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Constructing a mitotic spindle requires the coordinated actions of several kinesin motor proteins (e.g. Goshima and Vale, 2003). We visualized the dynamics of five green fluorescent protein (GFP)-tagged mitotic kinesins (class 5, 6, 8, 13, and 14) in live Drosophila Schneider cell line (S2), after first demonstrating that the GFP-tag does not interfere with the mitotic functions of these kinesins using an RNA interference (RNAi)-based rescue strategy.
Fig. 1 - Destabilization of cytoplasmic microtubules (bottom) by expression of Klp67A-GFP-NES (top). Klp67A-GFP-NES construct was transfected and transiently overexpressed, and the cells were fixed and stained with an anti-tubulin antibody. The cell with higher GFP intensity has fewer microtubules.
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 (Fig 1). 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]) seem 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 (Movie 1).
Movie 1 - Klp67A-GFP dynamics from metaphase to telophase. Images were taken every 5 sec by spinning-disk confocal microscope.
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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 (Fig 2).
Fig. 2 - Strategies to regulate mitotic kinesin activity in the cell cycle. (A) Nuclear sequestration of Klp67A or Ncd protects cytoplasmic microtubules from the undesired depolymerizing or cross-linking activities of these motors. Nuclear envelope breakdown enables the motors to perform their actions on microtubules. (B) Phosphorylation of Thr 933 residue by Cdc2 kinase is essential for Klp61F targeting to microtubules during mitosis. (C) Cdc2 phosphorylations around the motor domain of Pav prevent this motor for prematurely binding to the central spindle during metaphase. Dephosphorylation of these sites is required for central spindle targeting after anaphase. (D) Change of localization of Klp10A and Klp67A depolymerases after mitotic progression. Klp10A-GFP shows clear microtubule plus end tracking in interphase. Plus end tracking is still clearly detected during prophase, while centrosome/centromere localization also is seen. After nuclear envelope breakdown, plus end tracking becomes much less evident, and the majority of Klp10A localizes to centrosomes, centromeres, and interior pole regions. Klp67A enriched at the outer region of kinetochores during metaphase, followed by central spindle accumulation immediately upon anaphase onset.
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