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Michael Glotzer - One of the best experts on this subject based on the ideXlab platform.
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THE JOURNAL OF CELL BIOLOGY
2013Co-Authors: Ozlem Yuce, Alisa Piekny, Michael GlotzerAbstract:In anaphase, the Spindle dictates the site of contractile ring assembly. Assembly and ingression of the contractile ring involves activation of myosin-II and actin polymerization, which are triggered by the GTPase RhoA. In many cells, the Central Spindle affects division plane positioning via unknown molecular mechanisms. Here, we dissect furrow formation in human cells and show that the RhoGEF ECT2 is required for cortical localization of RhoA and contractile ring assembly. ECT2 concentrates on the Central Spindle by binding to Centralspindlin. Depletion of the Centralspindlin componen
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Clustering of Centralspindlin is essential for its accumulation to the Central Spindle and the midbody.
Current biology : CB, 2009Co-Authors: Andrea Hutterer, Michael Glotzer, Masanori MishimaAbstract:Summary Cytokinesis in animal cells requires the Central Spindle and midbody, which contain prominent microtubule bundles [1]. Centralspindlin, a heterotetrameric complex consisting of kinesin-6 and RhoGAP (Rho-family GTPase-activating protein) subunits, is essential for the formation of these structures [2]. Centralspindlin becomes precisely localized to the Central Spindle, where it promotes the equatorial recruitment of important cytokinetic regulators. These include ECT2, the activator of the small GTPase RhoA, which controls cleavage furrow formation and ingression [3–6]. Centralspindlin's own RhoGAP domain also contributes to furrow ingression [7–10]. Finally, Centralspindlin facilitates recruitment of the chromosome passenger complex [7, 8] and factors that control abscission [11, 12]. Despite the importance of localized accumulation of Centralspindlin, the mechanism by which this motor protein complex suddenly concentrates to the center of interpolar microtubule bundles during anaphase is unclear. Here, we show that Centralspindlin travels along Central Spindle microtubules as higher-order clusters. Clustering of Centralspindlin is critical for microtubule bundling and motility along microtubules in vitro and for midbody formation in vivo. These data support a positive feedback loop of Centralspindlin clustering and microtubule organization that may underlie its distinctive localization during cytokinesis.
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polo like kinase 1 directs assembly of the hscyk 4 rhogap ect2 rhogef complex to initiate cleavage furrow formation
PLOS Biology, 2009Co-Authors: Benjamin A Wolfe, Mark Petronczki, Tohru Takaki, Michael GlotzerAbstract:To complete cell division with high fidelity, cytokinesis must be coordinated with chromosome segregation. Mammalian Polo-like kinase 1, Plk1, may function as a critical link because it is required for chromosome segregation and establishment of the cleavage plane following anaphase onset. A Central Spindle–localized pool of the RhoGEF Ect2 promotes activation of the small GTPase RhoA, which drives contractile ring assembly at the equatorial cortex. Here, we have investigated how Plk1 promotes the Central Spindle recruitment of Ect2. Plk1 phosphorylates the noncatalytic N terminus of the RhoGAP HsCyk-4 at the Central Spindle, creating a phospho-epitope recognized by the BRCA1 C-terminal (BRCT) repeats of Ect2. Failure to phosphorylate HsCyk-4 blocks Ect2 recruitment to the Central Spindle and the subsequent induction of furrowing. Microtubules, as well as the microtubule-associated protein (MAP) Prc1, facilitate Plk1 phosphorylation of HsCyk-4. Characterization of a phosphomimetic version of HsCyk-4 indicates that Plk1 promotes Ect2 recruitment through multiple targets. Collectively, our data reveal that formation of the HsCyk-4-Ect2 complex is subject to multiple layers of regulation to ensure that RhoA activation occurs between the segregated sister chromatids during anaphase.
