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

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.

[1]  H. Bellen,et al.  A putative exchange factor for Rho1 GTPase is required for initiation of cytokinesis in Drosophila. , 1999, Genes & development.

[2]  Thomas D. Pollard,et al.  Actin, a Central Player in Cell Shape and Movement , 2009, Science.

[3]  Hongmao Sun,et al.  Selective small-molecule inhibitor reveals critical mitotic functions of human CDK1. , 2006, Proceedings of the National Academy of Sciences of the United States of America.

[4]  Y. Abe,et al.  AIP1/WDR1 supports mitotic cell rounding. , 2005, Biochemical and biophysical research communications.

[5]  A. Hyman,et al.  CDC-42 and RHO-1 coordinate acto-myosin contractility and PAR protein localization during polarity establishment in C. elegans embryos , 2006, Development.

[6]  Stefan Schinkinger,et al.  Characterizing single suspended cells by optorheology. , 2005, Acta Biomaterialia.

[7]  K. Shokat,et al.  Covalent capture of kinase-specific phosphopeptides reveals Cdk1-cyclin B substrates , 2008, Proceedings of the National Academy of Sciences.

[8]  J. Labbé,et al.  Microinjection of p34cdc2 kinase induces marked changes in cell shape, cytoskeletal organization, and chromatin structure in mammalian fibroblasts , 1990, Cell.

[9]  Glenn C. Simon,et al.  Sequential Cyk‐4 binding to ECT2 and FIP3 regulates cleavage furrow ingression and abscission during cytokinesis , 2008, The EMBO journal.

[10]  Yoshiharu Matsuura,et al.  Phosphorylation and Activation of Myosin by Rho-associated Kinase (Rho-kinase)* , 1996, The Journal of Biological Chemistry.

[11]  T. Fleming,et al.  Oncogene ect2 is related to regulators of small GTP-binding proteins , 1993, Nature.

[12]  T. Miki,et al.  Phosphorylation of the cytokinesis regulator ECT2 at G2/M phase stimulates association of the mitotic kinase Plk1 and accumulation of GTP-bound RhoA , 2006, Oncogene.

[13]  J. Labbé,et al.  In vitro disassembly of the nuclear lamina and M phase-specific phosphorylation of lamins by cdc2 kinase , 1990, Cell.

[14]  O. Gavet,et al.  Activation of cyclin B1–Cdk1 synchronizes events in the nucleus and the cytoplasm at mitosis , 2010, The Journal of cell biology.

[15]  Karl Mechtler,et al.  BAC TransgeneOmics: a high-throughput method for exploration of protein function in mammals , 2008, Nature Methods.

[16]  R. Saint,et al.  A RhoGEF and Rho family GTPase-activating protein complex links the contractile ring to cortical microtubules at the onset of cytokinesis. , 2003, Developmental cell.

[17]  F. Motegi,et al.  Sequential functioning of the ECT-2 RhoGEF, RHO-1 and CDC-42 establishes cell polarity in Caenorhabditis elegans embryos , 2006, Nature Cell Biology.

[18]  D. Wirtz,et al.  Interplay of RhoA and motility in the programmed spreading of daughter cells postmitosis. , 2010, Biophysical journal.

[19]  S. Gammeltoft,et al.  Proteomic screen defines the Polo‐box domain interactome and identifies Rock2 as a Plk1 substrate , 2007, The EMBO journal.

[20]  H. Lane,et al.  Phosphorylation by p34cdc2 regulates spindle association of human Eg5, a kinesin-related motor essential for bipolar spindle formation in vivo , 1995, Cell.

[21]  Péter Lénárt,et al.  Polo on the Rise-from Mitotic Entry to Cytokinesis with Plk1. , 2008, Developmental cell.

[22]  J. Pines,et al.  How cyclin A destruction escapes the spindle assembly checkpoint , 2010, The Journal of cell biology.

[23]  K. Hahn,et al.  Spatiotemporal dynamics of RhoA activity in migrating cells , 2006, Nature.

[24]  J. Tinevez,et al.  Polar actomyosin contractility destabilizes the position of the cytokinetic furrow , 2011, Nature.

[25]  Steven P Gygi,et al.  A probability-based approach for high-throughput protein phosphorylation analysis and site localization , 2006, Nature Biotechnology.

[26]  M. Gibson,et al.  Interkinetic Nuclear Migration Is a Broadly Conserved Feature of Cell Division in Pseudostratified Epithelia , 2011, Current Biology.

