keywords = {Actins, Animals, Bacterial Proteins, Blotting, Western, Cell Movement, Cell-Free System, Contractile Proteins, Female, HeLa Cells, Humans, Listeria monocytogenes, Membrane Proteins, Microfilament Proteins, Models, Biological, Oocytes, Profilins, Xenopus laevis},
	issn = {0092-8674},
	author = {Theriot, J A and Rosenblatt, J and Portnoy, D A and Goldschmidt-Clermont, P J and Mitchison, T. J.}
}
@article {573371,
	title = {Microtubule flux in mitosis is independent of chromosomes, centrosomes, and antiparallel microtubules.},
	journal = {Mol Biol Cell},
	volume = {5},
	number = {2},
	year = {1994},
	month = {1994 Feb},
	pages = {217-26},
	abstract = {We investigated the mechanism of poleward microtubule flux in the mitotic spindle by generating spindle subassemblies in Xenopus egg extracts in vitro and assaying their ability to flux by photoactivation of fluorescence and low-light multichannel fluorescence video-microscopy. We find that monopolar intermediates of in vitro spindle assembly (half-spindles) exhibit normal poleward flux, as do astral microtubule arrays induced by the addition of dimethyl sulfoxide to egg extracts in the absence of both chromosomes and conventional centrosomes. Immunodepletion of the kinesin-related microtubule motor protein Eg5, a candidate flux motor, suggests that Eg5 is not required for flux. These results suggest that poleward flux is a basic element of microtubule behavior exhibited by even simple self-organized microtubule arrays and presumably underlies the most elementary levels of spindle morphogenesis.},
	keywords = {Animals, Centromere, Chromosomes, Dimethyl Sulfoxide, Kinesin, Microtubules, Mitosis, Morphogenesis, Spindle Apparatus, Xenopus},
	issn = {1059-1524},
	author = {Sawin, K E and Mitchison, T. J.}
}
@article {573376,
	title = {Mitosis. Kinetochores pass the IQ test.},
	journal = {Curr Biol},
	volume = {4},
	number = {1},
	year = {1994},
	month = {1994 Jan 1},
	pages = {38-41},
	abstract = {Kinetochores oscillate to and fro on the mitotic spindle. The oscillations seem to be biased by the forces acting on the kinetochore, explaining the variety of chromosome movements seen at different stages of mitosis.},
	keywords = {Animals, Cell Cycle, Chromosomes, Kinetochores, Mitosis, Models, Biological, Spindle Apparatus},
	issn = {0960-9822},
	author = {AW Murray and Mitchison, T. J.}
}
@article {573356,
	title = {Towards a pharmacological genetics.},
	journal = {Chem Biol},
	volume = {1},
	number = {1},
	year = {1994},
	month = {1994 Sep},
	pages = {3-6},
	abstract = {In the past, pharmacological approaches have been instrumental in identifying proteins involved in biological processes. Combinatorial chemistry has the potential to make this type of approach even more powerful.},
	keywords = {Animals, Genetics, Humans, Pharmacology},
	issn = {1074-5521},
	author = {Mitchison, T. J.}
}
@article {573351,
	title = {A heterodimeric coiled-coil protein required for mitotic chromosome condensation in vitro.},
	journal = {Cell},
	volume = {79},
	number = {3},
	year = {1994},
	month = {1994 Nov 4},
	pages = {449-58},
	abstract = {We report here a chromosomal protein that plays an essential role in mitotic chromosome condensation in Xenopus egg extracts. Two polypeptides, designated XCAP-C and XCAP-E, were found to associate with each other in the extracts, presumably forming a heterodimer. During chromosome assembly in mitotic extracts, XCAP-C/E was recruited to the chromatin and formed a discrete internal structure within assembled chromosomes. Antibody blocking experiments showed that XCAP-C function is required for both assembly and structural maintenance of mitotic chromosomes in vitro. Deduced amino acid sequences revealed that the two polypeptides share common structural motifs, consisting of an N-terminal NTP-binding domain, two central coiled-coil regions, and a C-terminal conserved domain. These motifs are highly conserved in a protein family, members of which have been identified recently in both prokaryotes and eukaryotes.},
	keywords = {Amino Acid Sequence, Animals, Base Sequence, Carrier Proteins, Cell Compartmentation, Cell Cycle Proteins, Cell-Free System, Cells, Cultured, Chromosomal Proteins, Non-Histone, Chromosomes, Cloning, Molecular, Fluorescent Antibody Technique, Mitosis, Molecular Sequence Data, Morphogenesis, Movement, Nuclear Proteins, Ovum, Protein Binding, Protein Structure, Secondary, Recombinant Proteins, Sequence Homology, Amino Acid, Xenopus, Xenopus Proteins},
	issn = {0092-8674},
	author = {Hirano, T and Mitchison, T. J.}
}
@article {573401,
	title = {An assay for the activity of microtubule-based motors on the kinetochores of isolated Chinese hamster ovary chromosomes.},
	journal = {Methods Cell Biol},
	volume = {39},
	year = {1993},
	month = {1993},
	pages = {267-76},
	keywords = {Animals, CHO Cells, Chromosomes, Cricetinae, Microscopy, Microtubules, Movement, Video Recording},
	issn = {0091-679X},
	author = {Hyman, A A and Mitchison, T. J.}
}
@article {573386,
	title = {Localization of an exchangeable GTP binding site at the plus end of microtubules.},
	journal = {Science},
	volume = {261},
	number = {5124},
	year = {1993},
	month = {1993 Aug 20},
	pages = {1044-7},
	abstract = {Microtubule polarity arises from the head-to-tail orientation of alpha-beta tubulin heterodimers in the microtubule lattice. The identity of the polypeptide at each end of the microtubule is unknown, but structural models predict that the beta-tubulin end contains an exchangeable guanosine triphosphate (GTP) binding site. When GTP-coated fluorescent beads were incubated with microtubules, they bound specifically to plus ends, suggesting that tubulin is oriented in microtubules with beta-tubulin toward the plus end.},
	keywords = {Adenosine Triphosphate, Animals, Binding Sites, Binding, Competitive, Ethylmaleimide, Guanosine Triphosphate, Microtubules, Paclitaxel, Tetrahymena, Tubulin},
	issn = {0036-8075},
	author = {Mitchison, T. J.}
}
@article {573391,
	title = {Moving and stationary actin filaments are involved in spreading of postmitotic PtK2 cells.},
	journal = {J Cell Biol},
	volume = {122},
	number = {4},
	year = {1993},
	month = {1993 Aug},
	pages = {833-43},
	abstract = {We have investigated spreading of postmitotic PtK2 cells and the behavior of actin filaments in this system by time-lapse microscopy and photoactivation of fluorescence. During mitosis PtK2 cells round up and at cytokinesis the daughter cells spread back to regain their interphase morphology. Normal spreading edges are quite homogenous and are not comprised of two distinct areas (lamellae and lamellipodia) as found in moving edges of interphase motile cells. Spreading edges are connected to a network of long, thin, actin-rich fibers called retraction fibers. A role for retraction fibers in spreading was tested by mechanical disruption of fibers ahead of a spreading edge. Spreading is inhibited over the region of disruption, but not over neighboring intact fibers. Using photoactivation of fluorescence to mark actin filaments, we have determined that the majority of actin filaments move forward in spreading edges at the same rate as the edge. As far as we are aware, this is the first time that forward movement of a cell edge has been correlated with forward movement of actin filaments. In contrast, actin filaments in retraction fibers remain stationary with respect to the substrate. Thus there are at least two dynamic populations of actin polymer in spreading postmitotic cells. This is supported by the observation that actin filaments in some spreading edges not only move forward, but also separate into two fractions or broaden with time. A small fraction of postmitotic cells have a spreading edge with a distinct lamellipodium. In these edges, marked actin polymer fluxes backward with respect to substrate. We suggest that forward movement of actin filaments may participate in generating force for spreading in postmitotic cells and perhaps more generally for cell locomotion.},
