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A more recent version of this article appeared on August 1, 2005
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Submitted on February 11, 2005
Revised on May 31, 2005
Accepted on June 3, 2005
The Howard Hughes Medical Institute and the Department of Cellular and Molecular Pharmacology, University of California-San Francisco, CA 94107
Monitoring Editor: Ted Salmon
Constructing a mitotic spindle requires the coordinated actions of several kinesin motor proteins. Here, we have visualized the dynamics of five GFP-tagged mitotic kinesins (class 5,6,8,13,14) in live Drosophila S2 cells, after first demonstrating that the GFP-tag does not interfere with the mitotic functions of these kinesins using an RNAi-based rescue strategy. Class 8 (Klp67A) and class 14 (Ncd) kinesin are sequestered in an active form in the nucleus during interphase and engage their microtubule targets upon nuclear envelope breakdown (NEB). Relocalization of Klp67A to the cytoplasm using a nuclear export signal resulted in the disassembly of the interphase microtubule array, providing support for the hypothesis that this kinesin class possesses microtubule destabilizing activity. The interactions of Kinesin-5 (Klp61F) and -6 (Pavarotti) with microtubules, on the other hand, are activated and inactivated by Cdc2 phosphorylation respectively, as shown by examining localization after mutating Cdc2 consensus sites. The actions of microtubule destabilizing kinesins (class 8 and 13 (Klp10A)) appear to be controlled by cell cycle -dependent changes in their localizations. Klp10A, concentrated on microtubule plus ends in interphase and prophase, relocalizes to centromeres and spindle poles upon NEB and remains at these sites throughout anaphase. Consistent with this localization, RNAi analysis showed that this kinesin contributes to chromosome-to-pole movement during anaphase A. Klp67A also becomes kinetochore-associated upon NEB, but the majority of the population relocalizes to the central spindle by the timing of anaphase A onset, consistent with our RNAi result showing no effect of depleting this motor on anaphase A. These results reveal a diverse spectrum of regulatory mechanisms for controlling the localization and function of five mitotic kinesins at different stages of the cell cycle.
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