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Mesoscale macromolecular complexes and organelles, tens to hundreds of nanometers in size, crowd the eukaryotic cytoplasm. It is therefore unclear how mesoscale particles remain sufficiently mobile to regulate dynamic processes such as cell division. Here, we study mobility across dividing cells that contain densely packed, dynamic microtubules, comprising the metaphase spindle. In dividing human cells, we tracked 40 nm genetically encoded multimeric nanoparticles (GEMs), whose sizes are commensurate with the inter-filament spacing in metaphase spindles. Unexpectedly, the effective diffusivity of GEMs was similar inside the dense metaphase spindle and the surrounding cytoplasm. Eliminating microtubules or perturbing their polymerization dynamics decreased diffusivity by ~30%, suggesting that microtubule polymerization enhances random displacements to amplify diffusive-like motion. Our results suggest that microtubules effectively fluidize the mitotic cytoplasm to equalize mesoscale mobility across a densely packed, dynamic, non-uniform environment, thus spatially maintaining a key biophysical parameter that impacts biochemistry, ranging from metabolism to the nucleation of cytoskeletal filaments., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Cell division axes during development are specified in different orientations to establish multicellular assemblies, but the mechanisms that generate division axis diversity remain unclear. We show here that patterns of cell contact provide cues that diversify cell division orientation by modulating cortical non-muscle myosin flow. We reconstituted in vivo contact patterns using beads or isolated cells to show two findings. First, we identified three contact-dependent cues that pattern cell division orientation and myosin flow: physical contact, contact asymmetry, and a Wnt signal. Second, we experimentally demonstrated that myosin flow generates forces that trigger plasma membrane movements and propose that their anisotropy drives cell division orientation. Our data suggest that contact-dependent control of myosin specifies the division axes of Caenorhabditis elegans AB, ABa, EMS cells, and the mouse AB cell. The contact pattern-dependent generation of myosin flows, in concert with known microtubule/dynein pathways, may greatly expand division axis diversity during development., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The Golgi apparatus plays essential roles in the processing and sorting of proteins and lipids, but it can also act as a signalling hub and a microtubule-nucleation centre. The Golgi complex (GC) of mammalian cells is composed of stacks connected by tubular bridges to form a continuous membranous system. In spite of this structural complexity, the GC is highly dynamic, and this feature becomes particularly evident during mitosis, when the GC undergoes a multi-step disassembly process that allows its correct partitioning and inheritance by daughter cells. Strikingly, different steps of Golgi disassembly control mitotic entry and progression, indicating that cells actively monitor Golgi integrity during cell division. Here, we summarise the basic mechanisms and the molecular players that are involved in Golgi disassembly, focussing in particular on recent studies that have revealed the fundamental signalling pathways that connect Golgi inheritance to mitotic entry and progression., (© 2017 Société Française des Microscopies and Société de Biologie Cellulaire de France. Published by John Wiley & Sons Ltd.)

Correct orientation of the mitotic spindle in stem cells underlies organogenesis. Spindle abnormalities correlate with cancer progression in germ line-derived tumors. We discover a macromolecular complex between the scaffolding protein Gravin/AKAP12 and the mitotic kinases, Aurora A and Plk1, that is down regulated in human seminoma. Depletion of Gravin correlates with an increased mitotic index and disorganization of seminiferous tubules. Biochemical, super-resolution imaging, and enzymology approaches establish that this Gravin scaffold accumulates at the mother spindle pole during metaphase. Manipulating elements of the Gravin-Aurora A-Plk1 axis prompts mitotic delay and prevents appropriate assembly of astral microtubules to promote spindle misorientation. These pathological responses are conserved in seminiferous tubules from Gravin(-/-) mice where an overabundance of Oct3/4 positive germ line stem cells displays randomized orientation of mitotic spindles. Thus, we propose that Gravin-mediated recruitment of Aurora A and Plk1 to the mother (oldest) spindle pole contributes to the fidelity of symmetric cell division.

Cancerous inhibitor of protein phosphatase 2A (CIP2A) is overexpressed in most human cancers and has been described as being involved in the progression of several human malignancies via the inhibition of protein phosphatase 2A (PP2A) activity toward c-Myc. However, with the exception of this role, the cellular function of CIP2A remains poorly understood. On the basis of yeast two-hybrid and coimmunoprecipitation assays, we demonstrate here that NIMA (never in mitosis gene A)-related kinase 2 (NEK2) is a binding partner for CIP2A. CIP2A exhibited dynamic changes in distribution, including the cytoplasm and centrosome, depending on the cell cycle stage. When CIP2A was depleted, centrosome separation and the mitotic spindle dynamics were impaired, resulting in the activation of spindle assembly checkpoint signaling and, ultimately, extension of the cell division time. Our data imply that CIP2A strongly interacts with NEK2 during G2/M phase, thereby enhancing NEK2 kinase activity to facilitate centrosome separation in a PP1- and PP2A-independent manner. In conclusion, CIP2A is involved in cell cycle progression through centrosome separation and mitotic spindle dynamics.

Microtubules are cellular components that are required for a variety of essential processes such as cell motility, mitosis, and intracellular transport. This is possible because of the inherent dynamic properties of microtubules. Many of these properties are tightly regulated by a number of microtubule plus-end-binding proteins or +TIPs. These proteins recognize the distal end of microtubules and are thus in the right context to control microtubule dynamics. In this review, we address how microtubule dynamics are regulated by different +TIP families, focusing on how functionally diverse +TIPs spatially and temporally regulate microtubule dynamics during animal cell division., (© 2014 Elsevier Inc. All rights reserved.)

