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The Homotetrameric Kinesin-5 KLP61F Preferentially Crosslinks Microtubules into Antiparallel Orientations

2008; Elsevier BV; Volume: 18; Issue: 23 Linguagem: Inglês

10.1016/j.cub.2008.10.026

1879-0445

Siet van den Wildenberg, Tao Li, Lukas C. Kapitein, Christoph F. Schmidt, Jonathan M. Scholey, Erwin J.G. Peterman,

Genomics and Chromatin Dynamics

The segregation of genetic material during mitosis is coordinated by the mitotic spindle, whose action depends upon the polarity patterns of its microtubules (MTs) [1Brust-Mascher I. Scholey J.M. Mitotic spindle dynamics in Drosophila.Int. Rev. Cytol. 2007; 259: 139-172Crossref PubMed Scopus (21) Google Scholar, 2Walczak C.E. Heald R. Mechanisms of mitotic spindle assembly and function.Int. Rev. Cytol. 2008; 265: 111-158Crossref PubMed Scopus (275) Google Scholar]. Homotetrameric mitotic kinesin-5 motors can crosslink and slide adjacent spindle MTs [3Cole D.G. Saxton W.M. Sheehan K.B. Scholey J.M. A "slow" homotetrameric kinesin-related motor protein purified from Drosophila embryos.J. Biol. Chem. 1994; 269: 22913-22916Abstract Full Text PDF PubMed Google Scholar, 4Kapitein L.C. Peterman E.J. Kwok B.H. Kim J.H. Kapoor T.M. Schmidt C.F. The bipolar mitotic kinesin Eg5 moves on both microtubules that it crosslinks.Nature. 2005; 435: 114-118Crossref PubMed Scopus (473) Google Scholar, 5Kashina A.S. Baskin R.J. Cole D.G. Wedaman K.P. Saxton W.M. Scholey J.M. A bipolar kinesin.Nature. 1996; 379: 270-272Crossref PubMed Scopus (292) Google Scholar, 6Sawin K.E. LeGuellec K. Philippe M. Mitchison T.J. Mitotic spindle organization by a plus-end-directed microtubule motor.Nature. 1992; 359: 540-543Crossref PubMed Scopus (520) Google Scholar, 7Tao L. Mogilner A. Civelekoglu-Scholey G. Wollman R. Evans J. Stahlberg H. Scholey J.M. A homotetrameric kinesin-5, KLP61F, bundles microtubules and antagonizes Ncd in motility assays.Curr. Biol. 2006; 16: 2293-2302Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 8Hildebrandt E.R. Gheber L. Kingsbury T. Hoyt M.A. Homotetrameric form of Cin8p, a Saccharomyces cerevisiae kinesin-5 motor, is essential for its in vivo function.J. Biol. Chem. 2006; 281: 26004-26013Crossref PubMed Scopus (38) Google Scholar, 9Valentine M.T. Fordyce P.M. Block S.M. Eg5 steps it up!.Cell Div. 2006; 1: 31Crossref PubMed Scopus (50) Google Scholar, 10Civelekoglu-Scholey G. Scholey J.M. Mitotic motors: Kinesin-5 takes a brake.Curr. Biol. 2007; 17: R544-R547Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar, 11Cottingham F.R. Gheber L. Miller D.L. Hoyt M.A. Novel roles for saccharomyces cerevisiae mitotic spindle motors.J. Cell Biol. 1999; 147: 335-350Crossref PubMed Scopus (98) Google Scholar], but it is unknown whether they or other motors contribute to establishing these MT polarity patterns. Here, we explored whether the Drosophila embryo kinesin-5 KLP61F, which plausibly crosslinks both parallel and antiparallel MTs [7Tao L. Mogilner A. Civelekoglu-Scholey G. Wollman R. Evans J. Stahlberg H. Scholey J.M. A homotetrameric kinesin-5, KLP61F, bundles microtubules and antagonizes Ncd in motility assays.Curr. Biol. 2006; 16: 2293-2302Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 12Sharp D.J. McDonald K.L. Brown H.M. Matthies H.J. Walczak C. Vale R.D. Mitchison T.J. Scholey J.M. The bipolar kinesin, KLP61F, cross-links microtubules within interpolar microtubule bundles of Drosophila embryonic mitotic spindles.J. Cell Biol. 1999; 144: 125-138Crossref PubMed Scopus (245) Google Scholar], displays a preference for parallel or antiparallel MT orientation. In motility assays, KLP61F was observed to crosslink and slide adjacent MTs, as predicted. Remarkably, KLP61F displayed a 3-fold higher preference for crosslinking MTs in the antiparallel orientation. This polarity preference was observed in the presence of ADP or ATP plus AMPPNP, but not AMPPNP alone, which induces instantaneous rigor binding. Also, a purified motorless tetramer containing the C-terminal tail domains displayed an antiparallel orientation preference, confirming that motor activity is not required. The results suggest that, during morphogenesis of the Drosophila embryo mitotic spindle, KLP61F's crosslinking and sliding activities could facilitate the gradual accumulation of KLP61F within antiparallel interpolar MTs at the equator, where the motor could generate force to drive poleward flux and pole-pole separation.