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the 3ms of Central Spindle assembly microtubules motors and maps
Nature Reviews Molecular Cell Biology, 2009Co-Authors: Michael GlotzerAbstract:During anaphase, the mitotic Spindle reorganizes in preparation for cytokinesis. Kinesin motor proteins and microtubule-associated proteins (MAPs) bundle the interpolar microtubule plus ends and generate the Central Spindle, which regulates cleavage furrow initiation and the completion of cytokinesis. During metaphase, sister chromatids are positioned at the midpoint of the microtubule-based mitotic Spindle in preparation for their segregation. The onset of anaphase triggers inactivation of the key mitotic kinase cyclin-dependent kinase 1 (CDK1) and the polewards movement of sister chromatids. During anaphase, the mitotic Spindle reorganizes in preparation for cytokinesis. Kinesin motor proteins and microtubule-associated proteins bundle the plus ends of interpolar microtubules and generate the Central Spindle, which regulates cleavage furrow initiation and the completion of cytokinesis. Complementary approaches, including cell biology, genetics and computational modelling, have provided new insights into the mechanism and regulation of Central Spindle assembly.
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the 3ms of Central Spindle assembly microtubules motors and maps
Nature Reviews Molecular Cell Biology, 2009Co-Authors: Michael GlotzerAbstract:During anaphase, the mitotic Spindle reorganizes in preparation for cytokinesis. Kinesin motor proteins and microtubule-associated proteins (MAPs) bundle the interpolar microtubule plus ends and generate the Central Spindle, which regulates cleavage furrow initiation and the completion of cytokinesis.
Francis A Barr - One of the best experts on this subject based on the ideXlab platform.
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aurora a promotes chromosome congression by activating the condensin dependent pool of kif4a
bioRxiv, 2019Co-Authors: Elena Poser, Ulrike Gruneberg, Renaud Caous, Francis A BarrAbstract:Aurora kinases create phosphorylation gradients within the Spindle during prometaphase and anaphase. These locally regulate factors that promote Spindle organisation, chromosome condensation and movement, and cytokinesis. We show that one such factor is the kinesin KIF4A, which is present along the chromosome axes throughout mitosis and the Central Spindle in anaphase. These two pools of KIF4A depend on condensin I and PRC1, respectively. Previous work has shown KIF4A is activated by Aurora B at the anaphase Central Spindle. However, whether or not chromosome-associated KIF4A bound to condensin I is regulated by Aurora kinases remain unclear. To determine the roles of the two different pools of KIF4A, we generated specific point mutants that are unable to interact with either condensin I or PRC1, or are deficient for Aurora kinase regulation. By analysing these mutants, we show that Aurora kinases phosphorylate the condensin I dependent pool of KIF4A and thus actively promote chromosome congression from the Spindle poles to the metaphase plate.
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kif4a and pp2a b56 form a spatially restricted feedback loop opposing aurora b at the anaphase Central Spindle
Journal of Cell Biology, 2014Co-Authors: Ricardo Nunes Bastos, Michael J Cundell, Francis A BarrAbstract:The mitotic kinase Aurora B is concentrated at the anaphase Central Spindle by the kinesin MKlp2 during mitotic exit and cytokinesis. This pool of Aurora B phosphorylates substrates including the kinesin KIF4A to regulate Central Spindle length. In this paper, we identify a counteracting system in which PP2A–B56γ and -e, but not PP2A–B56α, -β, and -δ, are maintained at the Central Spindle by KIF4A. Biochemical assays show that PP2A–B56γ can dephosphorylate the T799 Aurora B site on KIF4A and thereby counteract the Aurora B– and microtubule-stimulated ATPase activity of KIF4A. In agreement with these observations, combined silencing of PP2A–B56γ and -e resulted in increased phosphorylation of KIF4A T799 and decreased Central Spindle growth in anaphase B. Furthermore, reduced turnover of regulatory phosphorylation on another Aurora B substrate MKlp1 was observed, suggesting that PP2A–B56γ and -e play a general role opposing Aurora B at the Central Spindle. KIF4A and PP2A–B56γ and -e therefore create a spatially restricted negative feedback loop counteracting Aurora B in anaphase.
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aurora b suppresses microtubule dynamics and limits Central Spindle size by locally activating kif4a
Journal of Cell Biology, 2013Co-Authors: Ricardo Nunes Bastos, Erich A. Nigg, Ulrike Gruneberg, Sapan R Gandhi, Ryan Baron, Francis A BarrAbstract:Anaphase Central Spindle formation is controlled by the microtubule-stabilizing factor PRC1 and the kinesin KIF4A. We show that an MKlp2-dependent pool of Aurora B at the Central Spindle, rather than global Aurora B activity, regulates KIF4A accumulation at the Central Spindle. KIF4A phosphorylation by Aurora B stimulates the maximal microtubule-dependent ATPase activity of KIF4A and promotes its interaction with PRC1. In the presence of phosphorylated KIF4A, microtubules grew more slowly and showed long pauses in growth, resulting in the generation of shorter PRC1-stabilized microtubule overlaps in vitro. Cells expressing only mutant forms of KIF4A lacking the Aurora B phosphorylation site overextended the anaphase Central Spindle, demonstrating that this regulation is crucial for microtubule length control in vivo. Aurora B therefore ensures that suppression of microtubule dynamic instability by KIF4A is restricted to a specific subset of microtubules and thereby contributes to Central Spindle size control in anaphase.
