Molecular Mechanism of Cytokinesis.

Division of amoebas, fungi, and animal cells into two daughter cells at the end of the cell cycle depends on a common set of ancient proteins, principally actin filaments and myosin-II motors. Anillin, formins, IQGAPs, and many other proteins regulate the assembly of the actin filaments into a contractile ring positioned between the daughter nuclei by different mechanisms in fungi and animal cells. Interactions of myosin-II with actin filaments produce force to assemble and then constrict the contractile ring to form a cleavage furrow. Contractile rings disassemble as they constrict. In some cases, knowledge about the numbers of participating proteins and their biochemical mechanisms has made it possible to formulate molecularly explicit mathematical models that reproduce the observed physical events during cytokinesis by computer simulations.

Chromosome Segregation Fidelity in Epithelia Requires Tissue Architecture.

Much of our understanding of chromosome segregation is based on cell culture systems. Here, we examine the importance of the tissue environment for chromosome segregation by comparing chromosome segregation fidelity across several primary cell types in native and nonnative contexts. We discover that epithelial cells have increased chromosome missegregation outside of their native tissues. Using organoid culture systems, we show that tissue architecture, specifically integrin function, is required for accurate chromosome segregation. We find that tissue architecture enhances the correction of merotelic microtubule-kinetochore attachments, and this is especially important for maintaining chromosome stability in the polyploid liver. We propose that disruption of tissue architecture could underlie the widespread chromosome instability across epithelial cancers. Moreover, our findings highlight the extent to which extracellular context can influence intrinsic cellular processes and the limitations of cell culture systems for studying cells that naturally function within a tissue.

Mitotic spindle assembly in animal cells: a fine balancing act.

The mitotic spindle has a crucial role in ensuring the accurate segregation of chromosomes into the two daughter cells during cell division, which is paramount for maintaining genome integrity. It is a self-organized and dynamic macromolecular structure that is constructed from microtubules, microtubule-associated proteins and motor proteins. Thirty years of research have led to the identification of centrosome-, chromatin- and microtubule-mediated microtubule nucleation pathways that each contribute to mitotic spindle assembly. Far from being redundant pathways, data are now emerging regarding how they function together to ensure the timely completion of mitosis. We are also beginning to comprehend the multiple mechanisms by which cells regulate spindle scaling. Together, this research has increased our understanding of how cells coordinate hundreds of proteins to assemble the dynamic, precise and robust structure that is the mitotic spindle.

Human kinesin-5 KIF11 drives the helical motion of anti-parallel and parallel microtubules around each other.

During mitosis, motor proteins and microtubule-associated protein organize the spindle apparatus by cross-linking and sliding microtubules. Kinesin-5 plays a vital role in spindle formation and maintenance, potentially inducing twist in the spindle fibers. The off-axis power stroke of kinesin-5 could generate this twist, but its implications in microtubule organization remain unclear. Here, we investigate 3D microtubule-microtubule sliding mediated by the human kinesin-5, KIF11, and found that the motor caused right-handed helical motion of anti-parallel microtubules around each other. The sidestepping ratio increased with reduced ATP concentration, indicating that forward and sideways stepping of the motor are not strictly coupled. Further, the microtubule-microtubule distance (motor extension) during sliding decreased with increasing sliding velocity. Intriguingly, parallel microtubules cross-linked by KIF11 orbited without forward motion, with nearly full motor extension. Altering the length of the neck linker increased the forward velocity and pitch of microtubules in anti-parallel overlaps. Taken together, we suggest that helical motion and orbiting of microtubules, driven by KIF11, contributes to flexible and context-dependent filament organization, as well as torque regulation within the mitotic spindle.

F-actin coordinates spindle morphology and function in Drosophila meiosis.

Meiosis is a highly conserved feature of sexual reproduction that ensures germ cells have the correct number of chromosomes prior to fertilization. A subset of microtubules, known as the spindle, are essential for accurate chromosome segregation during meiosis. Building evidence in mammalian systems has recently highlighted the unexpected requirement of the actin cytoskeleton in chromosome segregation; a network of spindle actin filaments appear to regulate many aspects of this process. Here we show that Drosophila oocytes also have a spindle population of actin that appears to regulate the formation of the microtubule spindle and chromosomal movements throughout meiosis. We demonstrate that genetic and pharmacological disruption of the actin cytoskeleton has a significant impact on spindle morphology, dynamics, and chromosome alignment and segregation during maturation and the metaphase-anaphase transition. We further reveal a role for calcium in maintaining the microtubule spindle and spindle actin. Together, our data highlights potential conservation of morphology and mechanism of the spindle actin during meiosis.

