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      Changes in Ect2 Localization Couple Actomyosin-Dependent Cell Shape Changes to Mitotic Progression

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          Summary

          As they enter mitosis, animal cells undergo profound actin-dependent changes in shape to become round. Here we identify the Cdk1 substrate, Ect2, as a central regulator of mitotic rounding, thus uncovering a link between the cell-cycle machinery that drives mitotic entry and its accompanying actin remodeling. Ect2 is a RhoGEF that plays a well-established role in formation of the actomyosin contractile ring at mitotic exit, through the local activation of RhoA. We find that Ect2 first becomes active in prophase, when it is exported from the nucleus into the cytoplasm, activating RhoA to induce the formation of a mechanically stiff and rounded metaphase cortex. Then, at anaphase, binding to RacGAP1 at the spindle midzone repositions Ect2 to induce local actomyosin ring formation. Ect2 localization therefore defines the stage-specific changes in actin cortex organization critical for accurate cell division.

          Highlights

          ► Ect2 drives dynamic changes in cell shape throughout mitosis ► Ect2 induces actin-dependent changes in cortical mechanics at mitotic onset ► Ect2's distinct functions are achieved through changes in subcellular localization ► Actin remodeling for animal cell division begins at mitotic entry

          Abstract

          Cell division is accompanied by dramatic changes in cell shape, which Matthews et al. show are regulated by the Cdk1 substrate Ect2. At mitotic onset, Ect2 leaves the nucleus to activate RhoA and cortical actomyosin, driving mitotic rounding. It is then repositioned at mitotic exit to drive actomyosin ring formation.

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          Most cited references60

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          A probability-based approach for high-throughput protein phosphorylation analysis and site localization.

          Data analysis and interpretation remain major logistical challenges when attempting to identify large numbers of protein phosphorylation sites by nanoscale reverse-phase liquid chromatography/tandem mass spectrometry (LC-MS/MS) (Supplementary Figure 1 online). In this report we address challenges that are often only addressable by laborious manual validation, including data set error, data set sensitivity and phosphorylation site localization. We provide a large-scale phosphorylation data set with a measured error rate as determined by the target-decoy approach, we demonstrate an approach to maximize data set sensitivity by efficiently distracting incorrect peptide spectral matches (PSMs), and we present a probability-based score, the Ascore, that measures the probability of correct phosphorylation site localization based on the presence and intensity of site-determining ions in MS/MS spectra. We applied our methods in a fully automated fashion to nocodazole-arrested HeLa cell lysate where we identified 1,761 nonredundant phosphorylation sites from 491 proteins with a peptide false-positive rate of 1.3%.
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            Phosphorylation and activation of myosin by Rho-associated kinase (Rho-kinase).

            The small GTPase Rho is implicated in physiological functions associated with actin-myosin filaments such as cytokinesis, cell motility, and smooth muscle contraction. We have recently identified and molecularly cloned Rho-associated serine/threonine kinase (Rho-kinase), which is activated by GTP Rho (Matsui, T., Amano, M., Yamamoto, T., Chihara, K., Nakafuku, M., Ito, M., Nakano, T., Okawa, K., Iwamatsu, A., and Kaibuchi, K. (1996) EMBO J. 15, 2208-2216). Here we found that Rho-kinase stoichiometrically phosphorylated myosin light chain (MLC). Peptide mapping and phosphoamino acid analyses revealed that the primary phosphorylation site of MLC by Rho-kinase was Ser-19, which is the site phosphorylated by MLC kinase. Rho-kinase phosphorylated recombinant MLC, whereas it failed to phosphorylate recombinant MLC, which contained Ala substituted for both Thr-18 and Ser-19. We also found that the phosphorylation of MLC by Rho-kinase resulted in the facilitation of the actin activation of myosin ATPase. Thus, it is likely that once Rho is activated, then it can interact with Rho-kinase and activate it. The activated Rho-kinase subsequently phosphorylates MLC. This may partly account for the mechanism by which Rho regulates cytokinesis, cell motility, or smooth muscle contraction.
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              Selective small-molecule inhibitor reveals critical mitotic functions of human CDK1.

              CDK1 is a nonredundant cyclin-dependent kinase (CDK) with an essential role in mitosis, but its multiple functions still are poorly understood at a molecular level. Here we identify a selective small-molecule inhibitor of CDK1 that reversibly arrests human cells at the G(2)/M border of the cell cycle and allows for effective cell synchronization in early mitosis. Inhibition of CDK1 during cell division revealed that its activity is necessary and sufficient for maintaining the mitotic state of the cells, preventing replication origin licensing and premature cytokinesis. Although CDK1 inhibition for up to 24 h is well tolerated, longer exposure to the inhibitor induces apoptosis in tumor cells, suggesting that selective CDK1 inhibitors may have utility in cancer therapy.
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                Author and article information

                Journal
                Dev Cell
                Dev. Cell
                Developmental Cell
                Cell Press
                1534-5807
                1878-1551
                14 August 2012
                14 August 2012
                : 23
                : 2
                : 371-383
                Affiliations
                [1 ]MRC Laboratory for Molecular Cell Biology, University College London, Gower St., London WC1E 6BT, UK
                [2 ]Department of Physics, Cavendish Laboratory, University of Cambridge, J.J. Thomson Avenue, Cambridge CB3 0HE, UK
                [3 ]PCC Curie, Institut Curie/CNRS/Université Paris 6 - UMR 168, 26 rue d'Ulm, 75248 Paris, France
                Author notes
                []Corresponding author b.baum@ 123456ucl.ac.uk
                Article
                DEVCEL2450
                10.1016/j.devcel.2012.06.003
                3763371
                22898780
                2e7dce64-2753-4652-b3af-8d1d079643c1
                © 2012 ELL & Excerpta Medica.

                This document may be redistributed and reused, subject to certain conditions.

                History
                : 7 December 2011
                : 4 April 2012
                : 5 June 2012
                Categories
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                Developmental biology
                Developmental biology

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