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kif14 and citron kinase act together to promote efficient cytokinesis
Journal of Cell Biology, 2006Co-Authors: Erich A. Nigg, Ravindra B. Chalamalasetty, Ulrike Gruneberg, Rudiger Neef, Eunice H Y Chan, Francis A BarrAbstract:Multiple mitotic kinesins and microtubule-associated proteins (MAPs) act in concert to direct cytokinesis (Glotzer, M. 2005. Science. 307:1735-1739). In anaphase cells, many of these proteins associate with an antiparallel array of microtubules termed the Central Spindle. The MAP and microtubule-bundling protein PRC1 (protein-regulating cytokinesis 1) is one of the key molecules required for the integrity of this structure (Jiang, W., G. Jimenez, N.J. Wells, T.J. Hope, G.M. Wahl, T. Hunter, and R. Fukunaga. 1998. Mol. Cell. 2:877-885; Mollinari, C., J.P. Kleman, W. Jiang, G. Schoehn, T. Hunter, and R.L. Margolis. 2002. J. Cell Biol. 157:1175-1186). In this study, we identify an interaction between endogenous PRC1 and the previously uncharacterized kinesin KIF14 as well as other mitotic kinesins (MKlp1/CHO1, MKlp2, and KIF4) with known functions in cytokinesis (Hill, E., M. Clarke, and F.A. Barr. 2000. EMBO J. 19:5711-5719; Matuliene, J., and R. Kuriyama. 2002. Mol. Biol. Cell. 13:1832-1845; Kurasawa, Y., W.C. Earnshaw, Y. Mochizuki, N. Dohmae, and K. Todokoro. 2004. EMBO J. 23:3237-3248). We find that KIF14 targets to the Central Spindle via its interaction with PRC1 and has an essential function in cytokinesis. In KIF14-depleted cells, citron kinase but not other components of the Central Spindle and cleavage furrow fail to localize. Furthermore, the localization of KIF14 and citron kinase to the Central Spindle and midbody is codependent, and they form a complex depending on the activation state of citron kinase. Contrary to a previous study (Di Cunto, F., S. Imarisio, E. Hirsch, V. Broccoli, A. Bulfone, A. Migheli, C. Atzori, E. Turco, R. Triolo, G.P. Dotto, et al. 2000. Neuron. 28:115-127), we find a general requirement for citron kinase in human cell division. Together, these findings identify a novel pathway required for efficient cytokinesis.
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relocation of aurora b from centromeres to the Central Spindle at the metaphase to anaphase transition requires mklp2
Journal of Cell Biology, 2004Co-Authors: Ulrike Gruneberg, Reiko Honda, Erich A. Nigg, Rudiger Neef, Francis A BarrAbstract:Mitotic kinases of the Polo and Aurora families are key regulators of chromosome segregation and cytokinesis. Here, we have investigated the role of MKlp1 and MKlp2, two vertebrate mitotic kinesins essential for cytokinesis, in the spatial regulation of the Aurora B kinase. Previously, we have demonstrated that MKlp2 recruits Polo-like kinase 1 (Plk1) to the Central Spindle in anaphase. We now find that in MKlp2 but not MKlp1-depleted cells the Aurora B–INCENP complex remains at the centromeres and fails to relocate to the Central Spindle. MKlp2 exerts dual control over Aurora B localization, because it is a binding partner for Aurora B, and furthermore for the phosphatase Cdc14A. Cdc14A can dephosphorylate INCENP and may contribute to its relocation to the Central Spindle in anaphase. We propose that MKlp2 is involved in the localization of Plk1, Aurora B, and Cdc14A to the Central Spindle during anaphase, and that the integration of signaling by these proteins is necessary for proper cytokinesis.
