title: Association of CD2AP with dynamic actin on vesicles in podocytes.
abstract: The docking protein CD2AP (CD2-associated protein) serves a nonredundant function in podocytes as CD2AP knockout mice die of renal failure at the age of 6-7 wk. Furthermore, haploinsufficiency due to mutation of the CD2AP gene is associated with focal segmental glomerulosclerosis in humans. Although CD2AP has been shown to interact with proteins regulating actin polymerization, with proteins of the slit diaphragm, and with the endocytic machinery, its critical function in podocytes remains unclear. In conditionally immortalized mouse podocytes, we demonstrate that CD2AP colocalizes with cortactin and F-actin in spots of < or =0.5-microm diameter. Confocal time-lapse microscopy in living podocytes expressing GFP-CD2AP or GFP-actin revealed that spots are motile, possess a limited lifetime, and are frequently associated with vesicles. A significant portion of spot-associated vesicles belongs to a later endosomal-sorting compartment, characterized by delayed uptake of fluorescent dextran (10 kDa) and by colocalization with Rab4, but not Rab5 and AP-2. Rapid accumulation of microinjected G-actin in spots and abrogation of spot motility by jasplakinolide demonstrate that spot movements depend on actin polymerization. Furthermore, a high turnover (half-time < 10 s) of CD2AP in spots was demonstrated by FRAP (fluorescence recovery after photobleaching). Our results demonstrate that CD2AP is associated with dynamic actin in a specific late endosomal compartment in podocytes, suggesting that CD2AP might be crucially involved in endosomal sorting and/or trafficking via regulation of actin assembly on vesicles.
authors.affiliation: Department of Anatomy and Cell Biology, University of Heidelberg, D-69120 Heidelberg, Germany.
authors.forename: Thilo
authors.initials: T
authors.lastname: Welsch
authors.forename: Nicole
authors.initials: N
authors.lastname: Endlich
authors.forename: Gökmen
authors.initials: G
authors.lastname: Gökce
authors.forename: Elena
authors.initials: E
authors.lastname: Doroshenko
authors.forename: Jeremy C
authors.initials: JC
authors.lastname: Simpson
authors.forename: Wilhelm
authors.initials: W
authors.lastname: Kriz
authors.forename: Andrey S
authors.initials: AS
authors.lastname: Shaw
authors.forename: Karlhans
authors.initials: K
authors.lastname: Endlich
mesh_terms: D000199:Actins / Q000378:metabolism*; D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D018929:Cell Culture Techniques; D003598:Cytoskeletal Proteins; D011992:Endosomes / Q000502:physiology; D049452:Green Fluorescent Proteins / Q000378:metabolism; D007678:Kidney Glomerulus / Q000166:cytology* / Q000502:physiology*; D051379:Mice; D018613:Microscopy, Confocal; D011506:Proteins / Q000378:metabolism / Q000502:physiology*
chemical_list: D000199:Actins; D048868:Adaptor Proteins, Signal Transducing; C114545:CD2-associated protein; D003598:Cytoskeletal Proteins; D011506:Proteins; D049452:Green Fluorescent Proteins