	keywords = {Actin Cytoskeleton, Actins, Animals, Cell Adhesion, Cell Line, Cell Movement, In Vitro Techniques, Macropodidae, Microscopy, Fluorescence, Video Recording},
	issn = {0021-9525},
	author = {Cramer, L and Mitchison, T. J.}
}
@article {573381,
	title = {The three faces of profilin.},
	journal = {Cell},
	volume = {75},
	number = {5},
	year = {1993},
	month = {1993 Dec 3},
	pages = {835-8},
	keywords = {Actin Cytoskeleton, Actins, Adenosine Triphosphate, Contractile Proteins, Microfilament Proteins, Profilins, Protein Binding},
	issn = {0092-8674},
	author = {Theriot, J A and Mitchison, T. J.}
}
@article {573396,
	title = {Topoisomerase II does not play a scaffolding role in the organization of mitotic chromosomes assembled in Xenopus egg extracts.},
	journal = {J Cell Biol},
	volume = {120},
	number = {3},
	year = {1993},
	month = {1993 Feb},
	pages = {601-12},
	abstract = {We have investigated the role of topoisomerase II (topo II) in mitotic chromosome assembly and organization in vitro using Xenopus egg extracts. When sperm chromatin was incubated with mitotic extracts, the highly compact chromatin rapidly swelled and concomitantly underwent local condensation. Further incubation induced the formation of entangled thin chromatin fibers that eventually resolved into highly condensed individual chromosomes. This in vitro system made it possible to manipulate mitotic chromosomes in their assembly condition without any isolation or stabilization steps. Two complementary approaches, immunodepletion and antibody blocking, demonstrated that topo II activity is required for chromosome assembly and condensation. Once condensation was completed, however, blocking of topo II activity had little effect on the chromosome morphology. Immunofluorescent studies showed that topo II was uniformly distributed throughout the condensed chromosomes and was not restricted to the chromosomal axis. Surprisingly, all detectable topo II molecules were easily extracted from the chromosomes under mild conditions where the shape of chromosomes was well preserved. Our results show that topo II is essential for mitotic chromosome assembly, but does not play a scaffolding role in the structural maintenance of chromosomes assembled in vitro. We also present evidence that changes of DNA topology affect the distribution of topo II in mitotic chromosomes in our system.},
	keywords = {Amino Acid Sequence, Animals, Antibodies, Base Sequence, Chromatin, Chromosomes, Cloning, Molecular, DNA, DNA Topoisomerases, Type II, Female, Male, Mitosis, Molecular Sequence Data, Oligodeoxyribonucleotides, Oligopeptides, Oocytes, Spermatozoa, Xenopus laevis},
	issn = {0021-9525},
	author = {Hirano, T and Mitchison, T. J.}
}
@article {573461,
	title = {Actin based motility on retraction fibers in mitotic PtK2 cells.},
	journal = {Cell Motil Cytoskeleton},
	volume = {22},
	number = {2},
	year = {1992},
	month = {1992},
	pages = {135-51},
	abstract = {When PtK2 cells round up in mitosis they leave retraction fibers attached between the substrate and the cell body. Retraction fibers and the region where they meet the cell body are rich in actin filaments as judged by phalloidin staining and electron microscopy. Video microscopy was used to study actin dependent motile processes on retraction fibers. Small, phase-dense nodules form spontaneously on the fibers, and move in to the cell body at a rate of 3 microns/minute. As they move in they increase progressively in phase-density. This movement appears to be related to actin dependent centripetal movement which has been previously studied in lamellipodia. Despite its generality, the mechanism of such movement is unknown, and retraction fibers present some special advantages for its study. Cytochalasin treatment causes nodules to stop moving and dissolve. Withdrawal of the drug causes them to reform and start moving. Surprisingly, movement after cytochalasin withdrawal was often outward, indicating a local reversal of cortical polarity. After a few minutes correct polarity is reestablished by a global control mechanism. The implications of these observations for the mechanism and polarity of actin dependent motility is discussed.},
	keywords = {Actins, Animals, Cell Line, Cell Movement, Cytochalasin D, Cytoplasm, Macropodidae, Microscopy, Electron, Mitosis},
	issn = {0886-1544},
	doi = {10.1002/cm.970220207},
	author = {Mitchison, T. J.}
}
@article {573456,
	title = {Evidence for kinesin-related proteins in the mitotic apparatus using peptide antibodies.},
	journal = {J Cell Sci},
	volume = {101 ( Pt 2)},
	year = {1992},
	month = {1992 Feb},
	pages = {303-13},
	abstract = {To identify kinesin-related proteins that may be important for mitotic function in embryonic and tissue culture cells we have generated polyclonal antibodies to two synthetic peptides corresponding to conserved regions of the kinesin motor domain. In Xenopus eggs we have identified a family of microtubule-binding proteins, recognized by one or both affinity-purified peptide antibodies but not by monoclonal antibodies that recognize conventional kinesin heavy chain. Like kinesin, most of these proteins bind to microtubules only upon addition of AMP-PNP or nucleotide depletion and are released upon subsequent addition of ATP. At least one protein, however, exhibits markedly distinct properties, binding readily to microtubules in the absence of AMP-PNP and/or nucleotide depletion. We also report that, unlike antibodies to conventional kinesin, the peptide antibodies to the kinesin motor domain immunofluorescently label spindles and kinetochores in mitotic tissue culture cells, suggesting that kinesin-like proteins may have important roles in chromosome movement and mitosis.},
	keywords = {Adenosine Triphosphate, Adenylyl Imidodiphosphate, Amino Acid Sequence, Animals, Antibody Formation, Centromere, CHO Cells, Cricetinae, Fluorescent Antibody Technique, Kinesin, Microtubule-Associated Proteins, Microtubules, Mitosis, Molecular Sequence Data, Oligopeptides, Ovum, Spindle Apparatus, Xenopus},
	issn = {0021-9533},
	author = {Sawin, K E and Mitchison, T. J. and Wordeman, L G}
}
@article {573406,
	title = {Compare and contrast actin filaments and microtubules.},
	journal = {Mol Biol Cell},
	volume = {3},
	number = {12},
	year = {1992},
	month = {1992 Dec},
	pages = {1309-15},
	keywords = {Actins, Animals, Cytoskeleton, Macromolecular Substances, Microtubules},
	issn = {1059-1524},
	author = {Mitchison, T. J.}
}
@article {573431,
	title = {Comparison of actin and cell surface dynamics in motile fibroblasts.},
	journal = {J Cell Biol},
	volume = {119},
	number = {2},
	year = {1992},
	month = {1992 Oct},
	pages = {367-77},
	abstract = {We have investigated the dynamic behavior of actin in fibroblast lamellipodia using photoactivation of fluorescence. Activated regions of caged resorufin (CR)-labeled actin in lamellipodia of IMR 90 and MC7 3T3 fibroblasts were observed to move centripetally over time. Thus in these cells, actin filaments move centripetally relative to the substrate. Rates were characteristic for each cell type; 0.66 +/- 0.27 microns/min in IMR 90 and 0.36 +/- 0.16 microns/min in MC7 3T3 cells. In neither case was there any correlation between the rate of actin movement and the rate of lamellipodial protrusion. The half-life of the activated CR-actin filaments was approximately 1 min in IMR 90 lamellipodia, and approximately 3 min in MC7 3T3 lamellipodia. Thus continuous filament turnover accompanies centripetal movement. In both cell types, the length of time required for a section of the actin meshwork to traverse the lamellipodium was several times longer than the filament half-life. The dynamic behavior of the dorsal surface of the cell was also observed by tracking lectin-coated beads on the surface and phase-dense features within lamellipodia of MC7 3T3 cells. The movement of these dorsal features occurred at rates approximately three times faster than the rate of movement of the underlying bulk actin cytoskeleton, even when measured in the same individual cells. Thus the transport of these dorsal features must occur by some mechanism other than simple attachment to the moving bulk actin cytoskeleton.},