Polarised epithelial cell divisions represent a fundamental mechanism for tissue maintenance and morphogenesis. Morphological and mechanical changes in the plasma membrane influence the organisation and crosstalk of microtubules and actin at the cell cortex, thereby regulating the mitotic spindle machinery and chromosome segregation. Yet, the precise mechanisms linking plasma membrane remodelling to cell polarity and cortical cytoskeleton dynamics to ensure accurate execution of mitosis in mammalian epithelial cells remain poorly understood. Here, we manipulated the density of mammary epithelial cells in culture, which led to several mitotic defects. Perturbation of cell-cell adhesion formation impairs the dynamics of the plasma membrane, affecting the shape and size of mitotic cells and resulting in defects in mitotic progression and the generation of daughter cells with aberrant architecture. In these conditions, F- actin-astral microtubule crosstalk is impaired, leading to mitotic spindle misassembly and misorientation, which in turn contributes to chromosome mis-segregation. Mechanistically, we identify S100 Ca2+-binding protein A11 (S100A11) as a key membrane-associated regulator that forms a complex with E-cadherin (CDH1) and the leucineglycine-asparagine repeat protein LGN (also known as GPSM2) to coordinate plasma membrane remodelling with E-cadherin-mediated cell adhesion and LGN-dependent mitotic spindle machinery. Thus, plasmamembrane-mediatedmaintenance of mammalian epithelial cell identity is crucial for correct execution of polarised cell divisions, genome maintenance and safeguarding tissue integrity. [ABSTRACT FROM AUTHOR]

Myosins are ATP-dependent actin-based molecular motors critical for diverse cellular processes like intracellular trafficking, cell motility, and cell invasion. During cell division, myosin MYO10 is important for proper mitotic spindle assembly, the anchoring of the spindle to the cortex, and positioning of the spindle to the cell mid-plane. However, myosins are regulated by myosin regulatory light chains (RLCs), and whether RLCs are important for cell division has remained unexplored. Here, we have determined that the previously uncharacterized myosin RLC Myl5 associates with the mitotic spindle and is required for cell division. We show that Myl5 localizes to the leading edge and filopodia during interphase and to mitotic spindle poles and spindle microtubules during early mitosis. Importantly, depletion of Myl5 led to defects in mitotic spindle assembly, chromosome congression, and chromosome segregation and to a slower transition through mitosis. Furthermore, Myl5 bound to MYO10 in vitro and co-localized with MYO10 at the spindle poles. These results suggest that Myl5 is important for cell division and that it may be performing its function through MYO10.

During mitosis, cells round up and utilize the interphase adhesion sites within the fibrous extracellular matrix (ECM) as guidance cues to orient the mitotic spindles. Here, using suspended ECM-mimicking nanofiber networks, we explore mitotic outcomes and error distribution for various interphase cell shapes. Elongated cells attached to single fibers through two focal adhesion clusters (FACs) at their extremities result in perfect spherical mitotic cell bodies that undergo significant 3-dimensional (3D) displacement while being held by retraction fibers (RFs). Increasing the number of parallel fibers increases FACs and retraction fiber-driven stability, leading to reduced 3D cell body movement, metaphase plate rotations, increased interkinetochore distances, and significantly faster division times. Interestingly, interphase kite shapes on a crosshatch pattern of four fibers undergo mitosis resembling single-fiber outcomes due to rounded bodies being primarily held in position by RFs from two perpendicular suspended fibers. We develop a cortex–astral microtubule analytical model to capture the retraction fiber dependence of the metaphase plate rotations. We observe that reduced orientational stability, on single fibers, results in increased monopolar mitotic defects, while multipolar defects become dominant as the number of adhered fibers increases. We use a stochastic Monte Carlo simulation of centrosome, chromosome, and membrane interactions to explain the relationship between the observed propensity of monopolar and multipolar defects and the geometry of RFs. Overall, we establish that while bipolar mitosis is robust in fibrous environments, the nature of division errors in fibrous microenvironments is governed by interphase cell shapes and adhesion geometries. [ABSTRACT FROM AUTHOR]

Kinetochores are molecular machines that power chromosome segregation during the mitotic and meiotic cell divisions of all eukaryotes. Aristotle explains how we think we have knowledge of a thing only when we have grasped its cause. In our case, to gain understanding of the kinetochore, the four causes correspond to questions that we must ask: (a) What are the constituent parts, (b) how does it assemble, (c) what is the structure and arrangement, and (d) what is the function? Here we outline the current blueprint for the assembly of a kinetochore, how functions are mapped onto this architecture, and how this is shaped by the underlying pericentromeric chromatin. The view of the kinetochore that we present is possible because an almost complete parts list of the kinetochore is now available alongside recent advances using in vitro reconstitution, structural biology, and genomics. In many organisms, each kinetochore binds to multiple microtubules, and we propose a model for how this ensemble-level architecture is organized, drawing on key insights from the simple one microtubule–one kinetochore setup in budding yeast and innovations that enable meiotic chromosome segregation. [ABSTRACT FROM AUTHOR]

