The molecular function of Ase1p

The midzone is the domain of the mitotic spindle that maintains spindle bipolarity during anaphase and generates forces required for spindle elongation (anaphase B). Although there is a clear role for microtubule (MT) motor proteins at the spindle midzone, less is known about how microtubule-associated proteins (MAPs) contribute to midzone organization and function. Here, we report that budding yeast Ase1p is a member of a conserved family of midzone-specific MAPs. By size exclusion chromatography and velocity sedimentation, both Ase1p in extracts and purified Ase1p behaved as a homodimer. Ase1p bound and bundled MTs in vitro. By live cell microscopy, loss of Ase1p resulted in a specific defect: premature spindle disassembly in mid-anaphase. Furthermore, when overexpressed, Ase1p was sufficient to trigger spindle elongation in S phase–arrested cells. FRAP revealed that Ase1p has both a very slow rate of turnover within the midzone and limited lateral diffusion along spindle MTs. We propose that Ase1p functions as an MT cross-bridge that imparts matrix-like characteristics to the midzone. MT-dependent networks of spindle midzone MAPs may be one molecular basis for the postulated spindle matrix.

[1]  H. Yin,et al.  Stu1p is physically associated with beta-tubulin and is required for structural integrity of the mitotic spindle. , 2002, Molecular biology of the cell.

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

[3]  J. Mcintosh The roles of microtubules in chromosome movement , 1994 .

[4]  M. Hoyt,et al.  A new view of the spindle checkpoint , 2001, The Journal of cell biology.

[5]  R. Cross,et al.  Kinetic evidence for low chemical processivity in ncd and Eg5. , 1997, Journal of molecular biology.

[6]  R. Baskin,et al.  A bipolar kinesin , 1996, Nature.

[7]  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.

[8]  M. Berns,et al.  Direct experimental evidence for the existence, structural basis and function of astral forces during anaphase B in vivo. , 1991, Journal of cell science.

[9]  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.

[10]  M. Kirschner,et al.  Tau protein binds to microtubules through a flexible array of distributed weak sites , 1991, The Journal of cell biology.

[11]  T. Hunter,et al.  PRC1 is a microtubule binding and bundling protein essential to maintain the mitotic spindle midzone , 2002, The Journal of cell biology.

[12]  Peter K Hepler,et al.  Cytokinesis in Higher Plants , 1996, Cell.

[13]  T. Huffaker,et al.  Astral microtubules are not required for anaphase B in Saccharomyces cerevisiae , 1992, The Journal of cell biology.

[14]  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.

[15]  J W Sedat,et al.  Mitosis in living budding yeast: anaphase A but no metaphase plate. , 1997, Science.

[16]  S F Altschul,et al.  Local alignment statistics. , 1996, Methods in enzymology.

[17]  Thomas L. Madden,et al.  BLAST 2 Sequences, a new tool for comparing protein and nucleotide sequences. , 1999, FEMS microbiology letters.

[18]  P. Hussey,et al.  A new class of microtubule-associated proteins in plants , 2000, Nature Cell Biology.

[19]  J. Pickett-Heaps,et al.  Traction fibre: toward a "tensegral" model of the spindle. , 1997, Cell motility and the cytoskeleton.

[20]  G. Lamers,et al.  The Saccharomyces cerevisiae Fin1 protein forms cell cycle-specific filaments between spindle pole bodies , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[21]  M W Berns,et al.  Mechanics of chromosome separation during mitosis in Fusarium (Fungi imperfecti): new evidence from ultrastructural and laser microbeam experiments , 1981, The Journal of cell biology.

[22]  M. Hoyt,et al.  Cell cycle-dependent degradation of the Saccharomyces cerevisiae spindle motor Cin8p requires APC(Cdh1) and a bipartite destruction sequence. , 2001, Molecular biology of the cell.

[23]  E. Salmon,et al.  Spindle dynamics and cell cycle regulation of dynein in the budding yeast, Saccharomyces cerevisiae , 1995, The Journal of cell biology.