	keywords = {3T3 Cells, Actins, Animals, Biological Transport, Cell Membrane, Cell Movement, Cytoskeleton, Fibroblasts, Fluorescence, Macromolecular Substances, Mice, Oxazines, Pseudopodia},
	issn = {0021-9525},
	author = {Theriot, J A and Mitchison, T. J.}
}
@article {573436,
	title = {A GTPase controls cell-substrate adhesion in Xenopus XTC fibroblasts.},
	journal = {J Cell Biol},
	volume = {118},
	number = {5},
	year = {1992},
	month = {1992 Sep},
	pages = {1235-44},
	abstract = {Cell-substrate adhesion is crucial at various stages of development and for the maintenance of normal tissues. Little is known about the regulation of these adhesive interactions. To investigate the role of GTPases in the control of cell morphology and cell-substrate adhesion we have injected guanine nucleotide analogs into Xenopus XTC fibroblasts. Injection of GTP gamma S inhibited ruffling and increased spreading, suggesting an increase in adhesion. To further investigate this, we made use of GRGDSP, a peptide which inhibits binding of integrins to vitronectin and fibronectin. XTC fibroblasts injected with non-hydrolyzable analogs of GTP took much more time to round up than mock-injected cells in response to treatment with GRGDSP, while GDP beta S-injected cells rounded up in less time than controls. Injection with GTP gamma S did not inhibit cell rounding induced by trypsin however, showing that cell contractility is not significantly affected by the activation of GTPases. These data provide evidence for the existence of a GTPase which can control cell-substrate adhesion from the cytoplasm. Treatment of XTC fibroblasts with the phorbol ester 12-o-tetradecanoylphorbol-13-acetate reduced cell spreading and accelerated cell rounding in response to GRGDSP, which is essentially opposite to the effect exerted by non-hydrolyzable GTP analogs. These results suggest the existence of at least two distinct pathways controlling cell-substrate adhesion in XTC fibroblasts, one depending on a GTPase and another one involving protein kinase C.},
	keywords = {Amino Acid Sequence, Animals, Cell Adhesion, Cell Line, Cytoskeleton, Dimethyl Sulfoxide, Fibroblasts, GTP Phosphohydrolases, Guanosine 5{\textquoteright}-O-(3-Thiotriphosphate), Guanosine Triphosphate, Molecular Sequence Data, Oligopeptides, Pseudopodia, Tetradecanoylphorbol Acetate, Xenopus},
	issn = {0021-9525},
	author = {Symons, M H and Mitchison, T. J.}
}
@article {573421,
	title = {Microtubule-motor activity of a yeast centromere-binding protein complex.},
	journal = {Nature},
	volume = {359},
	number = {6395},
	year = {1992},
	month = {1992 Oct 8},
	pages = {533-6},
	abstract = {During cell division, sister chromosomes segregate from each other on a microtubule-based structure called the mitotic spindle. Proteins bind to the centromere, a region of chromosomal DNA, to form the kinetochore, which mediates chromosome attachment to the mitotic spindle microtubules. In the budding yeast Saccharomyces cerevisiae, genetic analysis has shown that the 28-basepair (bp) CDEIII region of the 125-bp centromere DNA sequence (CEN sequence) is the main region controlling chromosome segregation in vivo. Therefore it is likely that proteins binding to the CDEIII region link the centromeres to the microtubules during mitosis. A complex of proteins (CBF3) that binds specifically to the CDEIII DNA sequence has been isolated by affinity chromatography. Here we describe kinetochore function in vitro. The CBF3 complex can link DNA to microtubules, and the complex contains a minus-end-directed microtubule-based motor. We suggest that microtubule-based motors form the fundamental link between microtubules and chromosomes at mitosis.},
	keywords = {Adenosine Triphosphate, Binding, Competitive, Centromere, Chromosomes, Fungal, DNA, Fungal, DNA-Binding Proteins, Fungal Proteins, Microtubules, Movement, Saccharomyces cerevisiae},
	issn = {0028-0836},
	doi = {10.1038/359533a0},
	author = {Hyman, A A and Middleton, K and Centola, M and Mitchison, T. J. and Carbon, J}
}
@article {573416,
	title = {Mitotic spindle organization by a plus-end-directed microtubule motor.},
	journal = {Nature},
	volume = {359},
	number = {6395},
	year = {1992},
	month = {1992 Oct 8},
	pages = {540-3},
	abstract = {Intracellular microtubule motor proteins may direct the motile properties and/or morphogenesis of the mitotic spindle (reviewed in ref. 3). The recent identification of kinesin-like proteins important for mitosis or meiosis indicates that kinesin-related proteins may play a universal role in eukaryotic cell division, but the precise function of such proteins in mitosis remains unknown. Here we use an in vitro assay for spindle assembly, derived from Xenopus egg extracts, to investigate the role of Eg5, a kinesin-like protein in Xenopus eggs. Eg5 is localized along spindle microtubules, and particularly enriched near spindle poles. Immunodepletion of Eg5 from egg extracts markedly reduces the extent of spindle formation in extracts, as does direct addition of anti-Eg5 antibodies. We also demonstrate that Eg5 is a plus-end-directed microtubule motor in vitro. Our results suggest a novel mechanism for the dynamic self-organization of spindle poles in mitosis.},
	keywords = {Animals, Antibodies, Fluorescent Antibody Technique, Microtubule-Associated Proteins, Microtubules, Mitosis, Molecular Structure, Movement, Ovum, Spindle Apparatus, Xenopus laevis},
	issn = {0028-0836},
	doi = {10.1038/359540a0},
	author = {Sawin, K E and LeGuellec, K and Philippe, M and Mitchison, T. J.}
}
@article {573441,
	title = {The nucleation-release model of actin filament dynamics in cell motility.},
	journal = {Trends Cell Biol},
	volume = {2},
	number = {8},
	year = {1992},
	month = {1992 Aug},
	pages = {219-22},
	abstract = {The actin cytoskeleton is intimately involved in the motile behaviour of animal cells. The structure and dynamic behaviour of actin and its binding proteins have been intensively studied in vitro over the past several decades, culminating in achievements such as an atomic model of the actin filament. Despite this progress, it is not yet clear how the behaviour of these purified proteins in vitro relates to the dynamic behaviour of actin inside living, moving cells. Here we discuss a new model that relates the known dynamic parameters for pure actin to the observed behaviour of actin filaments inside motile cells.},
	issn = {0962-8924},
	author = {Theriot, J A and Mitchison, T. J.}
}
@article {573411,
	title = {Poleward kinetochore fiber movement occurs during both metaphase and anaphase-A in newt lung cell mitosis.},
	journal = {J Cell Biol},
	volume = {119},
	number = {3},
	year = {1992},
	month = {1992 Nov},
	pages = {569-82},