Early cellular patterning is a critical step of embryonic development that determines the proper progression of morphogenesis in all metazoans. It relies on a series of rapid reductive divisions occurring simultaneously with the specification of the fate of different subsets of cells. Multiple species developmental strategies emerged in the form of a unique cleavage pattern with stereotyped division geometries. Cleavage geometries have long been associated to the emergence of canonical developmental features such as cell cycle asynchrony, zygotic genome activation and fate specification. Yet, the direct causal role of division positioning on blastomere cell behavior remain partially understood. Oriented and/or asymmetric divisions define blastomere cell sizes, contacts and positions, with potential immediate impact on cellular decisions, lineage specification and morphogenesis. Division positions also instruct daughter cells polarity, mechanics and geometries, thereby influencing subsequent division events, in an emergent interplay that may pattern early embryos independently of firm deterministic genetic programs. We here review the recent literature which helped to delineate mechanisms and functions of division positioning in early embryos. [ABSTRACT FROM AUTHOR]

Schwann cells (SCs) migrate along peripheral axons and divide intensively to generate the right number of cells prior to axonal ensheathment; however, little is known regarding the temporal and molecular control of their division and its impact on myelination. We report that Sil, a spindle pole protein associated with autosomal recessive primary microcephaly, is required for temporal mitotic exit of SCs. In sil-deficient Cassiopeia (csp-1-) mutants, SCs fail to radially sort and myelinate peripheral axons. Elevation of cAMP, but not Rac1 activity, in csp-1- restores myelin ensheathment. Most importantly, we show a significant decrease in laminin expression within csp-1- posterior lateral line nerve and that forcing Laminin 2 expression in csp-1- fully restores the ability of SCs to myelinate. Thus, we demonstrate an essential role for timely SC division in mediating laminin expression to orchestrate radial sorting and peripheral myelination in vivo. [ABSTRACT FROM AUTHOR]

The journey from a single fertilised cell to a multicellular organism is, at the most fundamental level, orchestrated by mitotic cell divisions. Both the rate and the orientation of cell divisions are important in ensuring the proper development of an embryo. Simultaneous with cell proliferation, embryonic cells constantly experience a wide range of mechanical forces from their surrounding tissue environment. Cells must be able to read and respond correctly to these forces since they are known to affect a multitude of biological functions, including cell divisions. The interplay between the mechanical environment and cell divisions is particularly crucial during embryogenesis when tissues undergo dynamic changes in their shape, architecture, and overall organisation to generate functional tissues and organs. Here we review our current understanding of the cellular mechanisms by which mechanical force regulates cell division and place this knowledge within the context of embryogenesis and tissue morphogenesis. [ABSTRACT FROM AUTHOR]

ABSTRACT Centrosomes are the main microtubule-organizing center of the cell. They are normally formed by two centrioles, embedded in a cloud of proteins known as pericentriolar material (PCM). The PCM ascribes centrioles with their microtubule nucleation capacity. Toxoplasma gondii, the causative agent of toxoplasmosis, divides by endodyogeny. Successful cell division is critical for pathogenesis. The centrosome, one of the microtubule organizing centers of the cell, plays central roles in orchestrating the temporal and physical coordination of major organelle segregation and daughter cell formation during endodyogeny. The Toxoplasma centrosome is constituted by multiple domains: an outer core, distal from the nucleus; a middle core; and an inner core, proximal to the nucleus. This modular organization has been proposed to underlie T. gondii’s cell division plasticity. However, the role of the inner core remains undeciphered. Here, we focus on understanding the function of the inner core by finely studying the localization and role of its only known molecular marker; TgCep250L1. We show that upon conditional degradation of TgCep250L1 parasites are unable to survive. Mutants exhibit severe nuclear segregation defects. In addition, the rest of the centrosome, defined by the position of the centrioles, disconnects from the nucleus. We explore the structural defects underlying these phenotypes by ultrastructure expansion microscopy. We show that TgCep250L1’s location changes with respect to other markers, and these changes encompass the formation of the mitotic spindle. Moreover, we show that in the absence of TgCep250L1, the microtubule binding protein TgEB1, fails to localize at the mitotic spindle, while unsegregated nuclei accumulate at the residual body. Overall, our data support a model in which the inner core of the T. gondii centrosome critically participates in cell division by directly impacting the formation or stability of the mitotic spindle. IMPORTANCE Toxoplasma gondii parasites cause toxoplasmosis, arguably the most widespread and prevalent parasitosis of humans and animals. During the clinically relevant stage of its life cycle, the parasites divide by endodyogeny. In this mode of division, the nucleus, containing loosely packed chromatin and a virtually intact nuclear envelope, parcels into two daughter cells generated within a common mother cell cytoplasm. The centrosome is a microtubule-organizing center critical for orchestrating the multiple simultaneously occurring events of endodyogeny. It is organized in two distinct domains: the outer and inner cores. We demonstrate here that the inner core protein TgCEP250L1 is required for replication of T. gondii. Lack of TgCEP250L1 renders parasites able to form daughter cells, while unable to segregate their nuclei. We determine that, in the absence of TgCEP250L1, the mitotic spindle, which is responsible for karyokinesis, does not assemble. Our results support a role for the inner core in nucleation or stabilization of the mitotic spindle in T. gondii.