Erich A. Nigg - One of the best experts on this subject based on the ideXlab platform.
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aurora b suppresses microtubule dynamics and limits Central Spindle size by locally activating kif4a
Journal of Cell Biology, 2013Co-Authors: Ricardo Nunes Bastos, Erich A. Nigg, Ulrike Gruneberg, Sapan R Gandhi, Ryan Baron, Francis A BarrAbstract:Anaphase Central Spindle formation is controlled by the microtubule-stabilizing factor PRC1 and the kinesin KIF4A. We show that an MKlp2-dependent pool of Aurora B at the Central Spindle, rather than global Aurora B activity, regulates KIF4A accumulation at the Central Spindle. KIF4A phosphorylation by Aurora B stimulates the maximal microtubule-dependent ATPase activity of KIF4A and promotes its interaction with PRC1. In the presence of phosphorylated KIF4A, microtubules grew more slowly and showed long pauses in growth, resulting in the generation of shorter PRC1-stabilized microtubule overlaps in vitro. Cells expressing only mutant forms of KIF4A lacking the Aurora B phosphorylation site overextended the anaphase Central Spindle, demonstrating that this regulation is crucial for microtubule length control in vivo. Aurora B therefore ensures that suppression of microtubule dynamic instability by KIF4A is restricted to a specific subset of microtubules and thereby contributes to Central Spindle size control in anaphase.
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structure of a survivin borealin incenp core complex reveals how chromosomal passengers travel together
Cell, 2007Co-Authors: Arockia A Jeyaprakash, Erich A. Nigg, Ulf R Klein, Doris Lindner, Judith Ebert, Elena ContiAbstract:The chromosomal passenger complex (CPC) is a key regulator of chromosome segregation and cytokinesis. CPC functions are connected to its localization. The complex first localizes to centromeres and later associates with the Central Spindle and midbody. Survivin, Borealin, and INCENP are the three components of the CPC that regulate the activity and localization of its enzymatic component, the kinase Aurora B. We determined the 1.4 A resolution crystal structure of the regulatory core of the CPC, revealing that Borealin and INCENP associate with the helical domain of Survivin to form a tight three-helical bundle. We used siRNA rescue experiments with structure-based mutants to explore the requirements for CPC localization. We show that the intertwined structural interactions of the core components lead to functional interdependence. Association of the core "passenger" proteins creates a single structural unit, whose composite molecular surface presents conserved residues essential for Central Spindle and midbody localization.
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Influence of human Ect2 depletion and overexpression on cleavage furrow formation and abscission.
Journal of Cell Science, 2006Co-Authors: Ravindra B. Chalamalasetty, Erich A. Nigg, Stefan Hümmer, Herman H W SilljéAbstract:The guanine nucleotide-exchange factor (GEF) Ect2 is essential for cytokinesis. Here we studied the subcellular localization of Ect2 and examined the consequences of either depleting or overexpressing Ect2 in human cells. We show that in mitotic cells Ect2 localizes to the Central Spindle and to the cell cortex. The latter association is mediated through a PH domain in Ect2 and Central Spindle localization requires the MKlp1-MgcRacGAP and MKlp2-Aurora-B complexes. Ect2 directly interacts with MKlp1-MgcRacGAP through its BRCT domain, whereas MKlp2-Aurora-B probably exerts a regulatory role in Ect2 Central Spindle targeting. Depletion of Ect2 impaired cleavage furrow formation and RhoA and Citron kinase failed to accumulate at the cleavage furrow. Ect2 displacement from the Central Spindle revealed that physiological levels of this protein in this location are not crucial for RhoA activation and cytokinesis. In cells overexpressing appropriate N-terminal Ect2 fragments, RhoA and Citron kinase localized to the cleavage furrow and ingression occurred, but abscission failed. This failure could be correlated with the persistence of these fragments at structures surrounding the midbody, suggesting that abscission requires the displacement of Ect2 from the contractile ring and its re-import into the nucleus.