	abstract = {Microtubules in the mitotic spindles of newt lung cells were marked using local photoactivation of fluorescence. The movement of marked segments on kinetochore fibers was tracked by digital fluorescence microscopy in metaphase and anaphase and compared to the rate of chromosome movement. In metaphase, kinetochore oscillations toward and away from the poles were coupled to kinetochore fiber shortening and growth. Marked zones on the kinetochore microtubules, meanwhile, moved slowly polewards at a rate of approximately 0.5 micron/min, which identifies a slow polewards movement, or "flux," of kinetochore microtubules accompanied by depolymerization at the pole, as previously found in PtK2 cells (Mitchison, 1989b). Marks were never seen moving away from the pole, indicating that growth of the kinetochore microtubules occurs only at their kinetochore ends. In anaphase, marked zones on kinetochore microtubules also moved polewards, though at a rate slower than overall kinetochore-to-pole movement. Early in anaphase-A, microtubule depolymerization at kinetochores accounted on average for 75\% of the rate of chromosome-to-pole movement, and depolymerization at the pole accounted for 25\%. When chromosome-to-pole movement slowed in late anaphase, the contribution of depolymerization at the kinetochores lessened, and flux became the dominant component in some cells. Over the whole course of anaphase-A, depolymerization at kinetochores accounted on average for 63\% of kinetochore fiber shortening, and flux for 37\%. In some anaphase cells up to 45\% of shortening resulted from the action of flux. We conclude that kinetochore microtubules change length predominantly through polymerization and depolymerization at the kinetochores during both metaphase and anaphase as the kinetochores move away from and towards the poles. Depolymerization, though not polymerization, also occurs at the pole during metaphase and anaphase, so that flux contributes to polewards chromosome movements throughout mitosis. Poleward force production for chromosome movements is thus likely to be generated by at least two distinct molecular mechanisms.},
	keywords = {Anaphase, Animals, Cells, Cultured, Chromosomes, Lung, Metaphase, Microscopy, Fluorescence, Microtubules, Mitosis, Salamandridae, Time Factors},
	issn = {0021-9525},
	author = {Mitchison, T. J. and Salmon, E D}
}
@article {573446,
	title = {The rate of actin-based motility of intracellular Listeria monocytogenes equals the rate of actin polymerization.},
	journal = {Nature},
	volume = {357},
	number = {6375},
	year = {1992},
	month = {1992 May 21},
	pages = {257-60},
	abstract = {The Gram-positive bacterium Listeria monocytogenes is a facultative intracellular pathogen capable of rapid movement through the host cell cytoplasm. The biophysical basis of the motility of L. monocytogenes is an interesting question in its own right, the answer to which may shed light on the general processes of actin-based motility in cells. Moving intracellular bacteria display phase-dense {\textquoteright}comet tails{\textquoteright} made of actin filaments, the formation of which is required for bacterial motility. We have investigated the dynamics of the actin filaments in the comet tails using the technique of photoactivation of fluorescence, which allows monitoring of the movement and turnover of labelled actin filaments after activation by illumination with ultraviolet light. We find that the actin filaments remain stationary in the cytoplasm as the bacterium moves forward, and that length of the comet tails is linearly proportional to the rate of movement. Our results imply that the motile mechanism involves continuous polymerization and release of actin filaments at the bacterial surface and that the rate of filament generation is related to the rate of movement. We suggest that actin polymerization provides the driving force for bacterial propulsion.},
	keywords = {Actins, Animals, Cell Line, Cell Movement, Flagella, Half-Life, Listeria monocytogenes, Macromolecular Substances},
	issn = {0028-0836},
	doi = {10.1038/357257a0},
	author = {Theriot, J A and Mitchison, T. J. and Tilney, L G and Portnoy, D A}
}
@article {573426,
	title = {Role of GTP hydrolysis in microtubule dynamics: information from a slowly hydrolyzable analogue, GMPCPP.},
	journal = {Mol Biol Cell},
	volume = {3},
	number = {10},
	year = {1992},
	month = {1992 Oct},
	pages = {1155-67},
	abstract = {The role of GTP hydrolysis in microtubule dynamics has been reinvestigated using an analogue of GTP, guanylyl-(alpha, beta)-methylene-diphosphonate (GMPCPP). This analogue binds to the tubulin exchangeable nucleotide binding site (E-site) with an affinity four to eightfold lower than GTP and promotes the polymerization of normal microtubules. The polymerization rate of microtubules with GMPCPP-tubulin is very similar to that of GTP-tubulin. However, in contrast to microtubules polymerized with GTP, GMPCPP-microtubules do not depolymerize rapidly after isothermal dilution. The depolymerization rate of GMPCPP-microtubules is 0.1 s-1 compared with 500 s-1 for GDP-microtubules. GMPCPP also completely suppresses dynamic instability. Contrary to previous work, we find that the beta--gamma bond of GMPCPP is hydrolyzed extremely slowly after incorporation into the microtubule lattice, with a rate constant of 4 x 10(-7) s-1. Because GMPCPP hydrolysis is negligible over the course of a polymerization experiment, it can be used to test the role of hydrolysis in microtubule dynamics. Our results provide strong new evidence for the idea that GTP hydrolysis by tubulin is not required for normal polymerization but is essential for depolymerization and thus for dynamic instability. Because GMPCPP strongly promotes spontaneous nucleation of microtubules, we propose that GTP hydrolysis by tubulin also plays the important biological role of inhibiting spontaneous microtubule nucleation.},
	keywords = {Animals, Binding Sites, Cattle, Guanosine Triphosphate, Hydrolysis, In Vitro Techniques, Kinetics, Microscopy, Electron, Microtubules, Polymers},
	issn = {1059-1524},
	author = {Hyman, A A and Salser, S and Drechsel, D N and Unwin, N and Mitchison, T. J.}
}
@article {573451,
	title = {Self-organization of polymer-motor systems in the cytoskeleton.},
	journal = {Philos Trans R Soc Lond B Biol Sci},
	volume = {336},
	number = {1276},
	year = {1992},
	month = {1992 Apr 29},
	pages = {99-106},
	abstract = {Microtubules and actin filaments are organized into dynamic arrays inside cells. In this paper I discuss in conceptual form the assembly mechanisms of three specific arrays: asters, spindles and leading edge structures. The role of energy transducing processes, particularly motor protein activity, in assembly is explored. I conclude that dynamic interaction between motor proteins and cytoskeletal polymers play a very general role in spatial organization of the cytoplasm.},
	keywords = {Actins, Animals, Cytoskeleton, Microtubules, Polymers, Proteins, Spindle Apparatus},
	issn = {0962-8436},
	doi = {10.1098/rstb.1992.0049},
	author = {Mitchison, T. J.}
}
@article {573481,
	title = {Actin microfilament dynamics in locomoting cells.},
	journal = {Nature},
	volume = {352},
	number = {6331},
	year = {1991},
	month = {1991 Jul 11},
	pages = {126-31},
	abstract = {The dynamic behaviour of actin filaments has been directly observed in living, motile cells using fluorescence photoactivation. In goldfish epithelial keratocytes, the actin microfilaments in the lamellipodium remain approximately fixed relative to the substrate as the cell moves over them, regardless of cell speed. The rate of turnover of actin subunits in the lamellipodium is remarkably rapid. Cell movement is directly and tightly coupled to the formation of new actin filaments at the leading edge.},
	keywords = {Actin Cytoskeleton, Actins, Animals, Cell Movement, Cells, Cultured, Epidermis, Fluorescent Dyes, Goldfish, Half-Life, Oxazines, Photochemistry, Polymers, Rabbits},
	issn = {0028-0836},
	doi = {10.1038/352126a0},
	author = {Theriot, J A and Mitchison, T. J.}
}
@article {573466,
	title = {Cell cycle control of higher-order chromatin assembly around naked DNA in vitro.},
	journal = {J Cell Biol},
	volume = {115},
	number = {6},
	year = {1991},
	month = {1991 Dec},
	pages = {1479-89},
