Rab3A and Rab27A cooperatively regulate the docking step of dense-core vesicle exocytosis in PC12 cells
Rab3A and Rab27A cooperatively regulate the docking step of dense-core vesicle exocytosis in PC12 cells
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DOI：
10.1242/jcs.02962
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发表时间：
2006-06-01
影响因子：
4
通讯作者：
Fukuda, Mitsunori
中科院分区：
文献类型：
--
作者：
Tsuboi, Takashi;Fukuda, Mitsunori
最近的研究表明，Rab 3A和Rab 27 A这两种小的GTP酶在内分泌细胞中致密核心囊泡胞吐的后期步骤中起关键作用;然而，这两种GTP酶调节致密核心囊泡胞吐的确切机制以及它们之间的功能关系都不清楚.在这项研究中，我们在PC 12细胞中表达了许多不同的Rab蛋白，从Rab 1到Rab 41，并系统地筛选了那些特异性定位于致密核心囊泡上的Rab蛋白。我们发现四种Rabs（Rab 3A、Rab 27 A、Rab 33 A、Rab 37）主要靶向PC 12细胞中的致密核心囊泡，并且其中三种Rabs（Rab 3A、Rab 27 A、Rab 33 A）在致密核心囊泡上内源性表达。我们进一步研究了在单个PC 12细胞中通过全内反射荧光显微镜用特异性小干扰RNA沉默每个Rab对囊泡动力学的影响。在PC 12细胞中沉默Rab 3A或Rab 27 A显著减少了与质膜对接的致密核心囊泡的数量，而不改变单个胞吐事件的动力学，而Rab 33 A的沉默根本没有影响。Rab 3A和Rab 27 A的同时沉默导致停靠在质膜上的囊泡数量显著减少。我们的研究结果表明Rab 3A和Rab 27 A协同调节致密核心囊泡与质膜的对接步骤。
Recent studies have suggested that two small GTPases, Rab3A and Rab27A, play a key role in the late steps of dense- core vesicle exocytosis in endocrine cells; however, neither the precise mechanisms by which these two GTPases regulate dense- core vesicle exocytosis nor the functional relationship between them is clear. In this study, we expressed a number of different Rab proteins, from Rab1 to Rab41 in PC12 cells and systematically screened them for those that are specifically localized on dense- core vesicles. We found that four Rabs ( Rab3A, Rab27A, Rab33A, Rab37) are predominantly targeted to dense- core vesicles in PC12 cells, and that three of them ( Rab3A, Rab27A, Rab33A) are endogenously expressed on densecore vesicles. We further investigated the effect of silencing each Rab with specific small interfering RNA on vesicle dynamics by total internal reflection fluorescence microscopy in a single PC12 cell. Silencing either Rab3A or Rab27A in PC12 cells significantly decreased the number of dense- core vesicles docked to the plasma membrane without altering the kinetics of individual exocytotic events, whereas silencing of Rab33A had no effect at all. Simultaneous silencing of Rab3A and Rab27A caused a significantly greater decrease in number of vesicles docked to the plasma membrane. Our findings indicate that Rab3A and Rab27A cooperatively regulate docking step( s) of dense- core vesicles to the plasma membrane.