[27]  Stefan Schinkinger,et al.  Quantifying the contribution of actin networks to the elastic strength of fibroblasts. , 2006, Journal of theoretical biology.

[28]  B. Baum,et al.  Myosin II-Dependent Cortical Movement Is Required for Centrosome Separation and Positioning during Mitotic Spindle Assembly , 2004, Cell.

[29]  S. Elledge,et al.  A quantitative atlas of mitotic phosphorylation , 2008, Proceedings of the National Academy of Sciences.

[30]  E. Nigg,et al.  Influence of human Ect2 depletion and overexpression on cleavage furrow formation and abscission , 2006, Journal of Cell Science.

[31]  Stefan Schinkinger,et al.  Optical deformability as an inherent cell marker for testing malignant transformation and metastatic competence. , 2005, Biophysical journal.

[32]  M. Matsuda,et al.  Activity of Rho-family GTPases during cell division as visualized with FRET-based probes , 2003, The Journal of cell biology.

[33]  J. Clarke,et al.  A Polarised Population of Dynamic Microtubules Mediates Homeostatic Length Control in Animal Cells , 2010, PLoS biology.

[34]  H. S. Roberts.  Mechanisms of Cytokinesis: A Critical Review , 1961, The Quarterly Review of Biology.

[35]  Stefan Schinkinger,et al.  The regulatory role of cell mechanics for migration of differentiating myeloid cells , 2009, Proceedings of the National Academy of Sciences.

[36]  K. Kamijo,et al.  Deregulation and Mislocalization of the Cytokinesis Regulator ECT2 Activate the Rho Signaling Pathways Leading to Malignant Transformation* , 2004, Journal of Biological Chemistry.

[37]  Frank Uhlmann,et al.  A Quantitative Model for Ordered Cdk Substrate Dephosphorylation during Mitotic Exit , 2011, Cell.

[38]  Junjie Chen,et al.  The Tandem BRCT Domains of Ect2 Are Required for Both Negative and Positive Regulation of Ect2 in Cytokinesis* , 2005, Journal of Biological Chemistry.

[39]  M. Glotzer,et al.  An ECT2–centralspindlin complex regulates the localization and function of RhoA , 2005, The Journal of cell biology.

[40]  M. Labouesse.  Faculty Opinions recommendation of Sequential functioning of the ECT-2 RhoGEF, RHO-1 and CDC-42 establishes cell polarity in Caenorhabditis elegans embryos. , 2006 .

[41]  V. Simanis,et al.  Dividing the Spoils of Growth and the Cell Cycle: The Fission Yeast as a Model for the Study of Cytokinesis , 2011, Cytoskeleton.

[42]  Stefan Schinkinger,et al.  High-throughput rheological measurements with an optical stretcher. , 2007, Methods in cell biology.

[43]  Keith Burridge,et al.  RhoA is required for cortical retraction and rigidity during mitotic cell rounding , 2003, The Journal of cell biology.

[44]  D. Gerlich,et al.  Aurora B-Mediated Abscission Checkpoint Protects against Tetraploidization , 2009, Cell.

[45]  A. Echard,et al.  Moesin and its activating kinase Slik are required for cortical stability and microtubule organization in mitotic cells , 2008, The Journal of cell biology.

[46]  J. de Gunzburg,et al.  Dynamic changes in Rap1 activity are required for cell retraction and spreading during mitosis , 2009, Journal of Cell Science.

[47]  R. Poon,et al.  How protein kinases co-ordinate mitosis in animal cells. , 2011, The Biochemical journal.

[48]  T. Mitchison,et al.  Investigation of the mechanism of retraction of the cell margin and rearward flow of nodules during mitotic cell rounding. , 1997, Molecular biology of the cell.

[49]  Kuan-Chung Su,et al.  Targeting of the RhoGEF Ect2 to the equatorial membrane controls cleavage furrow formation during cytokinesis. , 2011, Developmental cell.

[50]  O. Gavet,et al.  Progressive activation of CyclinB1-Cdk1 coordinates entry to mitosis. , 2010, Developmental cell.

[51]  Daniel J. Muller,et al.  Hydrostatic pressure and the actomyosin cortex drive mitotic cell rounding , 2011, Nature.

[52]  Buzz Baum,et al.  The actin cytoskeleton in spindle assembly and positioning. , 2009, Trends in cell biology.