	abstract = {We have developed an in vitro system in which higher-order chromatin structures are assembled around naked DNAs in a cell cycle-dependent manner. Membrane-free soluble extracts specific to interphase and mitotic states were prepared from Xenopus eggs. When high molecular weight DNA is incubated with interphase extracts, fluffy chromatin-like structures are assembled. In contrast, mitotic extracts produce highly condensed chromosome-like structures. Immunofluorescence studies show that a monoclonal antibody MPM-2, which recognizes a class of mitosis-specific phosphoproteins, stains the "core" or "axis" of condensed mitotic chromatin but not interphase chromatin. By adding mitotic extracts, interphase chromatin structures are synchronously converted into the condensed state. The increasingly condensed state of chromatin correlates with the appearance and structural rearrangements of the MPM-2-stained structures. These results suggest that mitosis-specific phosphoproteins recognized by MPM-2 may be directly involved in the assembly of the chromosome scaffold-like structures and chromatin condensation. Although both extracts promote nucleosome assembly at the same rate, topoisomerase II (topo II) activity is four to five times higher in mitotic extracts compared with interphase extracts. The addition of a topo II inhibitor VM-26 into mitotic assembly mixtures disturbs the organization of the MPM-2-stained structures and affects the final stage of chromatin condensation. This in vitro system should be useful for identifying cis- and trans-acting elements responsible for higher-order chromatin assembly and its structural changes in the cell cycle.},
	keywords = {Animals, Antibodies, Monoclonal, Cell Cycle, Chromatin, Chromosomes, DNA, Fluorescent Antibody Technique, Interphase, Mitosis, Nucleosomes, Oocytes, Topoisomerase II Inhibitors, Xenopus laevis},
	issn = {0021-9525},
	author = {Hirano, T and Mitchison, T. J.}
}
@article {573476,
	title = {Control of actin polymerization in live and permeabilized fibroblasts.},
	journal = {J Cell Biol},
	volume = {114},
	number = {3},
	year = {1991},
	month = {1991 Aug},
	pages = {503-13},
	abstract = {We have investigated the spatial control of actin polymerization in fibroblasts using rhodamine-labeled muscle actin in; (a) microinjection experiments to follow actin dynamics in intact cells, and (b) incubation with permeabilized cells to study incorporation sites. Rhodamine-actin was microinjected into NIH-3T3 cells which were then fixed and stained with fluorescein-phalloidin to visualize total actin filaments. The incorporation of newly polymerized actin was assayed using rhodamine/fluorescein ratio-imaging. The results indicated initial incorporation of the injected actin near the tip and subsequent transport towards the base of lamellipodia at rates greater than 4.5 microns/min. Furthermore, both fluorescein- and rhodamine-intensity profiles across lamellipodia revealed a decreasing density of actin filaments from tip to base. From this observation and the presence of centripetal flux of polymerized actin we infer that the actin cytoskeleton partially disassembles before it reaches the base of the lamellipodium. In permeabilized cells we found that, in agreement with the injection studies, rhodamine-actin incorporated predominantly in a narrow strip of less than 1-microns wide, located at the tip of lamellipodia. The critical concentration for the rhodamine-actin incorporation (0.15 microM) and its inhibition by CapZ, a barbed-end capping protein, indicated that the nucleation sites for actin polymerization most likely consist of free barbed ends of actin filaments. Because any potential monomer-sequestering system is bypassed by addition of exogenous rhodamine-actin to the permeabilized cells, these observations indicate that the localization of actin incorporation in intact cells is determined, at least in part, by the presence of specific elongation and/or nucleation sites at the tips of lamellipodia and not solely by localized desequestration of subunits. We propose that the availability of the incorporation sites at the tips of lamellipodia is because of capping activities which preferentially inhibit barbed-end incorporation elsewhere in the cell, but leave barbed ends at the tips of lamellipodia free to add subunits.},
	keywords = {Actins, Animals, Cell Line, Cell Membrane, Cell Membrane Permeability, Fibroblasts, Image Processing, Computer-Assisted, Kinetics, Mice, Microinjections, Microscopy, Fluorescence, Polymers, Rabbits, Rhodamines, Saponins},
	issn = {0021-9525},
	author = {Symons, M H and Mitchison, T. J.}
}
@article {573471,
	title = {Microtubule polymer assembly and transport during axonal elongation.},
	journal = {J Cell Biol},
	volume = {115},
	number = {2},
	year = {1991},
	month = {1991 Oct},
	pages = {365-79},
	abstract = {As axons elongate, tubulin, which is synthesized in the cell body, must be transported and assembled into new structures in the axon. The mechanism of transport and the location of assembly are presently unknown. We report here on the use of tubulin tagged with a photoactivatable fluorescent group to investigate these issues. Photoactivatable tubulin, microinjected into frog embryos at the two-cell stage, is incorporated into microtubules in neurons obtained from explants of the neural tube. When activated by light, a fluorescent mark is made on the microtubules in the axon, and transport and turnover can be visualized directly. We find that microtubules are generated in or near the cell body and continually transported distally as a coherent phase of polymer during axon elongation. This vectorial polymer movement was observed at all levels on the axon, even in the absence of axonal elongation. Measurements of the rate of polymer translocation at various places in the axon suggest that new polymer is formed by intercalary assembly along the axon and assembly at the growth cone in addition to transport of polymer from the cell body. Finally, polymer movement near the growth cone appeared to respond in a characteristic manner to growth cone behavior, while polymer proximally in the axon moved more consistently. These results suggest that microtubule translocation is the principal means of tubulin transport and that translocation plays an important role in generating new axon structure at the growth cone.},
	keywords = {Animals, Axons, Biological Transport, Dextrans, Fluorescein, Fluoresceins, Kinetics, Microscopy, Fluorescence, Microtubules, Neurons, Tubulin, Xenopus},
	issn = {0021-9525},
	author = {Reinsch, S S and Mitchison, T. J. and Kirschner, M}
}
@article {573496,
	title = {Mitotic spindle assembly by two different pathways in vitro.},
	journal = {J Cell Biol},
	volume = {112},
	number = {5},
	year = {1991},
	month = {1991 Mar},
	pages = {925-40},
	abstract = {We have used Xenopus egg extracts to study spindle morphogenesis in a cell-free system and have identified two pathways of spindle assembly in vitro using methods of fluorescent analogue cytochemistry. When demembranated sperm nuclei are added to egg extracts arrested in a mitotic state, individual nuclei direct the assembly of polarized microtubule arrays, which we term half-spindles; half-spindles then fuse pairwise to form bipolar spindles. In contrast, when sperm nuclei are added to extracts that are induced to enter interphase and arrested in the following mitosis, a single sperm nucleus can direct the assembly of a complete spindle. We find that microtubule arrays in vitro are strongly biased towards chromatin, but this does not depend on specific kinetochore-microtubule interactions. Indeed, although we have identified morphological and probably functional kinetochores in spindles assembled in vitro, kinetochores appear not to play an obligate role in the establishment of stable, bipolar microtubule arrays in either assembly pathway. Features of the two pathways suggest that spindle assembly involves a hierarchy of selective microtubule stabilization, involving both chromatin-microtubule interactions and antiparallel microtubule-microtubule interactions, and that fundamental molecular interactions are probably the same in both pathways. This in vitro reconstitution system should be useful for identifying the molecules regulating the generation of asymmetric microtubule arrays and for understanding spindle morphogenesis in general.},
