The rate of bipolar spindle assembly depends on the microtubule-gliding velocity of the mitotic kinesin Eg5.
During early embryonic cycles, the time required for mitotic spindle assembly must match the autonomous cell cycle oscillations because a lack of coordination between these two processes will result in chromosome segregation errors. Members of the widely conserved BimC kinesin family are essential for spindle formation in all eukaryotes, and complete loss of BimC function results in monopolar spindles that have two spindle poles that are not separated. However, the precise roles of BimC motor activity in the spindle assembly process are not known. To examine the contribution of BimC kinesin's motor activity to spindle assembly, we generated and characterized mutants of Eg5, a vertebrate BimC kinesin, with reduced in vitro microtubule-gliding velocities. In Xenopus egg extracts, we replaced endogenous Eg5 with recombinant wild-type or mutant motor proteins. By using centrosome-dependent and centrosome-independent spindle assembly assays, we found that mechanisms that determine spindle size and shape were robust to approximately 6-fold reductions in Eg5 motility. However, the spindle assembly process was slower when Eg5 motor function was impaired. This role of Eg5 was independent of its contribution to centrosome separation. We provide evidence that Eg5 is a rate-limiting component of the cellular machinery that drives spindle assembly in vertebrates.
Rockefeller University, 1230 York Avenue, New York, NY 10021, USA.
Benjamin H
BH
Kwok
Janet G
JG
Yang
Tarun M
TM
Kapoor
D000818:Animals; D018385:Centrosome / Q000378:metabolism; D016547:Kinesins / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D008870:Microtubules / Q000378:metabolism*; D016254:Mutagenesis, Insertional; D008941:Spindle Apparatus / Q000378:metabolism* / Q000502:physiology; D014981:Xenopus / Q000196:embryology*; D029867:Xenopus Proteins / Q000378:metabolism*
C088335:KIF11 protein, Xenopus; D029867:Xenopus Proteins; D016547:Kinesins