Microtubules tightly regulate various cellular activities. Our understanding of microtubules is largely based on experiments using microtubule‐targeting agents, which, however, are insufficient to dissect the dynamic mechanisms of specific microtubule populations, due to their slow effects on the entire pool of microtubules. To overcome this technological limitation, we have used chemo and optogenetics to disassemble specific microtubule subtypes, including tyrosinated microtubules, primary cilia, mitotic spindles, and intercellular bridges, by rapidly recruiting engineered microtubule‐cleaving enzymes onto target microtubules in a reversible manner. Using this approach, we show that acute microtubule disassembly swiftly halts vesicular trafficking and lysosomal dynamics. It also immediately triggers Golgi and ER reorganization and slows the fusion/fission of mitochondria without affecting mitochondrial membrane potential. In addition, cell rigidity is increased after microtubule disruption owing to increased contractile stress fibers. Microtubule disruption furthermore prevents cell division, but does not cause cell death during interphase. Overall, the reported tools facilitate detailed analysis of how microtubules precisely regulate cellular architecture and functions. SYNOPSIS: Characterization of microtubules in cells via microtubule‐targeting agents is not ideal for assessing dynamics of specific microtubule populations. Here, a novel approach using an engineered microtubule‐severing enzyme allows spatiotemporal manipulation of microtubule disassembly triggered by either chemicals or illumination. A triple glutamine mutation in Spastin (dNSpastin3Q) reduces its association with microtubules.Engineered dNSpastin3Q acts as a microtubule‐severing enzyme in living cells.Recruitment of dNSpastin3Q to microtubules can be controlled by chemical‐ or light‐induced dimerization.Induced dimerization of dNSpastin3Q leads to acute disassembly of target microtubule subtypes. [ABSTRACT FROM AUTHOR]

Red blood cells are produced by terminal erythroid differentiation, which involves the dramatic morphological transformation of erythroblasts into enucleated reticulocytes. Microtubules are important for enucleation, but it is not known if the centrosome, a key microtubule‐organizing center, is required as well. Mice lacking the conserved centrosome component, CDK5RAP2, are likely to have defective erythroid differentiation because they develop macrocytic anemia. Here, we show that fetal liver‐derived, CDK5RAP2‐deficient erythroid progenitors generate fewer and larger reticulocytes, hence recapitulating features of macrocytic anemia. In erythroblasts, but not in embryonic fibroblasts, loss of CDK5RAP2 or pharmacological depletion of centrosomes leads to highly aberrant spindle morphologies. Consistent with such cells exiting mitosis without chromosome segregation, tetraploidy is frequent in late‐stage erythroblasts, thereby giving rise to fewer but larger reticulocytes than normal. Our results define a critical role for CDK5RAP2 and centrosomes in spindle formation specifically during blood production. We propose that disruption of centrosome and spindle function could contribute to the emergence of macrocytic anemias, for instance, due to nutritional deficiency or exposure to chemotherapy. Synopsis: Immature erythroid cells differentiate into red blood cells by undergoing a defined number of cell divisions followed by ejection of their nuclei. This study investigates the role of centrosomes in this process using ex vivo differentiation of fetal liver‐derived erythroid progenitors in mice. The conserved centrosomal protein CDK5RAP2 facilitates erythroid differentiation both ex vivo and in vivoCentrosomes and CDK5RAP2 are crucial for normal spindle assembly in erythroblasts but not in embryonic fibroblastsErythroblasts lacking CDK5RAP2 or centrosomes develop tetraploidyTP53 is activated in erythroblasts lacking CDK5RAP2 or centrosomes but is not responsible for the observed defects in erythropoiesisUnlike embryonic fibroblasts, erythroblasts do not expand their centrosomes in mitosis [ABSTRACT FROM AUTHOR]

Uveal melanoma (UM) is the most common intraocular cancer in adults. Whilst treatment of primary UM (PUM) is often successful, around 50% of patients develop metastatic disease with poor outcomes, linked to chromosome 3 loss (monosomy 3, M3). Advances in understanding UM cell biology may indicate new therapeutic options. We report that UM exhibits centrosome abnormalities, which in other cancers are associated with increased invasiveness and worse prognosis, but also represent a potential Achilles' heel for cancer‐specific therapeutics. Analysis of 75 PUM patient samples revealed both higher centrosome numbers and an increase in centrosomes with enlarged pericentriolar matrix (PCM) compared to surrounding normal tissue, both indicative of centrosome amplification. The PCM phenotype was significantly associated with M3 (t‐test, p < 0.01). Centrosomes naturally enlarge as cells approach mitosis; however, whilst UM with higher mitotic scores had enlarged PCM regardless of genetic status, the PCM phenotype remained significantly associated with M3 in UM with low mitotic scores (ANOVA, p = 0.021) suggesting that this is independent of proliferation. Phenotypic analysis of patient‐derived cultures and established UM lines revealed comparable levels of centrosome amplification in PUM cells to archetypal triple‐negative breast cancer cell lines, whilst metastatic UM (MUM) cell lines had even higher levels. Importantly, many UM cells also exhibit centrosome clustering, a common strategy employed by other cancer cells with centrosome amplification to survive cell division. As UM samples with M3 display centrosome abnormalities indicative of amplification, this phenotype may contribute to the development of MUM, suggesting that centrosome de‐clustering drugs may provide a novel therapeutic approach. [ABSTRACT FROM AUTHOR]

Tissue morphogenesis requires the coordinated regulation of cellular behavior, which includes the orientation of cell division that defines the position of daughter cells in the tissue. Cell division orientation is instructed by biochemical and mechanical signals from the local tissue environment, but how those signals control mitotic spindle orientation is not fully understood. Here, we tested how mechanical tension across an epithelial monolayer is sensed to orient cell divisions. Tension across Madin-Darby canine kidney cell monolayers was increased by a low level of uniaxial stretch, which oriented cell divisions with the stretch axis irrespective of the orientation of the cell long axis. We demonstrate that stretch-induced division orientation required mechanotransduction through E-cadherin cell-cell adhesions. Increased tension on the E-cadherin complex promoted the junctional recruitment of the protein LGN, a core component of the spindle orientation machinery that binds the cytosolic tail of E-cadherin. Consequently, uniaxial stretch triggered a polarized cortical distribution of LGN. Selective disruption of trans engagement of E-cadherin in an otherwise cohesive cell monolayer, or loss of LGN expression, resulted in randomly oriented cell divisions in the presence of uniaxial stretch. Our findings indicate that E-cadherin plays a key role in sensing polarized tensile forces across the tissue and transducing this information to the spindle orientation machinery to align cell divisions.