	keywords = {Animals, Cell Nucleus, Cell-Free System, DNA Replication, Male, Microscopy, Electron, Microscopy, Fluorescence, Microtubules, Mitosis, Morphogenesis, Spermatozoa, Spindle Apparatus, Tubulin, Xenopus laevis},
	issn = {0021-9525},
	author = {Sawin, K E and Mitchison, T. J.}
}
@article {573491,
	title = {Poleward microtubule flux mitotic spindles assembled in vitro.},
	journal = {J Cell Biol},
	volume = {112},
	number = {5},
	year = {1991},
	month = {1991 Mar},
	pages = {941-54},
	abstract = {In the preceding paper we described pathways of mitotic spindle assembly in cell-free extracts prepared from eggs of Xenopus laevis. Here we demonstrate the poleward flux of microtubules in spindles assembled in vitro, using a photoactivatable fluorescein covalently coupled to tubulin and multi-channel fluorescence videomicroscopy. After local photoactivation of fluorescence by UV microbeam, we observed poleward movement of fluorescein-marked microtubules at a rate of 3 microns/min, similar to rates of chromosome movement and spindle elongation during prometaphase and anaphase. This movement could be blocked by the addition of millimolar AMP-PNP but was not affected by concentrations of vanadate up to 150 microM, suggesting that poleward flux may be driven by a microtubule motor similar to kinesin. In contrast to previous results obtained in vivo (Mitchison, T. J. 1989. J. Cell Biol. 109:637-652), poleward flux in vitro appears to occur independently of kinetochores or kinetochore microtubules, and therefore may be a general property of relatively stable microtubules within the spindle. We find that microtubules moving towards poles are dynamic structures, and we have estimated the average half-life of fluxing microtubules in vitro to be between approximately 75 and 100 s. We discuss these results with regard to the function of poleward flux in spindle movements in anaphase and prometaphase.},
	keywords = {Adenylyl Imidodiphosphate, Animals, Fluoresceins, Kinetics, Microscopy, Fluorescence, Microtubules, Spindle Apparatus, Tubulin, Vanadates, Xenopus laevis},
	issn = {0021-9525},
	author = {Sawin, K E and Mitchison, T. J.}
}
@article {573501,
	title = {Regulation of the direction of chromosome movement.},
	journal = {Cold Spring Harb Symp Quant Biol},
	volume = {56},
	year = {1991},
	month = {1991},
	pages = {745-50},
	keywords = {Adenosine Triphosphate, Animals, Chromosomes, Microtubules, Mitosis, Movement, Phosphorylation},
	issn = {0091-7451},
	author = {Hyman, A A and Mitchison, T. J.}
}
@article {573486,
	title = {Two different microtubule-based motor activities with opposite polarities in kinetochores.},
	journal = {Nature},
	volume = {351},
	number = {6323},
	year = {1991},
	month = {1991 May 16},
	pages = {206-11},
	abstract = {The movement of microtubules on the kinetochores of isolated chromosomes has been examined by video microscopy. Two different microtubule-based motors on the kinetochore were identified, which have opposite directions of movement. The activities of these two motors can be regulated by factors that can influence phosphorylation.},
	keywords = {Adenine, Adenosine Triphosphate, Animals, Cricetinae, Dose-Response Relationship, Drug, In Vitro Techniques, Microscopy, Fluorescence, Microtubules, Mitosis, Phosphorylation, Spindle Apparatus},
	issn = {0028-0836},
	doi = {10.1038/351206a0},
	author = {Hyman, A A and Mitchison, T. J.}
}
@article {573506,
	title = {Mitosis. The kinetochore in captivity.},
	journal = {Nature},
	volume = {348},
	number = {6296},
	year = {1990},
	month = {1990 Nov 1},
	pages = {14-5},
	keywords = {Chromosomes, Microtubules, Mitosis, Spindle Apparatus},
	issn = {0028-0836},
	doi = {10.1038/348014a0},
	author = {Mitchison, T. J.}
}
@article {573516,
	title = {Modulation of microtubule stability by kinetochores in vitro.},
	journal = {J Cell Biol},
	volume = {110},
	number = {5},
	year = {1990},
	month = {1990 May},
	pages = {1607-16},
	abstract = {The interface between kinetochores and microtubules in the mitotic spindle is known to be dynamic. Kinetochore microtubules can both polymerize and depolymerize, and their dynamic behavior is intimately related to chromosome movement. In this paper we investigate the influence of kinetochores on the inherent dynamic behavior of microtubules using an in vitro assay. The dynamics of microtubule plus ends attached to kinetochores are compared to those of free plus ends in the same solution. We show that microtubules attached to kinetochores exhibit the full range of dynamic instability behavior, but at altered transition rates. Surprisingly, we find that kinetochores increase the rate at which microtubule ends transit from growing to shrinking. This result contradicts our previous findings (Mitchison, T. J., and M. W. Kirschner, 1985b) for technical reasons which are discussed. We suggest that catalysis of the growing to shrinking transition by kinetochores may account for selective depolymerization of kinetochore microtubules during anaphase in vivo. We also investigate the effects of a nonhydrolyzable ATP analogue on kinetochore microtubule dynamics. We find that 5{\textquoteright} adenylylimido diphosphate induces a rigor state at the kinetochore-microtubule interface, which prevents depolymerization of the microtubule.},
	keywords = {Adenylyl Imidodiphosphate, Animals, Cell Line, Chromosomes, Fluorescent Antibody Technique, Microtubules, Spindle Apparatus},
	issn = {0021-9525},
	author = {Hyman, A A and Mitchison, T. J.}
}
@article {573511,
	title = {Real-time visualization of cell cycle-dependent changes in microtubule dynamics in cytoplasmic extracts.},
	journal = {Cell},
	volume = {62},
	number = {3},
	year = {1990},
	month = {1990 Aug 10},
	pages = {579-89},
	abstract = {Using Xenopus egg extracts arrested in interphase or mitosis, we directly observed differences in microtubule dynamics at different stages of the cell cycle. Interphase extracts were prepared from eggs in the first interphase after meiosis. Mitotic extracts were prepared by addition of purified cyclin to interphase extracts. Microtubules were nucleated by the addition of centrosomes and visualized by fluorescence video-microscopy in extracts to which rhodamine-labeled tubulin had been added. We found a striking difference in microtubule dynamics in mitotic versus interphase extracts. Quantitative analysis revealed that the rates of polymerization and depolymerization are similar in interphase and mitosis and that within the spatial and temporal resolution of our experiments the difference in dynamics is due almost entirely to an increase in the frequency of transition from growing to shrinking (catastrophe frequency) in the mitotic extracts.},
	keywords = {Animals, Cell Cycle, Cell Nucleus, Cycloheximide, Cytoplasm, Female, Interphase, Microtubules, Mitosis, Oocytes, Rhodamines, Time Factors, Tubulin, Video Recording, Xenopus laevis},
	issn = {0092-8674},
	author = {Belmont, L D and Hyman, A A and Sawin, K E and Mitchison, T. J.}
}
@article {573521,
	title = {Tubulin flux in the mitotic spindle: where does it come from, where is it going?},
	journal = {Cell Motil Cytoskeleton},
	volume = {16},
	number = {2},
	year = {1990},
	month = {1990},
	pages = {93-8},
	keywords = {Animals, Energy Metabolism, Fluorescent Dyes, Microtubule Proteins, Molecular Structure, Movement, Spindle Apparatus, Tubulin},
	issn = {0886-1544},
	doi = {10.1002/cm.970160202},
	author = {Mitchison, T. J. and Sawin, K E}
}
@article {573531,
	title = {Antigen processing: current issues, exceptional cases (Thy 1 alloantigen, MHC class-II-restricted cytolytic T cells), and implications for vaccine development.},
	journal = {J Autoimmun},
	volume = {2 Suppl},
	year = {1989},
	month = {1989 Jun},
	pages = {45-53},
	abstract = {A dogmatic view of antigen processing is presented in outline, followed by a survey of unresolved issues in the subject. The activity of Thy 1 as an alloantigen, and allospecific MHC Class-II-restricted cytolytic T cells offer examples of exceptional cases of antigen presentation. Implications for the design of vaccines are drawn.},