Prime movers: the mechanochemistry of mitotic kinesins.

Mitotic spindles are self-organizing protein machines that harness teams of multiple force generators to drive chromosome segregation. Kinesins are key members of these force-generating teams. Different kinesins walk directionally along dynamic microtubules, anchor, crosslink, align and sort microtubules into polarized bundles, and influence microtubule dynamics by interacting with microtubule tips. The mechanochemical mechanisms of these kinesins are specialized to enable each type to make a specific contribution to spindle self-organization and chromosome segregation.

Insights into the micromechanical properties of the metaphase spindle.

The microtubule-based metaphase spindle is subjected to forces that act in diverse orientations and over a wide range of timescales. Currently, we cannot explain how this dynamic structure generates and responds to forces while maintaining overall stability, as we have a poor understanding of its micromechanical properties. Here, we combine the use of force-calibrated needles, high-resolution microscopy, and biochemical perturbations to analyze the vertebrate metaphase spindle's timescale- and orientation-dependent viscoelastic properties. We find that spindle viscosity depends on microtubule crosslinking and density. Spindle elasticity can be linked to kinetochore and nonkinetochore microtubule rigidity, and also to spindle pole organization by kinesin-5 and dynein. These data suggest a quantitative model for the micromechanics of this cytoskeletal architecture and provide insight into how structural and functional stability is maintained in the face of forces, such as those that control spindle size and position, and can result from deformations associated with chromosome movement.

Spindle reorientation in response to mechanical stress is an emergent property of the spindle positioning mechanisms.

Proper orientation of the mitotic spindle plays a crucial role in embryos, during tissue development, and in adults, where it functions to dissipate mechanical stress to maintain tissue integrity and homeostasis. While mitotic spindles have been shown to reorient in response to external mechanical stresses, the subcellular cues that mediate spindle reorientation remain unclear. Here, we used a combination of optogenetics and computational modeling to investigate how mitotic spindles respond to inhomogeneous tension within the actomyosin cortex. Strikingly, we found that the optogenetic activation of RhoA only influences spindle orientation when it is induced at both poles of the cell. Under these conditions, the sudden local increase in cortical tension induced by RhoA activation reduces pulling forces exerted by cortical regulators on astral microtubules. This leads to a perturbation of the balance of torques exerted on the spindle, which causes it to rotate. Thus, spindle rotation in response to mechanical stress is an emergent phenomenon arising from the interaction between the spindle positioning machinery and the cell cortex.

Contribution of cytoplasm viscoelastic properties to mitotic spindle positioning.

Cells are filled with macromolecules and polymer networks that set scale-dependent viscous and elastic properties to the cytoplasm. Although the role of these parameters in molecular diffusion, reaction kinetics, and cellular biochemistry is being increasingly recognized, their contributions to the motion and positioning of larger organelles, such as mitotic spindles for cell division, remain unknown. Here, using magnetic tweezers to displace and rotate mitotic spindles in living embryos, we uncovered that the cytoplasm can impart viscoelastic reactive forces that move spindles, or passive objects with similar size, back to their original positions. These forces are independent of cytoskeletal force generators yet reach hundreds of piconewtons and scale with cytoplasm crowding. Spindle motion shears and fluidizes the cytoplasm, dissipating elastic energy and limiting spindle recoils with functional implications for asymmetric and oriented divisions. These findings suggest that bulk cytoplasm material properties may constitute important control elements for the regulation of division positioning and cellular organization.

Cross-linker design determines microtubule network organization by opposing motors.

