ARF6 requirement for Rac ruffling suggests a role for membrane trafficking in cortical actin rearrangements.

The ARF6 GTPase regulates a novel endosomal-plasma membrane recycling pathway and influences cortical actin remodeling. Here we examined the relationship between ARF6 and Rac1, a Rho family GTPase, implicated in cortical actin rearrangements. Endogenous Rac1 colocalized with ARF6 at the plasma membrane and on the ARF6 recycling endosome in untransfected HeLa and primary human fibroblast cells. In transfected HeLa cells Rac1 and ARF6 also colocalized. Cells expressing wild-type ARF6 or Rac1 formed actin-containing surface protrusions and membrane ruffles, respectively, upon treatment with the G protein activator aluminum fluoride. Aluminum fluoride-treatment of cells transfected with equivalent amounts of plasmid resulted in enhanced membrane ruffling, with protrusions appearing as Rac expression was lowered. Co-expression of the dominant negative, GTP binding-defective ARF6 T27N mutant inhibited the aluminum fluoride-induced ruffling observed in cells expressing Rac1, and the constitutive ruffling observed in cells expressing the activated Rac1 Q61L mutant. In contrast, co-expression of the GTP-binding-defective, T17N mutant of either Rac1 or Cdc42 with ARF6 did not inhibit the aluminum fluoride-induced surface protrusions, nor did inactivation of Rho with C3-transferase. These observations suggest that ARF6, a non-Rho family GTPase, can, by itself, alter cortical actin and can influence the ability of Rac1 to form lamellipodia, in part, by regulating its trafficking to the plasma membrane.

Localization of endogenous ARF6 to sites of cortical actin rearrangement and involvement of ARF6 in cell spreading.

To study the function of the endogenous ARF6 GTP binding protein in cells, we generated an antibody which specifically recognizes ARF6, and not the other ARF proteins. Using this antibody, ARF6 was detected in all mouse organs tested and in a variety of cultured cell lines including RBL, MDCK, NRK, BHK, COS, and HeLa cells. In NRK cells, by immunofluorescence, ARF6 localized to the plasma membrane, especially at regions exhibiting membrane ruffling, and was also concentrated in a fine punctate distribution in the juxtanuclear region. This pattern of localization of the endogenous protein was similar to the localization of ARF6 when overexpressed in NRK, or HeLa, cells. Treatments which perturb cortical actin in NRK cells, such as replating of cells after trypsinization or treatment with phorbol ester, resulted in the recruitment of endogenous ARF6 to the regions of cortical actin rearrangement. ARF6 activation and subsequent membrane recycling was required for cell spreading activity since expression of the dominant-negative, GTP-binding defective mutant of ARF6, T27N, previously shown to inhibit ARF6-regulated membrane recycling, inhibited cell attachment and spreading in HeLa cells. Furthermore, phorbol ester treatment enhanced the cell spreading activities in NRK cells, and in HeLa cells, but was not observed in cells expressing T27N. Taken together, these observations support a role for endogenous ARF6 in modeling the plasma membrane and cortical actin cytoskeleton.