	keywords = {Animals, Antigen-Presenting Cells, Antigens, Antigens, Surface, Antigens, Thy-1, Biological Transport, Active, Histocompatibility Antigens Class II, Humans, T-Lymphocytes, Cytotoxic, Vaccines},
	issn = {0896-8411},
	author = {Chain, B M and Mitchison, N A and Mitchison, T. J. and Davies, D H and Marcinkiewicz, J}
}
@article {573536,
	title = {Mitosis: basic concepts.},
	journal = {Curr Opin Cell Biol},
	volume = {1},
	number = {1},
	year = {1989},
	month = {1989 Feb},
	pages = {67-74},
	keywords = {Animals, Chromosomes, Microtubules, Mitosis, Spindle Apparatus},
	issn = {0955-0674},
	author = {Mitchison, T. J.}
}
@article {573526,
	title = {Polewards microtubule flux in the mitotic spindle: evidence from photoactivation of fluorescence.},
	journal = {J Cell Biol},
	volume = {109},
	number = {2},
	year = {1989},
	month = {1989 Aug},
	pages = {637-52},
	abstract = {I have synthesized a novel derivative of carboxyfluorescein that is nonfluorescent, but can be converted to a fluorescent form by exposure to 365-nm light. This photoactivable, fluorescent probe was covalently attached to tubulin and microinjected into mitotic tissue culture cells, where it incorporated into functional spindles. To generate a fluorescent bar across the mitotic spindle, metaphase cells were irradiated with a slit microbeam. This bar decreased in intensity over the first minute, presumably due to turnover of nonkinetochore microtubules. The remaining fluorescent zones, now presumably restricted to kinetochore microtubules, moved polewards at 0.3-0.7 microns/min. This result provides strong evidence for polewards flux in kinetochore microtubules. In conjunction with earlier biotin-tubulin incorporation experiments (Mitchison, T. J., L. Evans, E. Schulze, and M. Kirschner. 1986. Cell. 45:515-527), I conclude that microtubules polymerize at kinetochores and depolymerize near the poles throughout metaphase. The significance of this observation for spindle structure and function is discussed. Local photoactivation of fluorescence should be a generally useful method for following molecular dynamics inside living cells.},
	keywords = {Animals, Cytoskeleton, Epithelial Cells, Epithelium, Fluorescein, Fluoresceins, Fluorescent Dyes, Kidney, Microtubules, Spindle Apparatus, Swine, Tubulin},
	issn = {0021-9525},
	author = {Mitchison, T. J.}
}
@article {573541,
	title = {Behaviour of microtubules and actin filaments in living Drosophila embryos.},
	journal = {Development},
	volume = {103},
	number = {4},
	year = {1988},
	month = {1988 Aug},
	pages = {675-86},
	abstract = {We describe the preparation of novel fluorescent derivatives of rabbit muscle actin and bovine tubulin, and the use of these derivatives to study the behaviour of actin filaments and microtubules in living Drosophila embryos, in which the nuclei divide at intervals of 8 to 21 min. The fluorescently labelled proteins appear to function normally in vitro and in vivo, and they allow continuous observation of the cytoskeleton in living embryos without perturbing development. By coinjecting labelled actin and tubulin into the early syncytial embryo, the spatial relationships between the distinct filament networks that they form can be followed second by second. The dynamic rearrangements of actin filaments and microtubules observed confirms and extends results obtained from previous studies, in which fixation techniques and specific staining were used to visualize the cytoskeleton in the Drosophila embryo. However, no tested fixation method produces an exact representation of the in vivo microtubule distribution.},
	keywords = {Actins, Animals, Cattle, Cytoskeleton, Drosophila, Embryo, Nonmammalian, Microscopy, Fluorescence, Microtubules, Rabbits},
	issn = {0950-1991},
	author = {Kellogg, D R and Mitchison, T. J. and Alberts, B M}
}
@article {573551,
	title = {Microtubule dynamics and kinetochore function in mitosis.},
	journal = {Annu Rev Cell Biol},
	volume = {4},
	year = {1988},
	month = {1988},
	pages = {527-49},
	keywords = {Animals, Cell Movement, Chromosomes, In Vitro Techniques, Microtubules, Mitosis, Spindle Apparatus},
	issn = {0743-4634},
	doi = {10.1146/annurev.cb.04.110188.002523},
	author = {Mitchison, T. J.}
}
@article {573546,
	title = {Polewards chromosome movement driven by microtubule depolymerization in vitro.},
	journal = {Nature},
	volume = {331},
	number = {6156},
	year = {1988},
	month = {1988 Feb 11},
	pages = {499-504},
	abstract = {We constructed complexes between isolated chromosomes and microtubules made from purified tubulin to study the movement of chromosomes towards the {\textquoteright}minus{\textquoteright} end of microtubules in vitro, a process analogous to the movement of chromosomes towards the pole of the spindle at anaphase of mitosis. Our results show that the energy for this movement is derived solely from microtubule depolymerization, and indicate that anaphase movement of chromosomes is both powered and regulated by microtubule depolymerization at the kinetochore.},
	keywords = {Adenosine Triphosphate, Anaphase, Biotin, Chromosomes, Cross-Linking Reagents, Energy Metabolism, Guanosine Triphosphate, Microtubules, Movement, Polymers, Spindle Apparatus, Tubulin},
	issn = {0028-0836},
	doi = {10.1038/331499a0},
	author = {Koshland, D E and Mitchison, T. J. and Kirschner, M W}
}
@article {573561,
	title = {Cell motility: good things from slime.},
	journal = {Nature},
	volume = {328},
	number = {6132},
	year = {1987},
	month = {1987 Aug 20-26},
	pages = {665},
	keywords = {Amoeba, Animals, Axonal Transport, Cell Movement, Kinesin, Microtubules, Myxomycetes, Nerve Tissue Proteins},
	issn = {0028-0836},
	doi = {10.1038/328665a0},
	author = {Mitchison, T. J.}
}
@article {573556,
	title = {Some thoughts on the partitioning of tubulin between monomer and polymer under conditions of dynamic instability.},
	journal = {Cell Biophys},
	volume = {11},
	year = {1987},
	month = {1987 Dec},
	pages = {35-55},
	abstract = {We have considered the partitioning of tubulin between monomer and polymer in the cell under conditions of dynamic instability. Dynamic instability adds to the on and off rate constant of steady-state dynamics{\textquoteright} new parameters: (1) the rate at which growing microtubules transit to a shrinking phase; and (2) the rate at which shrinking microtubules transit to the growing phase. Under these conditions the free-monomer concentration in the cell increases with total tubulin if the number of nucleating sites is fixed. If the number of nucleating sites increases at fixed total tubulin, subunits shift from the monomer to the polymer phase. These important properties deviate from the traditional equilibrium and steady-state theories and have important implications for the biosynthetic regulation of tubulin.},
	keywords = {Biophysical Phenomena, Biophysics, Cell Physiological Phenomena, Guanosine Triphosphate, In Vitro Techniques, Kinetics, Microtubules, Polymers, Tubulin},
	issn = {0163-4992},
	author = {Mitchison, T. J. and Kirschner, M W}
}
@article {573566,
	title = {Isolation of mammalian centrosomes.},
	journal = {Methods Enzymol},
	volume = {134},
	year = {1986},
	month = {1986},
	pages = {261-8},
	keywords = {Animals, Cell Fractionation, Cell Line, Centrifugation, Density Gradient, Centrioles, Humans, Interphase, Microtubules, Organoids},
	issn = {0076-6879},
	author = {Mitchison, T. J. and Kirschner, M W}
}
@article {573571,
	title = {The role of microtubule polarity in the movement of kinesin and kinetochores.},
	journal = {J Cell Sci Suppl},
	volume = {5},
	year = {1986},
	month = {1986},
	pages = {121-8},
