Arf6 Regulation of Gyrating-Clathrin

'Gyrating-' or 'G'-clathrin are coated endocytic structures located near peripheral sorting endosomes (SEs), which exhibit highly dynamic but localized movements when visualized by live-cell microscopy. They have been implicated in recycling of transferrin from the sorting endosome directly to the cell surface, but there is no information about their formation or regulation. We show here that G-clathrin comprise a minority of clathrin-coated structures in the cell periphery and are brefeldin A (BFA)-resistant. Arf6-GTP substantially increases G-clathrin levels, probably by lengthening coated bud lifetimes as suggested by photobleaching and photoactivation results, and an Arf6(Q67L)-GTP mutant bearing an internal GFP tag can be directly visualized in G-clathrin structures in live cells. Upon siRNA-mediated depletion of Arf6 or expression of Arf6(T27N), G-clathrin levels rise and are primarily Arf1-dependent, yet still BFA-resistant. However, BFA-sensitive increased G-clathrin levels are observed upon acute incubation with cytohesin inhibitor SecinH3, indicating a shift in GEF usage. Depletion of both Arf6 and Arf1 abolishes G-clathrin, and results in partial inhibition of fast transferrin recycling consistent with the latter's participation in this pathway. Collectively, these results demonstrate that the dynamics of G-clathrin primarily requires completion of the Arf6 guanine nucleotide cycle, but can be regulated by multiple Arf and GEF proteins, reflecting both overlapping mechanisms operative in their regulation and the complexity of processes involved in endosomal sorting.