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

[25]  Frank Uhlmann,et al.  Orchestrating anaphase and mitotic exit: separase cleavage and localization of Slk19 , 2001, Nature Cell Biology.

[26]  I. Hagan,et al.  The S. pombe aurora-related kinase Ark1 associates with mitotic structures in a stage dependent manner and is required for chromosome segregation. , 2001, Journal of cell science.

[27]  Shmuel Pietrokovski,et al.  Increased coverage of protein families with the Blocks Database servers , 2000, Nucleic Acids Res..

[28]  David Pellman,et al.  Activity of the APCCdh1 form of the anaphase-promoting complex persists until S phase and prevents the premature expression of Cdc20p , 2001, The Journal of cell biology.

[29]  A. Hyman,et al.  Correct spindle elongation at the metaphase/anaphase transition is an APC-dependent event in budding yeast , 2001, The Journal of cell biology.

[30]  T. Mitchison,et al.  The Bipolar Kinesin, KLP61F, Cross-links Microtubules within Interpolar Microtubule Bundles of Drosophila Embryonic Mitotic Spindles , 1999, The Journal of cell biology.

[31]  B. Trus,et al.  The cytoplasmic matrix: its volume and surface area and the diffusion of molecules through it. , 1985, Proceedings of the National Academy of Sciences of the United States of America.

[32]  A. Straight,et al.  Microtubules, membranes and cytokinesis , 2000, Current Biology.

[33]  M. Hoyt,et al.  Mitotic motors in Saccharomyces cerevisiae. , 2000, Biochimica et biophysica acta.

[34]  D. Higgins,et al.  T-Coffee: A novel method for fast and accurate multiple sequence alignment. , 2000, Journal of molecular biology.

[35]  Clarence S. M. Chan,et al.  Sli15 Associates with the Ipl1 Protein Kinase to Promote Proper Chromosome Segregation in Saccharomyces cerevisiae , 1999, The Journal of cell biology.

[36]  D. Herlyn,et al.  Expression of an antigen homologous to the human CO17-1A/GA733 colon cancer antigen in animal tissues. , 1997, British Journal of Cancer.

[37]  M. Balasubramanian,et al.  Schizosaccharomyces pombe Bir1p, a nuclear protein that localizes to kinetochores and the spindle midzone, is essential for chromosome condensation and spindle elongation during mitosis. , 2002, Genetics.

[38]  K. Jacobson,et al.  The translational mobility of substances within the cytoplasmic matrix. , 1984, Proceedings of the National Academy of Sciences of the United States of America.

[39]  K. Loo,et al.  Two Saccharomyces cerevisiae kinesin-related gene products required for mitotic spindle assembly , 1992, The Journal of cell biology.

[40]  K. J. Monty,et al.  Determination of molecular weights and frictional ratios of proteins in impure systems by use of gel filtration and density gradient centrifugation. Application to crude preparations of sulfite and hydroxylamine reductases. , 1966, Biochimica et biophysica acta.

[41]  D. Roof,et al.  Degradation of the kinesin Kip1p at anaphase onset is mediated by the anaphase-promoting complex and Cdc20p , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[42]  G. Goshima,et al.  Bir1/Cut17 moving from chromosome to spindle upon the loss of cohesion is required for condensation, spindle elongation and repair , 2001, Genes to cells : devoted to molecular & cellular mechanisms.

[43]  Charles Boone,et al.  Control of Mitotic Spindle Position by the Saccharomyces cerevisiae Formin Bni1p , 1999, The Journal of cell biology.

[44]  D. Pellman,et al.  Analysis of the size and shape of protein complexes from yeast. , 2002, Methods in enzymology.

[45]  J. B. Rattner,et al.  Mapping the mammalian intercellular bridge. , 1992, Cell motility and the cytoskeleton.