[53]  R. Durbin,et al.  Systematic Analysis of Human Protein Complexes Identifies Chromosome Segregation Proteins , 2010, Science.

[54]  S. Carr,et al.  Plk1 Self-Organization and Priming Phosphorylation of HsCYK-4 at the Spindle Midzone Regulate the Onset of Division in Human Cells , 2009, PLoS biology.

[55]  Joshua T. Jones,et al.  Probing the precision of the mitotic clock with a live-cell fluorescent biosensor , 2004, Nature Biotechnology.

[56]  Li-quan Hong,et al.  Large-scale identification of novel mitosis-specific phosphoproteins. , 2008, Biochimica et biophysica acta.

[57]  T. Takaki,et al.  Polo-Like Kinase 1 Directs Assembly of the HsCyk-4 RhoGAP/Ect2 RhoGEF Complex to Initiate Cleavage Furrow Formation , 2009, PLoS biology.

[58]  J. Grosshans,et al.  Regulation of the Rac GTPase pathway by the multifunctional Rho GEF Pebble is essential for mesoderm migration in the Drosophila gastrula , 2009, Development.

[59]  A. Harris.  Location of cellular adhesions to solid substrata. , 1973, Developmental biology.

[60]  T. Miki,et al.  Human Ect2 Is an Exchange Factor for Rho Gtpases, Phosphorylated in G2/M Phases, and Involved in Cytokinesis , 1999, The Journal of cell biology.

[61]  S. Schmidt,et al.  APCcdh1 Mediates Degradation of the Oncogenic Rho-GEF Ect2 after Mitosis , 2011, PloS one.

[62]  R. Kobayashi,et al.  Phosphorylation and activation of 13S condensin by Cdc2 in vitro. , 1998, Science.

[63]  Jan-Michael Peters,et al.  Polo-like kinase 1 triggers the initiation of cytokinesis in human cells by promoting recruitment of the RhoGEF Ect2 to the central spindle. , 2007, Developmental cell.

[64]  J. Käs,et al.  The optical stretcher: a novel laser tool to micromanipulate cells. , 2001, Biophysical journal.

[65]  P. R. Elliott,et al.  Protein phosphatases and the regulation of mitosis , 2011, Journal of Cell Science.

[66]  J. Ellenberg,et al.  Fluorophores for live cell imaging of AGT fusion proteins across the visible spectrum. , 2006, BioTechniques.

[67]  Andrew E. Pelling,et al.  Moesin Controls Cortical Rigidity, Cell Rounding, and Spindle Morphogenesis during Mitosis , 2008, Current Biology.

[68]  Thomas D. Pollard,et al.  Understanding cytokinesis: lessons from fission yeast , 2010, Nature Reviews Molecular Cell Biology.

[69]  R. Medema,et al.  The decision to enter mitosis: feedback and redundancy in the mitotic entry network , 2009, The Journal of cell biology.

[70]  T. Veenstra,et al.  Cytokinesis regulator ECT2 changes its conformation through phosphorylation at Thr-341 in G2/M phase , 2006, Oncogene.

[71]  T. Holak,et al.  Lifeact: a versatile marker to visualize F-actin , 2008, Nature Methods.

[72]  S. Yonemura,et al.  Centralspindlin regulates ECT2 and RhoA accumulation at the equatorial cortex during cytokinesis , 2006, Journal of Cell Science.

[73]  Helen K. Matthews,et al.  Directional migration of neural crest cells in vivo is regulated by Syndecan-4/Rac1 and non-canonical Wnt signaling/RhoA , 2008, Development.

[74]  T. Murayama,et al.  Cell Cycle-Dependent Microtubule-Based Dynamic Transport of Cytoplasmic Dynein in Mammalian Cells , 2009, PloS one.

[75]  G. Schubiger,et al.  A genetic screen for suppressors and enhancers of the Drosophila cdk1-cyclin B identifies maternal factors that regulate microtubule and microfilament stability. , 2002, Genetics.

[76]  Timothy J Mitchison,et al.  Animal cytokinesis: from parts list to mechanisms. , 2006, Annual review of biochemistry.

[77]  K. Kamijo,et al.  Dissecting the role of Rho-mediated signaling in contractile ring formation. , 2005, Molecular biology of the cell.

[78]  J. Rubin,et al.  Rho exchange factor ECT2 is induced by growth factors and regulates cytokinesis through the N‐terminal cell cycle regulator‐related domains , 2003, Journal of cellular biochemistry.