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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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'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.
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.
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.
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.
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.
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.
We have developed a method to detect DNA synthesis in proliferating cells, based on the incorporation of 5-ethynyl-2'-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.