The mitotic spindle is a microtubule‐based machine that pulls the two identical sets of chromosomes to opposite ends of the cell during cell division. The fission yeast Schizosaccharomyces pombe is an important model organism for studying mitosis due to its simple, stereotyped spindle structure and well‐established genetic toolset. S. pombe spindle length is a useful metric for mitotic progression, but manually tracking spindle ends in each frame to measure spindle length over time is laborious and can limit experimental throughput. We have developed an ImageJ plugin that can automatically track S. pombe spindle length over time and replace manual or semi‐automated tracking of spindle elongation dynamics. Using an algorithm that detects the principal axis of the spindle and then finds its ends, we reliably track the length of the spindle as the cell divides. The plugin integrates with existing ImageJ features, exports its data for further analysis outside of ImageJ and does not require any programming by the user. Thus, the plugin provides an accessible tool for quantification of S. pombe spindle length that will allow automatic analysis of large microscopy data sets and facilitate screening for effects of cell biological perturbations on mitotic progression. [ABSTRACT FROM AUTHOR]

Leucine carboxyl methyltransferase-1 (LCMT1) and protein phosphatase methylesterase-1 (PME-1) are essential enzymes that regulate the methylation of the protein phosphatase 2A catalytic subunit (PP2AC). LCMT1 and PME-1 have been linked to the regulation of cell growth and proliferation, but the underlying mechanisms have remained elusive. We show here an important role for an LCMT1-PME-1 methylation equilibrium in controlling mitotic spindle size. Depletion of LCMT1 or overexpression of PME-1 led to long spindles. In contrast, depletion of PME-1, pharmacological inhibition of PME-1 or overexpression of LCMT1 led to short spindles. Furthermore, perturbation of the LCMT1-PME-1 methylation equilibrium led to mitotic arrest, spindle assembly checkpoint activation, defective cell divisions, induction of apoptosis and reduced cell viability. Thus, we propose that the LCMT1-PME-1 methylation equilibrium is critical for regulating mitotic spindle size and thereby proper cell division.

The journey from a single fertilised cell to a multicellular organism is, at the most fundamental level, orchestrated by mitotic cell divisions. Both the rate and the orientation of cell divisions are important in ensuring the proper development of an embryo. Simultaneous with cell proliferation, embryonic cells constantly experience a wide range of mechanical forces from their surrounding tissue environment. Cells must be able to read and respond correctly to these forces since they are known to affect a multitude of biological functions, including cell divisions. The interplay between the mechanical environment and cell divisions is particularly crucial during embryogenesis when tissues undergo dynamic changes in their shape, architecture, and overall organisation to generate functional tissues and organs. Here we review our current understanding of the cellular mechanisms by which mechanical force regulates cell division and place this knowledge within the context of embryogenesis and tissue morphogenesis.

Continuous poleward motion of microtubules in metazoan mitotic spindles has been fascinating generations of cell biologists over the last several decades. In human cells, this so‐called poleward flux was recently shown to be driven by the coordinated action of four mitotic kinesins. The sliding activities of kinesin‐5/EG5 and kinesin‐12/KIF15 are sequentially supported by kinesin‐7/CENP‐E at kinetochores and kinesin‐4/KIF4A on chromosome arms, with the individual contributions peaking during prometaphase and metaphase, respectively. Although recent data elucidate the molecular mechanism underlying this cellular phenomenon, the functional roles of microtubule poleward flux during cell division remain largely elusive. Here, we discuss potential contribution of microtubule flux engine to various essential processes at different stages of mitosis. [ABSTRACT FROM AUTHOR]

The mitotic spindle is a microtubule-based assembly that separates the chromosomes during cell division. As the spindle is basically a mechanical micro machine, the understanding of its functioning is constantly motivating the development of experimental approaches based on mechanical perturbations, which are complementary to and work together with the classical genetics and biochemistry methods. Recent data emerging from these approaches in combination with theoretical modeling led to novel ideas and significant revisions of the basic concepts in the field. In this Perspective, we discuss the advances in the understanding of spindle mechanics, focusing on microtubule forces that control chromosome movements. [ABSTRACT FROM AUTHOR]

The spindle shows remarkable diversity, and changes in an integrated fashion, as cells vary over evolution. Here, we provide a mechanistic explanation for variations in the first mitotic spindle in nematodes. We used a combination of quantitative genetics and biophysics to rule out broad classes of models of the regulation of spindle length and dynamics, and to establish the importance of a balance of cortical pulling forces acting in different directions. These experiments led us to construct a model of cortical pulling forces in which the stoichiometric interactions of microtubules and force generators (each force generator can bind only one microtubule), is key to explaining the dynamics of spindle positioning and elongation, and spindle final length and scaling with cell size. This model accounts for variations in all the spindle traits we studied here, both within species and across nematode species spanning over 100 million years of evolution.

