Impairing actin filament or syndapin functions promotes accumulation of clathrin-coated vesicles at the apical plasma membrane of acinar epithelial cells
Impairing actin filament or syndapin functions promotes accumulation of clathrin-coated vesicles at the apical plasma membrane of acinar epithelial cells
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DOI：
10.1091/mbc.e03-05-0315
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发表时间：
2003-11-01
影响因子：
3.3
通讯作者：
Hamm-Alvarez, SF
中科院分区：
文献类型：
--
作者：
da Costa, SR;Sou, E;Hamm-Alvarez, SF
在本文中，我们研究了肌动蛋白丝和辅助蛋白对原代兔泪腺腺泡顶端网格蛋白介导的内吞作用的贡献。共聚焦荧光和电子显微镜显示，细胞松弛素 D 促进了网格蛋白、α-适应蛋白、动力蛋白和 F-肌动蛋白在顶端的积累，并增加了顶端质膜上包被的凹坑和囊泡的数量。膜区室的山梨醇密度梯度分析表明，细胞松弛素 D 增加了 [C-14] 葡聚糖与受刺激腺泡顶膜的关联，这与顶端内吞作用的功能性抑制一致。重组 syndapin SH3 结构域与泪腺腺泡动力蛋白、神经元 Wiskott-Aldrich 综合征蛋白 (N-WASP) 和 synaptojanin 相互作用；通过电穿孔引入它们，引起了网格蛋白、辅助蛋白和顶端质膜上的包被凹坑的显着积累。这些 SH3 结构域还显着（p 小于或等于 0.05）增加 F-肌动蛋白，动力蛋白和 N-WASP 与附加细丝显着共定位。与 N-WASP 的 VCA 结构域共电穿孔可阻断 F-肌动蛋白的增加，并逆转表明顶端内吞作用受损的形态变化。我们认为，通过 N-WASP 激活 Arp2/3 复合物，联苯平 (syndapin) 对肌动蛋白聚合的瞬时调节协调了腺泡上皮顶端质膜处的动力介导的囊泡分裂。在此过程中，细胞松弛素 D 或联苯平 SH3 结构域捕获组装的 F-肌动蛋白中间体会损害内吞作用。
In this article, we investigate the contributions of actin filaments and accessory proteins to apical clathrin-mediated endocytosis in primary rabbit lacrimal acini. Confocal fluorescence and electron microscopy revealed that cytochalasin D promoted apical accumulation of clathrin, a-adaptin, dynamin, and F-actin and increased the amounts of coated pits and vesicles at the apical plasma membrane. Sorbitol density gradient analysis of membrane compartments showed that cytochalasin D increased [C-14]dextran association with apical membranes from stimulated acini, consistent with functional inhibition of apical endocytosis. Recombinant syndapin SH3 domains interacted with lacrimal acinar dynamin, neuronal Wiskott-Aldrich Syndrome protein (N-WASP), and synaptojanin; their introduction by electroporation elicited remarkable accumulation of clathrin, accessory proteins, and coated pits at the apical plasma membrane. These SH3 domains also significantly (p less than or equal to 0.05) increased F-actin, with substantial colocalization of dynamin and N-WASP with the additional filaments. Coelectroporation with the VCA domain of N-WASP blocked the increase in F-actin and reversed the morphological changes indicative of impaired apical endocytosis. We suggest that transient modulation of actin polymerization by syndapins through activation of the Arp2/3 complex via N-WASP coordinates dynamin-mediated vesicle fission at the apical plasma membrane of acinar epithelia. Trapping of assembled F-actin intermediates during this process by cytochalasin D or syndapin SH3 domains impairs endocytosis.