title: Filamin A regulates caveolae internalization and trafficking in endothelial cells.
abstract: 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 approximately 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.
authors.affiliation: Department of Pharmacology, University of Illinois at Chicago, Chicago, IL 60612, USA.
authors.forename: Maria
authors.initials: M
authors.lastname: Sverdlov
authors.forename: Vasily
authors.initials: V
authors.lastname: Shinin
authors.forename: Aaron T
authors.initials: AT
authors.lastname: Place
authors.forename: Maricela
authors.initials: M
authors.lastname: Castellon
authors.forename: Richard D
authors.initials: RD
authors.lastname: Minshall
mesh_terms: D000199:Actins / Q000378:metabolism; D022005:Adherens Junctions / Q000378:metabolism; D000818:Animals; D001692:Biological Transport / Q000502:physiology; D021941:Caveolae / Q000378:metabolism*; D051242:Caveolin 1 / Q000235:genetics / Q000378:metabolism; D002478:Cells, Cultured; D003285:Contractile Proteins / Q000235:genetics / Q000378:metabolism*; D003599:Cytoskeleton / Q000378:metabolism; D004705:Endocytosis / Q000502:physiology; D042783:Endothelial Cells / Q000166:cytology / Q000378:metabolism*; D064448:Filamins; D036681:Fluorescence Recovery After Photobleaching; D006801:Humans; D008168:Lung / Q000098:blood supply; D051379:Mice; D018345:Mice, Knockout; D008840:Microfilament Proteins / Q000235:genetics / Q000378:metabolism*; D008856:Microscopy, Fluorescence / Q000379:methods; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D000199:Actins; D051242:Caveolin 1; D003285:Contractile Proteins; D064448:Filamins; D008840:Microfilament Proteins; D034741:RNA, Small Interfering; D011993:Recombinant Fusion Proteins