The phenotypic spectrum of colorectal cancer (CRC) is remarkably diverse, with seemingly endless variations in cell shape, mitotic figures and multicellular configurations. Despite this morphological complexity, histological grading of collective phenotype patterns provides robust prognostic stratification in CRC. Although mechanistic understanding is incomplete, previous studies have shown that the cortical protein ezrin controls diversification of cell shape, mitotic figure geometry and multicellular architecture, in 3D organotypic CRC cultures. Because ezrin is a substrate of Src tyrosine kinase that is frequently overexpressed in CRC, we investigated Src regulation of ezrin and morphogenic growth in 3D CRC cultures. Here we show that Src perturbations disrupt CRC epithelial spatial organisation. Aberrant Src activity suppresses formation of the cortical ezrin cap that anchors interphase centrosomes. In CRC cells with a normal centrosome number, these events lead to mitotic spindle misorientation, perturbation of cell cleavage, abnormal epithelial stratification, apical membrane misalignment, multilumen formation and evolution of cribriform multicellular morphology, a feature of low‐grade cancer. In isogenic CRC cells with centrosome amplification, aberrant Src signalling promotes multipolar mitotic spindle formation, pleomorphism and morphological features of high‐grade cancer. Translational studies in archival human CRC revealed associations between Src intensity, multipolar mitotic spindle frequency and high‐grade cancer morphology. Collectively, our study reveals Src regulation of CRC morphogenic growth via ezrin–centrosome engagement and uncovers combined perturbations underlying transition to high‐grade CRC morphology. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland. [ABSTRACT FROM AUTHOR]

The DNA damage response recognizes DNA lesions and coordinates a cell cycle arrest with the repair of the damaged DNA, or removal of the affected cells to prevent the passage of genetic alterations to the next generation. The mitotic cell division, on the other hand, is a series of processes that aims to accurately segregate the genomic material from the maternal to the two daughter cells. Despite their great importance in safeguarding genomic integrity, the DNA damage response and the mitotic cell division were long viewed as unrelated processes, mainly because animal cells that are irradiated during mitosis continue cell division without repairing the broken chromosomes. However, recent studies have demonstrated that DNA damage proteins play an important role in mitotic cell division. This is performed through regulation of the onset of mitosis, mitotic spindle formation, correction of misattached kinetochore–microtubules, spindle checkpoint signaling, or completion of cytokinesis (abscission), in the absence of DNA damage. In this review, we summarize the roles of DNA damage proteins in unperturbed mitosis, analyze the molecular mechanisms involved, and discuss the potential implications of these findings in cancer therapy. [ABSTRACT FROM AUTHOR]

Mitotic division maintains genetic identity of any multicellular organism throughout an entire lifetime. Each time a parent cell divides, chromosomes are equally distributed between the daughter cells due to the action of mitotic spindle. Mitotic spindle is formed by the microtubules that represent dynamic polymers of tubulin protein. Spindle microtubules are attached end-on to kinetochores – large multi-protein complexes on chromosomes. This review focuses on the four-subunit NDC80 complex, one of the most important kinetochore elements that plays a major role in the attachment of assembling/disassembling microtubule ends to the chromosomes. Here, we summarize published data on the structure, properties, and regulation of the NDC80 complex and discuss possible relationship between changes in the expression of genes coding for the NDC80 complex components, mitotic disorders, and oncogenesis with special emphasis on the diagnostic and therapeutic potential of NDC80. [ABSTRACT FROM AUTHOR]

Background: Astral microtubules emanating from the mitotic centrosomes play pivotal roles in defining cell division axis and tissue morphogenesis. Previous studies have demonstrated that human transforming acidic coiledcoil 3 (TACC3), the most conserved TACC family protein, regulates formation of astral microtubules at centrosomes in vertebrate cells by affecting γ-tubulin ring complex (γ-TuRC) assembly. However, the molecular mechanisms underlying such function were not completely understood. Results: Here, we show that Aurora A site-specific phosphorylation in TACC3 regulates formation of astral microtubules by stabilizing γ-TuRC assembly in human cells. Mutation of the most conserved Aurora A targeting site, Ser 558 to alanine (S558A) in TACC3 results in robust loss of astral microtubules and disrupts localization of the γ-tubulin ring complex (γ-TuRC) proteins at the spindle poles. Under similar condition, phospho-mimicking S558D mutation retains astral microtubules and the γ-TuRC proteins in a manner similar to control cells expressed with wild type TACC3. Time-lapse imaging reveals that S558A mutation leads to defects in positioning of the spindlepoles and thereby causes delay in metaphase to anaphase transition. Biochemical results determine that the Ser 558-phosphorylated TACC3 interacts with the γ-TuRC proteins and further, S558A mutation impairs the interaction. We further reveal that the mutation affects the assembly of γ-TuRC from the small complex components. Conclusions: The results demonstrate that TACC3 phosphorylation stabilizes γ-tubulin ring complex assembly and thereby regulates formation of centrosomal asters. They also implicate a potential role of TACC3 phosphorylation in the functional integrity of centrosomes/spindle poles. [ABSTRACT FROM AUTHOR]

The length of the mitotic spindle scales with cell size in a wide range of organisms during embryonic development. Interestingly, in C. elegans embryos, this goes along with temporal regulation: larger cells speed up spindle assembly and elongation. We demonstrate that, similarly in fission yeast, spindle length and spindle dynamics adjust to cell size, which allows to keep mitosis duration constant. Since prolongation of mitosis was shown to affect cell viability, this may resemble a mechanism to regulate mitosis duration. We further reveal how the velocity of spindle elongation is regulated: coupled to cell size, the amount of kinesin-6 Klp9 molecules increases, resulting in an acceleration of spindle elongation in anaphase B. In addition, the number of Klp9 binding sites to microtubules increases overproportionally to Klp9 molecules, suggesting that molecular crowding inversely correlates to cell size and might have an impact on spindle elongation velocity control.