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kif14 and citron kinase act together to promote efficient cytokinesis
Journal of Cell Biology, 2006Co-Authors: Erich A. Nigg, Ravindra B. Chalamalasetty, Ulrike Gruneberg, Rudiger Neef, Eunice H Y Chan, Francis A BarrAbstract:Multiple mitotic kinesins and microtubule-associated proteins (MAPs) act in concert to direct cytokinesis (Glotzer, M. 2005. Science. 307:1735-1739). In anaphase cells, many of these proteins associate with an antiparallel array of microtubules termed the Central Spindle. The MAP and microtubule-bundling protein PRC1 (protein-regulating cytokinesis 1) is one of the key molecules required for the integrity of this structure (Jiang, W., G. Jimenez, N.J. Wells, T.J. Hope, G.M. Wahl, T. Hunter, and R. Fukunaga. 1998. Mol. Cell. 2:877-885; Mollinari, C., J.P. Kleman, W. Jiang, G. Schoehn, T. Hunter, and R.L. Margolis. 2002. J. Cell Biol. 157:1175-1186). In this study, we identify an interaction between endogenous PRC1 and the previously uncharacterized kinesin KIF14 as well as other mitotic kinesins (MKlp1/CHO1, MKlp2, and KIF4) with known functions in cytokinesis (Hill, E., M. Clarke, and F.A. Barr. 2000. EMBO J. 19:5711-5719; Matuliene, J., and R. Kuriyama. 2002. Mol. Biol. Cell. 13:1832-1845; Kurasawa, Y., W.C. Earnshaw, Y. Mochizuki, N. Dohmae, and K. Todokoro. 2004. EMBO J. 23:3237-3248). We find that KIF14 targets to the Central Spindle via its interaction with PRC1 and has an essential function in cytokinesis. In KIF14-depleted cells, citron kinase but not other components of the Central Spindle and cleavage furrow fail to localize. Furthermore, the localization of KIF14 and citron kinase to the Central Spindle and midbody is codependent, and they form a complex depending on the activation state of citron kinase. Contrary to a previous study (Di Cunto, F., S. Imarisio, E. Hirsch, V. Broccoli, A. Bulfone, A. Migheli, C. Atzori, E. Turco, R. Triolo, G.P. Dotto, et al. 2000. Neuron. 28:115-127), we find a general requirement for citron kinase in human cell division. Together, these findings identify a novel pathway required for efficient cytokinesis.
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Cell cycle regulation of Central Spindle assembly
Nature, 2004Co-Authors: Masanori Mishima, Erich A. Nigg, Ulrike Gruneberg, Visnja Pavicic, Michael GlotzerAbstract:The bipolar mitotic Spindle is responsible for segregating sister chromatids at anaphase. Microtubule motor proteins generate Spindle bipolarity and enable the Spindle to perform mechanical work^ 1 . A major change in Spindle architecture occurs at anaphase onset when Central Spindle assembly begins. This structure regulates the initiation of cytokinesis and is essential for its completion^ 2 . Central Spindle assembly requires the Centralspindlin complex composed of the Caenorhabditis elegans ZEN-4 (mammalian orthologue MKLP1) kinesin-like protein and the Rho family GAP CYK-4 (MgcRacGAP). Here we describe a regulatory mechanism that controls the timing of Central Spindle assembly. The mitotic kinase Cdk1/cyclin B phosphorylates the motor domain of ZEN-4 on a conserved site within a basic amino-terminal extension characteristic of the MKLP1 subfamily. Phosphorylation by Cdk1 diminishes the motor activity of ZEN-4 by reducing its affinity for microtubules. Preventing Cdk1 phosphorylation of ZEN-4/MKLP1 causes enhanced metaphase Spindle localization and defects in chromosome segregation. Thus, phosphoregulation of the motor domain of MKLP1 kinesin ensures that Central Spindle assembly occurs at the appropriate time in the cell cycle and maintains genomic stability.
Masanori Mishima - One of the best experts on this subject based on the ideXlab platform.
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preparation of Centralspindlin as an active heterotetramer of kinesin and gtpase activating protein subunits for in vitro structural and functional assays
Methods in Cell Biology, 2017Co-Authors: Masanori MishimaAbstract:Centralspindlin is a crucial regulator of animal cytokinesis, consisting of MKLP1 kinesin-6 and CYK4 Rho-family GTPase activating protein (RhoGAP). As a microtubule-bundling protein, it plays a crucial role in the formation of the Central Spindle. Through distinct accumulation to the antiparallel microtubule overlaps at the Central Spindle and the midbody, it recruits various downstream factors to the site of cell division as well as anchors the plasma membrane to maintain the narrow intercellular channels between the daughter cells until their final separation (abscission). A unique and functionally important feature of Centralspindlin as a kinesin-containing protein complex is that the nonmotor component, CYK4, is not a passive cargo of the MKLP1 motor, but an integrated component of a microtubule-organizing machinery. Thus, for in vitro structural and functional assays, it is pivotal to prepare active stoichiometric complexes of the two components. Discussed here are two complimentary approaches, (1) reconstitution of the complex in bacterial extracts (in extract reconstitution) and (2) purification of a native complex from a mammalian cell line using a localization and affinity purification (LAP) tag.
