PRC1 is a microtubule binding and bundling protein essential to maintain the mitotic spindle midzone

Midzone microtubules of mammalian cells play an essential role in the induction of cell cleavage, serving as a platform for a number of proteins that play a part in cytokinesis. We demonstrate that PRC1, a mitotic spindle-associated Cdk substrate that is essential to cell cleavage, is a microtubule binding and bundling protein both in vivo and in vitro. Overexpression of PRC1 extensively bundles interphase microtubules, but does not affect early mitotic spindle organization. PRC1 contains two Cdk phosphorylation motifs, and phosphorylation is possibly important to mitotic suppression of bundling, as a Cdk phosphorylation-null mutant causes extensive bundling of the prometaphase spindle. Complete suppression of PRC1 by siRNA causes failure of microtubule interdigitation between half spindles and the absence of a spindle midzone. Truncation mutants demonstrate that the NH2-terminal region of PRC1, rich in α-helical sequence, is important for localization to the cleavage furrow and to the center of the midbody, whereas the central region, with the highest sequence homology between species, is required for microtubule binding and bundling activity. We conclude that PRC1 is a microtubule-associated protein required to maintain the spindle midzone, and that distinct functions are associated with modular elements of the primary sequence.

[1]  M. Jordan,et al.  Phase dynamics at microtubule ends: the coexistence of microtubule length changes and treadmilling , 1987, The Journal of cell biology.

[2]  C. Sellitto,et al.  Distribution of a matrix component of the midbody during the cell cycle in Chinese hamster ovary cells , 1988, The Journal of cell biology.

[3]  Y. Wang,et al.  Mechanism of the formation of contractile ring in dividing cultured animal cells. I. Recruitment of preexisting actin filaments into the cleavage furrow , 1990, The Journal of cell biology.

[4]  A. Murray,et al.  Cyclin is degraded by the ubiquitin pathway , 1991, Nature.

[5]  R. Margolis,et al.  Telophase disc: a new mammalian mitotic organelle that bisects telophase cells with a possible function in cytokinesis. , 1991, Journal of cell science.

[6]  A. Matus,et al.  Reorganisation of the microtubular cytoskeleton by embryonic microtubule-associated protein 2 (MAP2c). , 1992, Development.

[7]  B. Schaar,et al.  CENP-E is a putative kinetochore motor that accumulates just before mitosis , 1992, Nature.

[8]  Corey Nislow,et al.  A plus-end-directed motor enzyme that moves antiparallel microtubules in vitro localizes to the interzone of mitotic spindles , 1992, Nature.

[9]  T. Sasaki,et al.  Regulation of cytoplasmic division of Xenopus embryo by rho p21 and its inhibitory GDP/GTP exchange protein (rho GDI) , 1993, The Journal of cell biology.

[10]  D. Mastronarde,et al.  Interpolar spindle microtubules in PTK cells , 1993, The Journal of cell biology.

[11]  M. Gonzalez-Garay,et al.  Stable expression of heterologous microtubule-associated proteins (MAPs) in Chinese hamster ovary cells: evidence for differing roles of MAPs in microtubule organization , 1994, The Journal of cell biology.

[12]  M. Gatti,et al.  The Drosophila kinesin-like protein KLP3A is a midbody component required for central spindle assembly and initiation of cytokinesis , 1995, The Journal of cell biology.

[13]  C. Waterman-Storer,et al.  The p150Glued component of the dynactin complex binds to both microtubules and the actin-related protein centractin (Arp-1). , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[14]  R. Erikson,et al.  Plk is an M-phase-specific protein kinase and interacts with a kinesin-like protein, CHO1/MKLP-1 , 1995, Molecular and cellular biology.

[15]  G. Fink,et al.  Two microtubule-associated proteins required for anaphase spindle movement in Saccharomyces cerevisiae [published erratum appears in J Cell Biol 1995 Oct;131(2):561] , 1995, The Journal of cell biology.

[16]  G. Drewes,et al.  Structure, Microtubule Interactions, and Phosphorylation of Tau Protein a , 1996, Annals of the New York Academy of Sciences.

[17]  M. Hyvönen,et al.  T7 vectors with modified T7lac promoter for expression of proteins in Escherichia coli. , 1996, Analytical biochemistry.

[18]  Y. Wang,et al.  Signals from the spindle midzone are required for the stimulation of cytokinesis in cultured epithelial cells. , 1996, Molecular biology of the cell.

[19]  R. Ryo,et al.  The involvement of the rho gene product, a small molecular weight GTP-binding protein, in polyploidization of a human megakaryocytic cell line, CMK. , 1996, Experimental hematology.

[20]  J. Peters,et al.  APC-Mediated Proteolysis of Ase1 and the Morphogenesis of the Mitotic Spindle , 1997, Science.

[21]  I. Kiss,et al.  Mutation of a gene for a Drosophila kinesin-like protein, Klp38B, leads to failure of cytokinesis. , 1997, Journal of cell science.

