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.
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 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.
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.
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' 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.
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.
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.
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.
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.
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.
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.
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 'end-on' to 'side-on'. 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.
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's role in cell motility is to govern the alignment of traction stresses that permit single-cell migration.
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.
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.
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.
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.
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.
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.
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' 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.