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Central Spindle robustness by PRC1-Centralspindlin interaction.
Cell cycle (Georgetown Tex.), 2015Co-Authors: Masanori Mishima, Kianyong LeeAbstract:Mitotic apparatus (MA) plays Central roles in cell division for both mitosis and cytokinesis. It achieves these mechanical tasks by changing its morphology under the control of cell cycle machinery. This relies on dynamic polymerization and depolymerization cycles of microtubules and their assembly into higher order structures such as bundles involving various microtubule regulators. A dramatic remodeling of the MA occurs at the metaphase to anaphase transition (Fig. 1). Before this, 2 Spindle poles are connected both by interpolar microtubules and by kinetochore microtubules attaching the unsegregated chromatids. After anaphase onset, the link via kinetochore microtubules and chromosomes disappears due to loss of chromosome cohesion. As a consequence, the interpolar microtubules, which have now developed into a more prominent structure termed the Central Spindle, become the sole mechanical link between the 2 poles (Fig. 1, case i). Metaphase-anaphase transition also promotes the growth of astral microtubules. Dynein anchored at the cell cortex interacts with the astral microtubules and generates mechanical forces (cortical pulling forces) that pull Spindle poles toward the cell cortex.1 Grill SW, et al. Nature 2001; 409(6820):630-3; PMID:11214323; http://dx.doi.org/10.1038/35054572 [CrossRef], [PubMed], [Web of Science ®] In some cell types such as the C. elegans embryos, cortical pulling force is the major driving force for chromosome separation via elongation of the pole-to-pole distance (anaphase B). In this situation, the Central Spindle is dispensable for chromosome segregation; it rather works as a brake against the cortical pulling force. Indeed, in C. elegans embryos, chromosome separation is accelerated when the Central Spindle is severed by laser manipulation or by genetic perturbation.2 Saunders AM, et al. Curr Biol 2007; 17(12):R453-4; PMID:17580072; http://dx.doi.org/10.1016/j.cub.2007.05.001 [CrossRef], [PubMed], [Web of Science ®] So, why does a cell form the Central Spindle? Well, this is because it has an important role in cytokinesis.
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Direct interaction between Centralspindlin and PRC1 reinforces mechanical resilience of the Central Spindle
Nature communications, 2015Co-Authors: Kianyong Lee, Behrooz Esmaeili, Ben Zealley, Masanori MishimaAbstract:During animal cell division, the Central Spindle, an anti-parallel microtubule bundle structure formed between segregating chromosomes during anaphase, cooperates with astral microtubules to position the cleavage furrow. Because the Central Spindle is the only structure linking the two halves of the mitotic Spindle, it is under mechanical tension from dynein-generated cortical pulling forces, which determine Spindle positioning and drive chromosome segregation through Spindle elongation. The Central Spindle should be flexible enough for efficient chromosome segregation while maintaining its structural integrity for reliable cytokinesis. How the cell balances these potentially conflicting requirements is poorly understood. Here, we demonstrate that the Central Spindle in C. elegans embryos has a resilient mechanism for recovery from perturbations by excess tension derived from cortical pulling forces. This mechanism involves the direct interaction of two different types of conserved microtubule bundlers that are crucial for Central Spindle formation, PRC1 and Centralspindlin.
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still entangled assembly of the Central Spindle by multiple microtubule modulators
Seminars in Cell & Developmental Biology, 2010Co-Authors: Max E Douglas, Masanori MishimaAbstract:The Central Spindle is a microtubule-based structure that assembles during anaphase of mitosis in animal cells and is essential for multiple steps of cytokinesis. Central Spindle assembly occurs by the cooperative action of multiple microtubule motors and modulators. Here, we review the mechanism by which the Central Spindle is formed, the role of several key proteins in this process and how Central Spindle assembly is temporally and spatially coordinated with mitosis.