Cancer cells are characterized by extensive reprogramming of metabolic pathways in order to promote cell division and survival. However, the growth promotion effects of metabolic reprogramming can be due to moonlighting functions of metabolic enzymes as well as the redirection of flux through particular pathways. To identify metabolic enzymes that might have potential moonlighting functions in oncogenesis, we have examined recent screens of the yeast GFP strain collection for metabolic enzymes that have been implicated in cancer metabolism with an unusual subcellular localization. Asparagine synthetase forms filaments in yeast in response to nutrient limitation and is part of a pathway that is a chemotherapy target in acute lymphoblastic leukemia. Interestingly, while yeast asparagine synthetase forms cytoplasmic filaments in response to nutrient stress, human asparagine synthetase is associated with the centrosomes and mitotic spindles. This localization is disrupted by both nocodazole and asparaginase treatments. This failure to localize occurs even though asparagine synthetase is highly upregulated in response to asparaginase treatment. Together, these results argue that human asparagine synthetase undergoes regulated recruitment to the mitotic spindles and that it may have acquired a second role in mitosis similar to other metabolic enzymes that contribute to metabolic reprogramming in cancer cells.

Progression of mitosis and cytokinesis depends on the reorganization of cytoskeleton, with microtubules driving the segregation of chromosomes and their partitioning to two daughter cells. In dividing plant cells, microtubules undergo global reorganization throughout mitosis and cytokinesis, and with the aid of various microtubule-associated proteins (MAPs), they form unique systems such as the preprophase band (PPB), the acentrosomal mitotic spindle, and the phragmoplast. Such proteins include nucleators of de novo microtubule formation, plus end binding proteins involved in the regulation of microtubule dynamics, crosslinking proteins underlying microtubule bundle formation and members of the kinesin superfamily with microtubule-dependent motor activities. The coordinated function of such proteins not only drives the continuous remodeling of microtubules during mitosis and cytokinesis but also assists the positioning of the PPB, the mitotic spindle, and the phragmoplast, affecting tissue patterning by controlling cell division plane (CDP) orientation. The affinity and the function of such proteins is variably regulated by reversible phosphorylation of serine and threonine residues within the microtubule binding domain through a number of protein kinases and phosphatases which are differentially involved throughout cell division. The purpose of the present review is to provide an overview of the function of protein kinases and protein phosphatases involved in cell division regulation and to identify cytoskeletal substrates relevant to the progression of mitosis and cytokinesis and the regulation of CDP orientation. [ABSTRACT FROM AUTHOR]

Chromosome segregation during cell division requires a bipolar mitotic spindle. Therefore, how the spindle is formed, maintained, and functions is of fundamental importance for all eukaryotic cells. Members of the evolutionarily conserved kinesin-5 family of motor proteins have been shown to play an essential role in spindle formation by generating forces that establish and maintain spindle bipolarity and contribute to spindle elongation. Recent work demonstrates that accessory proteins and post-translational modifications regulate the localization and activity of kinesin-5 motors in cells. In addition, some kinesin-5 motors can move toward the microtubule plus-or-minus end. This new information provides insight into how these motors function during mitosis. Highlights Kinesin-5 motors are phosphorylated at multiple sites within the protein; phosphorylation impacts motor activity, localization, and function in mitosis. Kinesin-5 motors walk toward the microtubule plus end. In yeast, kinesin-5 additionally shows minus-end-directed motion that affects mitosis. Localization of vertebrate kinesin-5 motors to spindle microtubules and the centrosome requires a microtubule-associated protein,TPX2. Spindle bipolarity requires kinesin-5 activity in diverse cells; in some cases kinesin-12 can substitute for kinesin-5 and drive spindle formation. [ABSTRACT FROM AUTHOR]

Kinesins are widely expressed, microtubule‐dependent motors that play vital roles in microtubule‐associated cellular activities, such as cell division and intracellular transport. Eg5, also known as kinesin‐5 or kinesin spindle protein, is a member of the kinesin family that contributes to the formation and maintenance of the bipolar mitotic spindle during cell division. Small‐molecule compounds that inhibit Eg5 activity have been shown to impair spindle assembly, block mitotic progression, and possess anti‐cancer activity. Recent studies focusing on the localization and functions of Eg5 in plants have demonstrated that in addition to spindle organization, this motor protein has non‐canonical functions, such as chromosome segregation and cytokinesis, that have not been observed in animals. In this review, we discuss the structure, function, and localization of Eg5 in various organisms, highlighting the specific role of this protein in plants. We also propose directions for the future studies of novel Eg5 functions based on the lessons learned from plants. [ABSTRACT FROM AUTHOR]

Cell division is fundamental to life and its perturbation can disrupt organismal development, alter tissue homeostasis, and cause disease. Analysis of mitotic abnormalities provides insight into how certain perturbations affect the fidelity of cell division and how specific cellular structures, molecules, and enzymatic activities contribute to the accuracy of this process. However, accurate classification of mitotic defects is instrumental for correct interpretation of data and formulation of new hypotheses. In this article, we provide guidelines for identifying specific mitotic stages and for classifying normal and deviant mitotic phenotypes. We hope this will clarify confusion about how certain defects are classified and help investigators avoid misnomers, misclassification, and/or misinterpretation, thus leading to a unified and standardized system to classify mitotic defects. [ABSTRACT FROM AUTHOR]