During cell division, cross-linking motors determine the architecture of the spindle, a dynamic microtubule network that segregates the chromosomes in eukaryotes. It is unclear how motors with opposite directionality coordinate to drive both contractile and extensile behaviors in the spindle. Particularly, the impact of different cross-linker designs on network self-organization is not understood, limiting our understanding of self-organizing structures in cells but also our ability to engineer new active materials. Here, we use experiment and theory to examine active microtubule networks driven by mixtures of motors with opposite directionality and different cross-linker design. We find that although the kinesin-14 HSET causes network contraction when dominant, it can also assist the opposing kinesin-5 KIF11 to generate extensile networks. This bifunctionality results from HSET's asymmetric design, distinct from symmetric KIF11. These findings expand the set of rules underlying patterning of active microtubule assemblies and allow a better understanding of motor cooperation in the spindle.

NuMA assemblies organize microtubule asters to establish spindle bipolarity in acentrosomal human cells.

In most animal cells, mitotic spindle formation is mediated by coordination of centrosomal and acentrosomal pathways. At the onset of mitosis, centrosomes promote spindle bipolarization. However, the mechanism through which the acentrosomal pathways facilitate the establishment of spindle bipolarity in early mitosis is not completely understood. In this study, we show the critical roles of nuclear mitotic apparatus protein (NuMA) in the generation of spindle bipolarity in acentrosomal human cells. In acentrosomal human cells, we found that small microtubule asters containing NuMA formed at the time of nuclear envelope breakdown. In addition, these asters were assembled by dynein and the clustering activity of NuMA. Subsequently, NuMA organized the radial array of microtubules, which incorporates Eg5, and thus facilitated spindle bipolarization. Importantly, in cells with centrosomes, we also found that NuMA promoted the initial step of spindle bipolarization in early mitosis. Overall, these data suggest that canonical centrosomal and NuMA-mediated acentrosomal pathways redundantly promote spindle bipolarity in human cells.

Microtubule poleward flux in human cells is driven by the coordinated action of four kinesins.

Mitotic spindle microtubules (MTs) undergo continuous poleward flux, whose driving force and function in humans remain unclear. Here, we combined loss-of-function screenings with analysis of MT-dynamics in human cells to investigate the molecular mechanisms underlying MT-flux. We report that kinesin-7/CENP-E at kinetochores (KTs) is the predominant driver of MT-flux in early prometaphase, while kinesin-4/KIF4A on chromosome arms facilitates MT-flux during late prometaphase and metaphase. Both these activities work in coordination with kinesin-5/EG5 and kinesin-12/KIF15, and our data suggest that the MT-flux driving force is transmitted from non-KT-MTs to KT-MTs by the MT couplers HSET and NuMA. Additionally, we found that the MT-flux rate correlates with spindle length, and this correlation depends on the establishment of stable end-on KT-MT attachments. Strikingly, we find that MT-flux is required to regulate spindle length by counteracting kinesin 13/MCAK-dependent MT-depolymerization. Thus, our study unveils the long-sought mechanism of MT-flux in human cells as relying on the coordinated action of four kinesins to compensate for MT-depolymerization and regulate spindle length.

RanGTP and importin ß regulate meiosis I spindle assembly and function in mouse oocytes.

Homologous chromosome segregation during meiosis I (MI) in mammalian oocytes is carried out by the acentrosomal MI spindles. Whereas studies in human oocytes identified Ran GTPase as a crucial regulator of the MI spindle function, experiments in mouse oocytes questioned the generality of this notion. Here, we use live-cell imaging with fluorescent probes and Förster resonance energy transfer (FRET) biosensors to monitor the changes in Ran and importin ß signaling induced by perturbations of Ran in mouse oocytes while examining the MI spindle dynamics. We show that unlike RanT24N employed in previous studies, a RanT24N, T42A double mutant inhibits RanGEF without perturbing cargo binding to importin ß and disrupts MI spindle function in chromosome segregation. Roles of Ran and importin ß in the coalescence of microtubule organizing centers (MTOCs) and MI spindle assembly are further supported by the use of the chemical inhibitor importazole, whose effects are partially rescued by the GTP hydrolysis-resistant RanQ69L mutant. These results indicate that RanGTP is essential for MI spindle assembly and function both in humans and mice.

Live imaging of the Drosophila ovarian niche shows spectrosome and centrosome dynamics during asymmetric germline stem cell division.