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Clustering of Centralspindlin is essential for its accumulation to the Central Spindle and the midbody.
Current biology : CB, 2009Co-Authors: Andrea Hutterer, Michael Glotzer, Masanori MishimaAbstract:Summary Cytokinesis in animal cells requires the Central Spindle and midbody, which contain prominent microtubule bundles [1]. Centralspindlin, a heterotetrameric complex consisting of kinesin-6 and RhoGAP (Rho-family GTPase-activating protein) subunits, is essential for the formation of these structures [2]. Centralspindlin becomes precisely localized to the Central Spindle, where it promotes the equatorial recruitment of important cytokinetic regulators. These include ECT2, the activator of the small GTPase RhoA, which controls cleavage furrow formation and ingression [3–6]. Centralspindlin's own RhoGAP domain also contributes to furrow ingression [7–10]. Finally, Centralspindlin facilitates recruitment of the chromosome passenger complex [7, 8] and factors that control abscission [11, 12]. Despite the importance of localized accumulation of Centralspindlin, the mechanism by which this motor protein complex suddenly concentrates to the center of interpolar microtubule bundles during anaphase is unclear. Here, we show that Centralspindlin travels along Central Spindle microtubules as higher-order clusters. Clustering of Centralspindlin is critical for microtubule bundling and motility along microtubules in vitro and for midbody formation in vivo. These data support a positive feedback loop of Centralspindlin clustering and microtubule organization that may underlie its distinctive localization during cytokinesis.
David M Glover - One of the best experts on this subject based on the ideXlab platform.
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the drosophila phosphatidylinositol transfer protein encoded by vibrator is essential to maintain cleavage furrow ingression in cytokinesis
Journal of Cell Science, 2006Co-Authors: Melanie K Gatt, David M GloverAbstract:Cytokinesis requires the coordination of cytoskeletal and plasma membrane dynamics. A role for phosphatidylinositol lipids has been proposed for the successful completion of cytokinesis but this is still poorly characterised. Here, we show mutants of the gene vibrator, previously found to encode the Drosophila phosphatidylinositol transfer protein, produce multinucleate cells indicative of cytokinesis failure in male meiosis. Examination of fixed preparations of mutant spermatocytes showed contractile rings of anillin and actin that were of normal appearance at early stages but were larger and less well organised at later stages of cytokinesis than in wild-type cells. Time-lapse imaging revealed sequential defects in cytokinesis of vibrator spermatocytes. In cells that fail cytokinesis, Central Spindle formation occurred correctly, but furrow ingression was delayed and the Central Spindle did not become compressed to the extent seen in wild-type cells. Cells then stalled at this point before the apparent connection between the constricted cytoskeleton and the plasma membrane was lost; the furrow then underwent elastic regression. We discuss these defects in relation to multiple functions of phosphoinositol lipids in regulating actin dynamics and membrane synthesis.
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klp67a destabilises pre anaphase microtubules but subsequently is required to stabilise the Central Spindle
Journal of Cell Science, 2005Co-Authors: Melanie K Gatt, Matthew S Savoian, Maria Giovanna Riparbelli, Chiara Massarelli, Giuliano Callaini, David M GloverAbstract:Klp67A is a member of the Kip3 subfamily of microtubule destabilising kinesins, the loss of which results in abnormally long and stable pre-anaphase microtubules. Here we examine its role during cytokinesis in Drosophila primary spermatocytes that require the coordinated interaction of an interior and peripheral set of Central Spindle microtubules. In mutants anaphase B Spindles elongated with normal kinetics but bent towards the cortex. Both peripheral and interior Spindle microtubules then formed diminished bundles of abnormally positioned Central Spindle microtubules associated with the pavarotti-KLP and KLP3A motor proteins. The minus ends of these were poorly aligned as revealed by Asp protein localisation. Furrows always initiated at the sites of Central Spindle bundles but could be unilateral or nonequatorially positioned. Ectopic furrows were stimulated by the interior Central Spindle and formed only after this structure buckled and contacted the cortex. Furrows often halted and regressed as they could not be sustained by the Central Spindles that became increasing unstable over time and often completely degraded. Consistent with this, actin and anillin failed to form homogenous bands. Thus, the Klp67A microtubule catastrophe factor is required for cytokinesis by regulating both the formation and stability of the Central Spindle.