[46]  G. Fink,et al.  Methods in yeast genetics , 1979 .

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

[48]  Timothy J. Mitchison,et al.  Eg5 is static in bipolar spindles relative to tubulin , 2001, The Journal of cell biology.

[49]  D N Mastronarde,et al.  Three-dimensional ultrastructural analysis of the Saccharomyces cerevisiae mitotic spindle , 1995, The Journal of cell biology.

[50]  Peter A. Jones,et al.  Identification of DNA methylation differences during tumorigenesis by methylation-sensitive arbitrarily primed polymerase chain reaction. , 2002, Methods.

[51]  T. Mitchison,et al.  Mitosis: a history of division , 2001, Nature Cell Biology.

[52]  K. Bloom Yeast weighs in on the elusive spindle matrix: New filaments in the nucleus , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[53]  Kim Nasmyth,et al.  Cleavage of Cohesin by the CD Clan Protease Separin Triggers Anaphase in Yeast , 2000, Cell.

[54]  J. McIntosh,et al.  Three-dimensional reconstruction and analysis of mitotic spindles from the yeast, Schizosaccharomyces pombe , 1993, The Journal of cell biology.

[55]  M. Berns,et al.  Astral and spindle forces in PtK2 cells during anaphase B: a laser microbeam study. , 1993, Journal of cell science.

[56]  J. White,et al.  A genetic screen for temperature-sensitive cell-division mutants of Caenorhabditis elegans. , 1998, Genetics.

[57]  M. Hoyt,et al.  Kinesin-related proteins required for structural integrity of the mitotic spindle , 1992, Cell.

[58]  Timothy J. Mitchison,et al.  Kin I Kinesins Are Microtubule-Destabilizing Enzymes , 1999, Cell.

[59]  W. Earnshaw,et al.  Chromosomal passengers and the (aurora) ABCs of mitosis. , 2001, Trends in cell biology.

[60]  A. Lupas,et al.  Predicting coiled coils from protein sequences , 1991, Science.

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

[62]  S. Kuo,et al.  Motile Properties of the Kinesin-related Cin8p Spindle Motor Extracted from Saccharomyces cerevisiae Cells* , 1999, The Journal of Biological Chemistry.

[63]  J. McIntosh,et al.  Model for Mitosis , 1969, Nature.

[64]  S. Feinstein,et al.  Identification of a novel microtubule binding and assembly domain in the developmentally regulated inter-repeat region of tau , 1994, The Journal of cell biology.

[65]  A. Murray,et al.  Time-Lapse Microscopy Reveals Unique Roles for Kinesins during Anaphase in Budding Yeast , 1998, The Journal of cell biology.

[66]  J. R. McIntosh,et al.  Anaphase motions in dilute colchicine. Evidence of two phases in chromosome segregation. , 1973, Experimental cell research.

[67]  E. Salmon,et al.  The polarity and dynamics of microtubule assembly in the budding yeast Saccharomyces cerevisiae , 1999, Nature Cell Biology.

[68]  J. McIntosh,et al.  On the mechanism of anaphase spindle elongation in Diatoma vulgare , 1977, The Journal of cell biology.

[69]  E. O'Toole,et al.  Yeast Bim1p Promotes the G1-specific Dynamics of Microtubules , 1999, The Journal of cell biology.

[70]  G. C. Rogers,et al.  Mitosis, microtubules, and the matrix , 2001, The Journal of cell biology.

[71]  G. Lawler,et al.  Effect of cytoskeletal geometry on intracellular diffusion. , 1989, Biophysical journal.

[72]  K. Johansen,et al.  Skeletor, a Novel Chromosomal Protein That Redistributes during Mitosis Provides Evidence for the Formation of a Spindle Matrix , 2000, The Journal of cell biology.

[73]  P. Silver,et al.  Kinetics of spindle pole body separation in budding yeast. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[74]  G. C. Rogers,et al.  Microtubule motors in mitosis , 2000, Nature.