Filamin A Regulates Caveolae Internalization and Trafficking in Endothelial Cells
Filamin A Regulates Caveolae Internalization and Trafficking in Endothelial Cells
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DOI：
10.1091/mbc.e08-10-0997
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发表时间：
2009-11-01
影响因子：
3.3
通讯作者：
Minshall, Richard D.
中科院分区：
文献类型：
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作者：
Sverdlov, Maria;Shinin, Vasily;Minshall, Richard D.
通过小凹的转胞吞作用对于通过调节大分子、激素和脂质的组织递送来维持血管稳态至关重要。在本研究中，我们测试了以下假设：F-肌动蛋白交联蛋白 filamin A 和 Caveolin-1 之间的相互作用促进小凹的内化和运输。小干扰RNA介导的细丝蛋白A（而非细丝蛋白B）的敲低，使白蛋白的摄取和转胞吞作用分别减少了大约35%和60%，而不改变肌动蛋白细胞骨架结构或细胞-细胞粘附连接。在细丝蛋白 A 表达减少的细胞中，通过光漂白后的荧光恢复测量的细胞内 Caveolin-1-绿色荧光蛋白 (GFP) 标记的囊泡和通过全内反射荧光显微镜测量的膜相关囊泡的迁移率均降低。此外，在缺乏细丝蛋白A的黑色素瘤细胞（M2细胞）中，大多数caveolin-1-GFP位于质膜上，而在重构细丝蛋白A表达的细胞（用细丝蛋白A-RFP转染的A7细胞和M2细胞）中，caveolin-1-GFP集中在细胞内囊泡中。通过密度梯度中这些蛋白质的共分级以及免疫共沉淀证实了细丝蛋白 A 与内皮细胞中的 Caveolin-1 的关联。此外，这种相互作用通过 Src 激活而增强，与 Caveolin-1 磷酸化增加相关，并通过 Src 抑制而阻断。综上所述，这些数据表明细丝蛋白 A 与 Caveolin-1 的结合通过调节囊泡内化、聚集和运输来促进小凹介导的运输。
Transcytosis via caveolae is critical for maintaining vascular homeostasis by regulating the tissue delivery of macromolecules, hormones, and lipids. In the present study, we test the hypothesis that interactions between F-actin cross-linking protein filamin A and caveolin-1 facilitate the internalization and trafficking of caveolae. Small interfering RNA-mediated knockdown of filamin A, but not filamin B, reduced the uptake and transcytosis of albumin by similar to 35 and 60%, respectively, without altering the actin cytoskeletal structure or cell-cell adherens junctions. Mobility of both intracellular caveolin-1-green fluorescent protein (GFP)-labeled vesicles measured by fluorescence recovery after photobleaching and membrane-associated vesicles measured by total internal reflection-fluorescence microscopy was decreased in cells with reduced filamin A expression. In addition, in melanoma cells that lack filamin A (M2 cells), the majority of caveolin-1-GFP was localized on the plasma membrane, whereas in cells in which filamin A expression was reconstituted (A7 cells and M2 cells transfected with filamin A-RFP), caveolin-1-GFP was concentrated in intracellular vesicles. Filamin A association with caveolin-1 in endothelial cells was confirmed by cofractionation of these proteins in density gradients, as well as by coimmunoprecipitation. Moreover, this interaction was enhanced by Src activation, associated with increased caveolin-1 phosphorylation, and blocked by Src inhibition. Taken together, these data suggest that filamin A association with caveolin-1 promotes caveolae-mediated transport by regulating vesicle internalization, clustering, and trafficking.