Department of Genetic Disease Research, Osaka City University, Graduate School of Medicine, Asahi-machi 1-4-3, Abeno, Osaka, Japan.
Nat Commun. 2013;4:2033. doi: 10.1038/ncomms3033.
Cytoplasmic dynein drives the movement of a wide range of cargoes towards the minus ends of microtubules. We previously demonstrated that LIS1 forms an idling complex with dynein, which is transported to the plus ends of microtubules by kinesin motors. Here we report that the small GTPase Rab6a is essential for activation of idling dynein. Immunoprecipitation and microtubule pull-down assays reveal that the GTP bound mutant, Rab6a(Q72L), dissociates LIS1 from a LIS1-dynein complex, activating dynein movement in in vitro microtubule gliding assays. We monitor transient interaction between Rab6a(Q72L) and dynein in vivo using dual-colour fluorescence cross-correlation spectroscopy in dorsal root ganglion (DRG) neurons. Finally, we demonstrate that Rab6a(Q72L) mediates LIS1 release from a LIS1-dynein complex followed by dynein activation through an in vitro single-molecule assay using triple-colour quantum dots. Our findings reveal a surprising function for GTP bound Rab6a as an activator of idling dynein.
细胞质动力蛋白驱动着各种货物向微管的负端移动。我们之前的研究表明，LIS1 与动力蛋白形成空闲复合物，由驱动蛋白马达运输到微管的正端。在这里，我们报告小 GTPase Rab6a 对于激活空闲动力蛋白是必不可少的。免疫沉淀和微管下拉实验表明，结合 GTP 的突变体 Rab6a(Q72L)使 LIS1 从 LIS1-动力蛋白复合物中解离，在体外微管滑行实验中激活动力蛋白的运动。我们使用双荧光荧光相关光谱技术在背根神经节（DRG）神经元中监测 Rab6a(Q72L)与动力蛋白之间的瞬时相互作用。最后，我们通过使用三色量子点的体外单分子实验证明，Rab6a(Q72L)通过从 LIS1-动力蛋白复合物中释放 LIS1 来介导 LIS1 的释放，从而激活动力蛋白。我们的研究结果揭示了 GTP 结合的 Rab6a 作为空闲动力蛋白激活剂的惊人功能。