Drosophila female germline stem cells (GSCs) are found inside the cellular niche at the tip of the ovary. They undergo asymmetric divisions to renew the stem cell lineage and to produce sibling cystoblasts that will in turn enter differentiation. GSCs and cystoblasts contain spectrosomes, membranous structures essential for orientation of the mitotic spindle and that, particularly in GSCs, change shape depending on the cell cycle phase. Using live imaging and a fusion protein of GFP and the spectrosome component Par-1, we follow the complete spectrosome cycle throughout GSC division and quantify the relative duration of the different spectrosome shapes. We also determine that the Par-1 kinase shuttles between the spectrosome and the cytoplasm during mitosis and observe the continuous addition of new material to the GSC and cystoblast spectrosomes. Next, we use the Fly-FUCCI tool to define, in live and fixed tissues, that GSCs have a shorter G1 compared with the G2 phase. The observation of centrosomes in dividing GSCs allowed us to determine that centrosomes separate very early in G1, before centriole duplication. Furthermore, we show that the anterior centrosome associates with the spectrosome only during mitosis and that, upon mitotic spindle assembly, it translocates to the cell cortex, where it remains anchored until centrosome separation. Finally, we demonstrate that the asymmetric division of GSCs is not an intrinsic property of these cells, as the spectrosome of GSC-like cells located outside of the niche can divide symmetrically. Thus, GSCs display unique properties during division, a behaviour influenced by the surrounding niche.

The chromosomal passenger complex (CPC): from easy rider to the godfather of mitosis.

Successful cell division requires the precise and timely coordination of chromosomal, cytoskeletal and membrane trafficking events. These processes are regulated by the competing actions of protein kinases and phosphatases. Aurora B is one of the most intensively studied kinases. In conjunction with inner centromere protein (INCENP), borealin (also known as Dasra) and survivin it forms the chromosomal passenger complex (CPC). This complex targets to different locations at differing times during mitosis, where it regulates key mitotic events: correction of chromosome-microtubule attachment errors; activation of the spindle assembly checkpoint; and construction and regulation of the contractile apparatus that drives cytokinesis. Our growing understanding of the CPC has seen it develop from a mere passenger riding on the chromosomes to one of the main controllers of mitosis.

Relaxation and Noise-Driven Oscillations in a Model of Mitotic Spindle Dynamics.

During cell division, the mitotic spindle moves dynamically through the cell to position the chromosomes and determine the ultimate spatial position of the two daughter cells. These movements have been attributed to the action of cortical force generators which pull on the astral microtubules to position the spindle, as well as pushing events by these same microtubules against the cell cortex and plasma membrane. Attachment and detachment of cortical force generators working antagonistically against centring forces of microtubules have been modelled previously (Grill et al. in Phys Rev Lett 94:108104, 2005) via stochastic simulations and mean-field Fokker-Planck equations (describing random motion of force generators) to predict oscillations of a spindle pole in one spatial dimension. Using systematic asymptotic methods, we reduce the Fokker-Planck system to a set of ordinary differential equations (ODEs), consistent with a set proposed by Grill et al., which can provide accurate predictions of the conditions for the Fokker-Planck system to exhibit oscillations. In the limit of small restoring forces, we derive an algebraic prediction of the amplitude of spindle-pole oscillations and demonstrate the relaxation structure of nonlinear oscillations. We also show how noise-induced oscillations can arise in stochastic simulations for conditions in which the mean-field Fokker-Planck system predicts stability, but for which the period can be estimated directly by the ODE model and the amplitude by a related stochastic differential equation that incorporates random binding kinetics.

Wnt signaling recruits KIF2A to the spindle to ensure chromosome congression and alignment during mitosis.

