Cell cycle-dependent dynamics and regulation of mitotic kinesins in Drosophila S2 cells
Cell cycle-dependent dynamics and regulation of mitotic kinesins in Drosophila S2 cells
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DOI：
10.1091/mbc.e05-02-0118
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发表时间：
2005-08-01
影响因子：
3.3
通讯作者：
Vale, RD
中科院分区：
文献类型：
--
作者：
Goshima, G;Vale, RD
构建有丝分裂纺锤体需要几个驱动蛋白马达蛋白的协调作用。在这里，我们已经可视化了五个绿色荧光蛋白（GFP）标记的有丝分裂驱动蛋白（类5，6，8，13，和14）在活的果蝇Schneider细胞系（S2）的动力学，在第一次证明，GFP标签不干扰这些驱动蛋白的有丝分裂功能，使用RNA干扰（RNAi）为基础的救援策略。8类（Klp67A）和14类（Ncd）驱动蛋白在间期期间以活性形式被隔离在细胞核中，并在核膜破裂（NEB）时与它们的微管靶结合。Klp67 A的细胞质中使用的核输出信号的重新定位导致在分裂间期微管阵列，提供支持的假设，这类驱动蛋白具有微管不稳定的活动。另一方面，驱动蛋白-5（Klp61F）和-6（Pavarotti）与微管的相互作用分别通过Cdc2磷酸化激活和失活，如通过检测突变Cdc2共有位点后的定位所示。微管不稳定驱动蛋白（8类和13类[KIp10A]）的作用似乎是由细胞周期依赖性变化控制的。KIp10A，集中在微管加两端的间期和前期，重新定位到着丝粒和纺锤体极后NEB和保持在这些网站在整个后期。与这种定位一致，RNAi分析表明，这种驱动蛋白有助于后期A期间染色体向极的移动。Klp67 A也成为NEB上相关的动粒，但大多数群体通过后期A开始的时间重新定位到中央纺锤体，这与我们的RNAi结果一致，该结果显示耗尽该马达对后期A没有影响。这些结果揭示了一个不同的频谱的调控机制控制的定位和功能的有丝分裂驱动蛋白在细胞周期的不同阶段。
Constructing a mitotic spindle requires the coordinated actions of several kinesin motor proteins. Here, we have 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. 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 [KIp10A]) seem to be controlled by cell cycle-dependent changes in their localizations. KIp10A, 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.