Exophilin8 transiently clusters insulin granules at the actin-rich cell cortex prior to exocytosis
Exophilin8 transiently clusters insulin granules at the actin-rich cell cortex prior to exocytosis
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DOI：
10.1091/mbc.e10-05-0404
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发表时间：
2011-05-15
影响因子：
3.3
通讯作者：
Izumi, Tetsuro
中科院分区：
文献类型：
--
作者：
Mizuno, Kouichi;Ramalho, Jose S.;Izumi, Tetsuro
Exophilin8/MyRIP/Slac2-c 是小 GTP 酶 Rab27a 的效应蛋白，特异性定位于视网膜黑素体和分泌颗粒上。我们研究了 exophilin8 在胰岛素颗粒运输中的作用。胰腺β细胞或其细胞系MIN6中外源性表达exophilin8，细胞角处存在极化（exophilin8阳性）胰岛素颗粒，其中存在皮质肌动蛋白和微管正端结合蛋白EB1。突变分析表明，exophilin8 作为 Rab27a 和肌球蛋白 Va 之间的连接物的能力对其颗粒聚类活性至关重要。此外，exophilin8 和 exophilin8 相关胰岛素颗粒明显稳定且不可移动。全内反射荧光显微镜表明，exophilin8 限制胰岛素颗粒运动的区域比另一种 Rab27a 效应子 granuphilin 积累的区域更深，该区域直接附着在质膜上。然而，外亲蛋白8诱导的胰岛素颗粒的不动性在促分泌素刺激后被消除，并且不抑制诱发的胞吐作用。此外，exophilin8 的消耗会阻止胰岛素颗粒被运输到质膜附近并抑制它们的融合。这些发现表明，exophilin8 短暂地将胰岛素颗粒捕获到靠近微管正端的皮质肌动蛋白网络中，并在刺激期间供其释放。
Exophilin8/MyRIP/Slac2-c is an effector protein of the small GTPase Rab27a and is specifically localized on retinal melanosomes and secretory granules. We investigated the role of exophilin8 in insulin granule trafficking. Exogenous expression of exophilin8 in pancreatic beta cells or their cell line, MIN6, polarized (exophilin8-positive) insulin granules at the cell corners, where both cortical actin and the microtubule plus-end-binding protein, EB1, were present. Mutation analyses indicated that the ability of exophilin8 to act as a linker between Rab27a and myosin Va is essential for its granule-clustering activity. Moreover, exophilin8 and exophilin8-associated insulin granules were markedly stable and immobile. Total internal reflection fluorescence microscopy indicated that exophilin8 restricts the motion of insulin granules at a region deeper than that where another Rab27a effector, granuphilin, accumulates docked granules directly attached to the plasma membrane. However, the exophilin8-induced immobility of insulin granules was eliminated upon secretagogue stimulation and did not inhibit evoked exocytosis. Furthermore, exophilin8 depletion prevents insulin granules from being transported close to the plasma membrane and inhibits their fusion. These findings indicate that exophilin8 transiently traps insulin granules into the cortical actin network close to the microtubule plus-ends and supplies them for release during the stimulation.