[22]  M. Glotzer.  The mechanism and control of cytokinesis. , 1997, Current opinion in cell biology.

[23]  A. Hyman,et al.  A requirement for Rho and Cdc42 during cytokinesis in Xenopus embryos , 1997, Current Biology.

[24]  K. Fujisawa,et al.  Role of citron kinase as a target of the small GTPase Rho in cytokinesis , 1998, Nature.

[25]  J Hardin,et al.  Cytokinesis and midzone microtubule organization in Caenorhabditis elegans require the kinesin-like protein ZEN-4. , 1998, Molecular biology of the cell.

[26]  R. Margolis,et al.  Colocalization of TD-60 and INCENP throughout G2 and mitosis: evidence for their possible interaction in signalling cytokinesis , 1998, Chromosoma.

[27]  G. Wahl,et al.  PRC1: a human mitotic spindle-associated CDK substrate protein required for cytokinesis. , 1998, Molecular cell.

[28]  H. Saya,et al.  Roles of Rho-associated Kinase in Cytokinesis; Mutations in Rho-associated Kinase Phosphorylation Sites Impair Cytokinetic Segregation of Glial Filaments , 1998, The Journal of cell biology.

[29]  D. Eckley,et al.  A Dominant Mutant of Inner Centromere Protein (INCENP), a Chromosomal Protein, Disrupts Prometaphase Congression and Cytokinesis , 1998, The Journal of cell biology.

[30]  M. Tatsuka,et al.  AIM‐1: a mammalian midbody‐associated protein required for cytokinesis , 1998, The EMBO journal.

[31]  H. Bellen,et al.  pavarotti encodes a kinesin-like protein required to organize the central spindle and contractile ring for cytokinesis. , 1998, Genes & development.

[32]  G. Wiche,et al.  Novel Features of the Light Chain of Microtubule-associated Protein MAP1B: Microtubule Stabilization, Self Interaction, Actin Filament Binding, and Regulation by the Heavy Chain , 1998, The Journal of cell biology.

[33]  J. Wehland,et al.  Suppression of tubulin tyrosine ligase during tumor growth. , 1998, Journal of cell science.

[34]  D. Glover,et al.  Drosophila Polo Kinase Is Required for Cytokinesis , 1998, The Journal of cell biology.

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

[36]  J. Schwartz,et al.  Dynein motor regulation stabilizes interphase microtubule arrays and determines centrosome position , 1999, The EMBO journal.

[37]  J. Chan,et al.  The 65-kDa carrot microtubule-associated protein forms regularly arranged filamentous cross-bridges between microtubules. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[38]  Yu-Li Wang,et al.  The Small GTP-binding Protein Rho Regulates Cortical Activities in Cultured Cells during Division , 1999, The Journal of cell biology.

[39]  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.

[40]  J. Schumacher,et al.  The Aurora-related kinase AIR-2 recruits ZEN-4/CeMKLP1 to the mitotic spindle at metaphase and is required for cytokinesis , 2000, Current Biology.

[41]  C. Mollinari,et al.  Human Survivin Is a Kinetochore-Associated Passenger Protein , 2000, The Journal of cell biology.

[42]  D. Vaux,et al.  Survivin and the inner centromere protein INCENP show similar cell-cycle localization and gene knockout phenotype , 2000, Current Biology.

[43]  J. Spudich,et al.  Towards a molecular understanding of cytokinesis. , 2000, Trends in cell biology.

[44]  L. Tsai,et al.  Regulation of cytoplasmic dynein behaviour and microtubule organization by mammalian Lis1 , 2000, Nature Cell Biology.

[45]  M. Kirschner,et al.  The KEN box: an APC recognition signal distinct from the D box targeted by Cdh1. , 2000, Genes & development.

[46]  F. Barr,et al.  The Rab6‐binding kinesin, Rab6‐KIFL, is required for cytokinesis , 2000, The EMBO journal.

[47]  Samantha G. Zeitlin,et al.  Animal cytokinesis: Breaking up is hard to do , 2001, Current Biology.

[48]  T. Tuschl,et al.  Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells , 2001, Nature.

[49]  Sally P. Whea.  Midzone Microtubule Bundles Are Continuously Required for Cytokinesis in Cultured Epithelial Cells , 2002 .

[50]  Longzhi Cao.  Mechanism of the Formation of Contractile Ring in Dividing Cultured Animal Cells. II. Cortical Movement of Microinjected Actin Filaments , 2002 .

[51]  Y. Terada,et al.  CHO1, a mammalian kinesin-like protein, interacts with F-actin and is involved in the terminal phase of cytokinesis , 2002, The Journal of cell biology.

[52]  M. Glotzer,et al.  Central spindle assembly and cytokinesis require a kinesin-like protein/RhoGAP complex with microtubule bundling activity. , 2002, Developmental cell.