Canonical Wnt signaling plays critical roles in development and tissue renewal by regulating ß-catenin target genes. Recent evidence showed that ß-catenin-independent Wnt signaling is also required for faithful execution of mitosis. However, the targets and specific functions of mitotic Wnt signaling still remain uncharacterized. Using phosphoproteomics, we identified that Wnt signaling regulates the microtubule depolymerase KIF2A during mitosis. We found that Dishevelled recruits KIF2A via its N-terminal and motor domains, which is further promoted upon LRP6 signalosome formation during cell division. We show that Wnt signaling modulates KIF2A interaction with PLK1, which is critical for KIF2A localization at the spindle. Accordingly, inhibition of basal Wnt signaling leads to chromosome misalignment in somatic cells and pluripotent stem cells. We propose that Wnt signaling monitors KIF2A activity at the spindle poles during mitosis to ensure timely chromosome alignment. Our findings highlight a function of Wnt signaling during cell division, which could have important implications for genome maintenance, notably in stem cells.

CRMP5 participates in oocyte meiosis by regulating spastin to correct microtubule-kinetochore misconnection.

Our previous studies have suggested that spastin, which aggregates on spindle microtubules in oocytes, may promote the assembly of mouse oocyte spindles by cutting microtubules. This action may be related to CRMP5, as knocking down CRMP5 results in reduced spindle microtubule density and maturation defects in oocytes. In this study, we found that, after knocking down CRMP5 in oocytes, spastin distribution shifted from the spindle to the spindle poles and errors in microtubule-kinetochore attachment appeared in oocyte spindles. However, CRMP5 did not interact with the other two microtubule-severing proteins, katanin-like-1 (KATNAL1) and fidgetin-like-1 (FIGNL1), which aggregate at the spindle poles. We speculate that, in oocytes, due to the reduction of spastin distribution on chromosomes after knocking down CRMP5, microtubule-kinetochore errors cannot be corrected through severing, resulting in meiotic division abnormalities and maturation defects in oocytes. This finding provides new insights into the regulatory mechanisms of spastin in oocytes and important opportunities for the study of meiotic division mechanisms.

Asymmetry is defined during meiosis in the oocyte of the parthenogenetic nematode Diploscapter pachys.

Asymmetric cell division is an essential feature of normal development and certain pathologies. The process and its regulation have been studied extensively in the Caenorhabditis elegans embryo, particularly how symmetry of the actomyosin cortical cytoskeleton is broken by a sperm-derived signal at fertilization, upstream of polarity establishment. Diploscapter pachys is the closest parthenogenetic relative to C. elegans, and D. pachys one-cell embryos also divide asymmetrically. However how polarity is triggered in the absence of sperm remains unknown. In post-meiotic embryos, we find that the nucleus inhabits principally one embryo hemisphere, the future posterior pole. When forced to one pole by centrifugation, the nucleus returns to its preferred pole, although poles appear identical as concerns cortical ruffling and actin cytoskeleton. The location of the meiotic spindle also correlates with the future posterior pole and slight actin enrichment is observed at that pole in some early embryos along with microtubule structures emanating from the meiotic spindle. Polarized location of the nucleus is not observed in pre-meiotic D. pachys oocytes. All together our results are consistent with the idea that polarity of the D. pachys embryo is attained during meiosis, seemingly based on the location of the meiotic spindle, by a mechanism that may be present but suppressed in C. elegans.

Analysis of the potential role of fission yeast PP2A in spindle assembly checkpoint inactivation.

As a surveillance mechanism, the activated spindle assembly checkpoint (SAC) potently inhibits the E3 ubiquitin ligase APC/C (anaphase-promoting complex/cyclosome) to ensure accurate chromosome segregation. Although the protein phosphatase 2A (PP2A) has been proposed to be both, directly and indirectly, involved in spindle assembly checkpoint inactivation in mammalian cells, whether it is similarly operating in the fission yeast Schizosaccharomycer pombe has never been demonstrated. Here, we investigated whether fission yeast PP2A is involved in SAC silencing by following the rate of cyclin B (Cdc13) destruction at SPBs during the recovery phase in nda3-KM311 cells released from the inhibition of APC/C by the activated spindle checkpoint. The timing of the SAC inactivation is only slightly delayed when two B56 regulatory subunits (Par1 and Par2) of fission yeast PP2A are absent. Overproduction of individual PP2A subunits either globally in the nda3-KM311 arrest-and-release system or locally in the synthetic spindle checkpoint activation system only slightly suppresses the SAC silencing defects in PP1 deletion (dis2Δ) cells. Our study thus demonstrates that the fission yeast PP2A is not a key regulator actively involved in SAC inactivation.