	abstract = {Microtubules are important for organizing and directing many types of intracellular motility. Recently progress has been made in the analysis of two types of motility at the molecular level: the movement of axonal vesicles driven by kinesin, and the movement of chromosomes driven by the kinetochore. Both require ATP for movement in vitro. Kinesin-driven movement is unidirectional, towards the microtubule plus end, while movement of the kinetochore is bidirectional. These similarities and differences are discussed and incorporated into a new model for the kinetochore-microtubule interface.},
	keywords = {Animals, Centromere, Chromosomes, Kinesin, Microtubule Proteins, Microtubules, Models, Biological, Movement, Nerve Tissue Proteins},
	issn = {0269-3518},
	author = {Mitchison, T. J.}
}
@article {573581,
	title = {Properties of the kinetochore in vitro. I. Microtubule nucleation and tubulin binding.},
	journal = {J Cell Biol},
	volume = {101},
	number = {3},
	year = {1985},
	month = {1985 Sep},
	pages = {755-65},
	abstract = {We have isolated chromosomes from Chinese hamster ovary cells arrested in mitosis with vinblastine and examined the interactions of their kinetochores with purified tubulin in vitro. The kinetochores nucleate microtubule (MT) growth with complex kinetics. After an initial lag phase, MTs are continuously nucleated with both plus and minus ends distally localized. This mixed polarity seems inconsistent with the formation of an ordered, homopolar kinetochore fiber in vivo. As isolated from vinblastine-arrested cells, kinetochores contain no bound tubulin. The kinetochores of chromosomes isolated from colcemid-arrested cells or of chromosomes incubated with tubulin in vitro are brightly stained after anti-tubulin immunofluorescence. This bound tubulin is probably not in the form of MTs. It is localized to the corona region by immunoelectron microscopy, where it may play a role in MT nucleation in vitro.},
	keywords = {Animals, Cell-Free System, Centromere, Chromosomes, Cricetinae, Cricetulus, Demecolcine, Female, In Vitro Techniques, Microscopy, Electron, Microtubules, Ovary, Protein Binding, Spindle Apparatus, Tubulin, Vinblastine},
	issn = {0021-9525},
	author = {Mitchison, T. J. and Kirschner, M W}
}
@article {573576,
	title = {Properties of the kinetochore in vitro. II. Microtubule capture and ATP-dependent translocation.},
	journal = {J Cell Biol},
	volume = {101},
	number = {3},
	year = {1985},
	month = {1985 Sep},
	pages = {766-77},
	abstract = {We have studied the interaction of preformed microtubules (MTs) with the kinetochores of isolated chromosomes. This reaction, which we call MT capture, results in MTs becoming tightly bound to the kinetochore, with their ends capped against depolymerization. These observations, combined with MT dynamic instability, suggest a model for spindle morphogenesis. In addition, ATP appears to mobilize dynamic processes at captured MT ends. We used biotin-labeled MT seeds to follow assembly dynamics at the kinetochore. In the presence of ATP and unlabeled tubulin, labeled MT segments translocate away from the kinetochore by polymerization of subunits at the attached end. We have termed this reaction proximal assembly. Further studies demonstrated that translocation could be uncoupled from MT assembly. We suggest that the kinetochore contains an ATPase activity that walks along the MT lattice toward the plus end. This activity may be responsible for the movement of chromosomes away from the pole in prometaphase.},
	keywords = {Adenosine Triphosphate, Animals, Centromere, Chromosomes, Cricetinae, Cricetulus, Female, In Vitro Techniques, Kinetics, Microtubules, Morphogenesis, Movement, Ovary, Spindle Apparatus, Tubulin},
	issn = {0021-9525},
	author = {Mitchison, T. J. and Kirschner, M W}
}
@article {573586,
	title = {Assembly properties of fluorescein-labeled tubulin in vitro before and after fluorescence bleaching.},
	journal = {J Cell Biol},
	volume = {99},
	number = {6},
	year = {1984},
	month = {1984 Dec},
	pages = {2146-56},
	abstract = {Brain tubulin has been conjugated with dichlorotriazinyl-aminofluorescein (DTAF) to form a visualizable complex for the study of tubulin dynamics in living cells. By using several assays we confirm the finding of Keith et al. (Keith, C. H., J. R. Feramisco, and M. Shelanski, 1981, J. Cell Biol., 88:234-240) that DTAF-tubulin polymerizes like control tubulin in vitro. The fluorescein moiety of the complex is readily bleached by the 488-nm line from an argon ion laser. When irradiations are performed over short times (less than 1 s) and in the presence of 2 mM glutathione, a mixture of DTAF-tubulin and control protein (as occurs after microinjection of the fluorescent conjugate into living cells) will retain full polymerization activity. Slow bleaching (approximately 5 min) or bleaching without glutathione promotes formation of covalent cross-links between neighboring polypeptides and kills the polymerization activity of DTAF-tubulin, including some molecules that are neither cross-linked nor bleached. Even under conditions that damage DTAF-tubulin, however, DTAF-microtubules are not destroyed by bleaching. They will continue to elongate by addition of DTAF-tubulin subunits to their free ends, and they neither bind nor exchange subunits along their lateral surfaces. These results suggest that DTAF-tubulin is a suitable analog for tubulin, both in studies of protein incorporation and for investigations of fluorescence redistribution after photobleaching.},
	keywords = {Animals, Brain, Cattle, Flagella, Fluorescein-5-isothiocyanate, Fluoresceins, Fluorescent Dyes, Kinetics, Lasers, Macromolecular Substances, Microtubules, Sea Urchins, Thiocyanates, Tubulin},
	issn = {0021-9525},
	author = {Leslie, R J and Saxton, W M and Mitchison, T. J. and Neighbors, B and Salmon, E D and McIntosh, J R}
}
@article {573591,
	title = {Localization of antigenic determinants in whole Drosophila embryos.},
	journal = {Dev Biol},
	volume = {99},
	number = {1},
	year = {1983},
	month = {1983 Sep},
	pages = {261-4},
	abstract = {Procedures are described for removal of the vitelline membrane from gently fixed Drosophila embryos en masse. The resulting embryos retain excellent structural integrity and are now suitable for a variety of immunocytochemical and biochemical characterizations.},
	keywords = {Animals, Antibodies, Monoclonal, Drosophila melanogaster, Embryo, Nonmammalian, Epitopes, Fixatives, Fluorescent Antibody Technique},
	issn = {0012-1606},
	author = {Mitchison, T. J. and Sedat, J}
}
@article {1433280,
	title = {Comparison between procaine and isocarboxazid metabolism in vitro by a liver microsomal amidase-esterase},
	journal = {Biochem Pharmacol},
	volume = {24},
	number = {16},
	year = {1975},
	month = {1975 Aug 15},
	pages = {1517-21},
	keywords = {Amidohydrolases, Animals, Esterases, Hydrogen-Ion Concentration, In Vitro Techniques, Isocarboxazid, Kinetics, Male, Metals, Microsomes, Liver, Phospholipids, Procaine, Proteins, Rats, Subcellular Fractions, Temperature},
	issn = {1873-2968},
	doi = {10.1016/0006-2952(75)90029-5},
	author = {Moroi, K and Sato, T}
}
@article {1433279,
	title = {Formate assay in body fluids: application in methanol poisoning},
	journal = {Biochem Med},
	volume = {13},
	number = {2},
	year = {1975},
	month = {1975 Jun},
	pages = {117-26},
	keywords = {Aldehyde Oxidoreductases, Animals, Body Fluids, Carbon Dioxide, Formates, Haplorhini, Humans, Hydrogen-Ion Concentration, Kinetics, Methanol, Methods, Pseudomonas},
	issn = {0006-2944},
	doi = {10.1016/0006-2944(75)90147-7},
	author = {Makar, A B and McMartin, K E and Palese, M and Tephly, T R}
}
@article {1433281,
	title = {Metal substitutions incarbonic anhydrase: a halide ion probe study},
	journal = {Biochem Biophys Res Commun},
	volume = {66},
	number = {4},
	year = {1975},
	month = {1975 Oct 27},
	pages = {1281-6},
	keywords = {Animals, Binding Sites, Cadmium, Carbonic Anhydrases, Cattle, Humans, Hydrogen-Ion Concentration, Magnetic Resonance Spectroscopy, Mercury, Protein Binding, Protein Conformation, Zinc},
	issn = {0006-291X},
	doi = {10.1016/0006-291x(75)90498-2},
	author = {Smith, R J and Bryant, R G}
}
