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Vol. 20, Issue 21, 4531-4540, November 1, 2009

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Filamin A Regulates Caveolae Internalization and Trafficking in Endothelial Cells

Maria Sverdlov^*, Vasily Shinin^*, Aaron T. Place^*, Maricela Castellon^*,, and Richard D. Minshall^*,,

Departments of *Pharmacology and Anesthesiology and Center for Lung and Vascular Biology, University of Illinois at Chicago, Chicago, IL 60612

Submitted October 6, 2008; Revised July 31, 2009; Accepted September 9, 2009
Monitoring Editor: Sean Munro

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 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.

Address correspondence to: Richard D. Minshall (rminsh{at}uic.edu).

Abbreviations used: BSA, bovine serum albumin; Cav-1, caveolin-1; CTB, cholera toxin subunit B; DAPI, 4,6-diamidino-2-phenylindole; FLN, filamin; FRAP, fluorescence recovery after photobleaching; GFP, green fluorescent protein; HMVEC, human microvascular endothelial cell(s); Lat B, latrinculin B; PP2, 4-Amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine; TIR-FM, total internal reflection fluorescence microscopy; WT, wild type; YFP, yellow fluorescent protein.

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