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mutations in orbit mast reveal that the Central Spindle is comprised of two microtubule populations those that initiate cleavage and those that propagate furrow ingression
Journal of Cell Biology, 2004Co-Authors: Yoshihiro H Inoue, Matthew S Savoian, Takao Suzuki, Endre Mathe, Masatoshi Yamamoto, David M GloverAbstract:We address the relative roles of astral and Central Spindle microtubules (MTs) in cytokinesis of Drosophila melanogaster primary spermatocytes. Time-lapse imaging studies reveal that the Central Spindle is comprised of two MT populations, “interior” Central Spindle MTs found within the Spindle envelope and “peripheral” astral MTs that probe the cytoplasm and initiate cleavage furrows where they contact the cortex and form overlapping bundles. The MT-associated protein Orbit/Mast/CLASP concentrates on interior rather than peripheral Central Spindle MTs. Interior MTs are preferentially affected in hypomorphic orbit mutants, and consequently the interior Central Spindle fails to form or is unstable. In contrast, peripheral MTs still probe the cortex and form regions of overlap that recruit the Pav-KLP motor and Aurora B kinase. orbit mutants have disorganized or incomplete anillin and actin rings, and although cleavage furrows initiate, they ultimately regress. Our work identifies a new function for Orbit/Mast/CLASP and identifies a novel MT population involved in cleavage furrow initiation.
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drosophila dd4 mutants reveal that gammaturc is required to maintain juxtaposed half Spindles in spermatocytes
Journal of Cell Science, 2003Co-Authors: Vitor Barbosa, Elena Rebollo, Melanie K Gatt, Cayetano Gonzalez, David M GloverAbstract:The weak Spindle integrity checkpoint in Drosophila spermatocytes has revealed a novel function of the γ-tubulin ring complex (γTuRC) in maintaining Spindle bipolarity throughout meiosis. Bipolar and bi-astral Spindles could form in Drosophila mutants for dd4, the gene encoding the 91 kDa subunit of γTuRC. However, these Spindles collapsed around metaphase and began to elongate as if attempting anaphase B. The microtubules of the collapsing Spindle folded back on themselves, their putative plus ends forming the focused apexes of biconical figures. Cells with such Spindles were unable to undergo cytokinesis. A second type of Spindle, monopolar hemi-Spindles, also formed as a result of either Spindle collapse at an earlier stage or failure of centrosome separation. Multiple centrosome-like bodies at the foci of hemi-Spindles nucleated robust asters of microtubules in the absence of detectable γ-tubulin. Time-lapse imaging revealed these to be intermediates that developed into cones, structures that also had putative plus ends of microtubules focused at their tips. Unlike biconical figures, however, cones seemed to contain a Central Spindle-like structure at their apexes and undergo cytokinesis. We conclude that spermatocytes do not need astral microtubules nucleated by opposite poles to intersect in order to form a Central Spindle and a cleavage furrow.
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A requirement for the Abnormal Spindle protein to organise microtubules of the Central Spindle for cytokinesis in Drosophila.
Journal of cell science, 2002Co-Authors: Maria Giovanna Riparbelli, David M Glover, Giuliano Callaini, Maria Do Carmo AvidesAbstract:Drosophila abnormal Spindle (asp) mutants exhibit a mitotic metaphase checkpoint arrest with abnormal Spindle poles, which reflects a requirement for Asp for the integrity of microtubule organising centres (MTOCs). In male meiosis, the absence of a strong Spindle integrity checkpoint enables asp mutant cells to proceed through anaphase and telophase. However, the Central Spindle region is not correctly organised and cells frequently fail to complete cytokinesis. This contrasts with meiosis in wild-type males where at late anaphase a dense array of microtubules forms in the Central Spindle region that has Asp localised at its border. We speculate that Asp is associated with the minus ends of microtubules that have been released from the Spindle poles to form the Central Spindle. A parallel situation arises in female meiosis where Asp not only associates with the minus ends of microtubules at the acentriolar poles but also with the Central Spindle pole body that forms between the two tandem Spindles of meiosis II. Upon fertilisation, Asp is also recruited to the MTOC that nucleates the sperm aster. Asp is required for growth of the microtubules of the sperm aster, which in asp mutants remains diminutive and so prevents migration of the pronuclei.