Control of microtubule dynamics underlies several fundamental processes such as cell polarity, cell division, and cell motility. To gain insights into the mechanisms that control microtubule dynamics during cell motility, we investigated the interactome of the microtubule plus-end-binding protein end-binding 1 (EB1). Via molecular mapping and cross-linking mass spectrometry we identified and characterized a large complex associating a specific isoform of myomegalin termed "SMYLE" (for short myomegalin-like EB1 binding protein), the PKA scaffolding protein AKAP9, and the pericentrosomal protein CDK5RAP2. SMYLE was associated through an evolutionarily conserved N-terminal domain with AKAP9, which in turn was anchored at the centrosome via CDK5RAP2. SMYLE connected the pericentrosomal complex to the microtubule-nucleating complex (?-TuRC) via Galectin-3-binding protein. SMYLE associated with nascent centrosomal microtubules to promote microtubule assembly and acetylation. Disruption of SMYLE interaction with EB1 or AKAP9 prevented microtubule nucleation and their stabilization at the leading edge of migrating cells. In addition, SMYLE depletion led to defective astral microtubules and abnormal orientation of the mitotic spindle and triggered G1 cell-cycle arrest, which might be due to defective centrosome integrity. As a consequence, SMYLE loss of function had a profound impact on tumor cell motility and proliferation, suggesting that SMYLE might be an important player in tumor progression. [ABSTRACT FROM AUTHOR]

Oncogenic Ras has been shown to change the way cancer cells divide by increasing the forces generated during mitotic rounding. In this way, RasV12 enables cancer cells to divide across a wider range of mechanical environments than normal cells. Here, we identify a further role for oncogenic Ras-ERK signaling in division by showing that RasV12 expression alters the shape, division orientation, and respreading dynamics of cells as they exit mitosis. Many of these effects appear to result from the impact of RasV12 signaling on actomyosin contractility, because RasV12 induces the severing of retraction fibers that normally guide spindle positioning and provide a memory of the interphase cell shape. In support of this idea, the RasV12 phenotype is reversed by inhibition of actomyosin contractility and can be mimicked by the loss of cell-substrate adhesion during mitosis. Finally, we show that RasV12 activation also perturbs division orientation in cells cultured in 2D epithelial monolayers and 3D spheroids. Thus, the induction of oncogenic Ras-ERK signaling leads to rapid changes in division orientation that, along with the effects of RasV12 on cell growth and cell-cycle progression, are likely to disrupt epithelial tissue organization and contribute to cancer dissemination. [Display omitted] • Oncogenic Ras alters cell shape dynamics during post-mitotic respreading • High contractility in Ras-activated cells breaks mitotic substrate contacts • Loss of substrate cues lead to defects in mitotic spindle orientation • Ras induces out-of-plane divisions in epithelial monolayers and spheroids Cell division is dysregulated in cancer. Ganguli et al. show that activation of an oncogene, Ras, directly alters cell-substrate adhesion and cell shape during progression through mitosis. These changes are accompanied by alterations in mitotic spindle orientation, which lead to out-of-plane divisions and loss of epithelial tissue architecture. [ABSTRACT FROM AUTHOR]

We present a novel platform for the large-scale simulation of three-dimensional fibrous structures immersed in a Stokesian fluid and evolving under confinement or in free-space in three dimensions. One of the main motivations for this work is to study the dynamics of fiber assemblies within biological cells. For this, we also incorporate the key biophysical elements that determine the dynamics of these assemblies, which include the polymerization and depolymerization kinetics of fibers, their interactions with molecular motors and other objects, their flexibility, and hydrodynamic coupling. This work, to our knowledge, is the first technique to include many-body hydrodynamic interactions (HIs), and the resulting fluid flows, in cellular assemblies of flexible fibers. We use non-local slender body theory to compute the fluid–structure interactions of the fibers and a second-kind boundary integral formulation for other rigid bodies and the confining boundary. A kernel-independent implementation of the fast multipole method is utilized for efficient evaluation of HIs. The deformation of the fibers is described by nonlinear Euler–Bernoulli beam theory and their polymerization is modeled by the reparametrization of the dynamic equations in the appropriate non-Lagrangian frame. We use a pseudo-spectral representation of fiber positions and implicit time-stepping to resolve large fiber deformations, and to allow time-steps not excessively constrained by temporal stiffness or fiber–fiber interactions. The entire computational scheme is parallelized, which enables simulating assemblies of thousands of fibers. We use our method to investigate two important questions in the mechanics of cell division: (i) the effect of confinement on the hydrodynamic mobility of microtubule asters; and (ii) the dynamics of the positioning of mitotic spindle in complex cell geometries. Finally to demonstrate the general applicability of the method, we simulate the sedimentation of a cloud of semi-flexible fibers. [ABSTRACT FROM AUTHOR]

The ascidian embryo is an ideal system to investigate how cell position is determined during embryogenesis. Using 3D timelapse imaging and computational methods we analyzed the planar cell divisions in ascidian early embryos and found that spindles in every cell tend to align at metaphase in the long length of the apical surface except in cells undergoing unequal cleavage. Furthermore, the invariant and conserved cleavage pattern of ascidian embryos was found to consist in alternate planar cell divisions between ectoderm and endomesoderm. In order to test the importance of alternate cell divisions we manipulated zygotic transcription induced by β-catenin or downregulated wee1 activity, both of which abolish this cell cycle asynchrony. Crucially, abolishing cell cycle asynchrony consistently disrupted the spindle orienting mechanism underpinning the invariant cleavage pattern. Our results demonstrate how an evolutionary conserved cell cycle asynchrony maintains the invariant cleavage pattern driving morphogenesis of the ascidian blastula. [ABSTRACT FROM AUTHOR]