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 ce lls, 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 includ ing RBL, MDCK, NRK, BHK, COS, and HeLa cells. In NRK cells, by immunof luorescence, ARF6 Localized to the plasma membrane, especially at regi ons exhibiting membrane ruffling, and was also concentrated in a fine punctate distribution in the juxtanuclear region. This pattern of loca lization of the endogenous protein was similar to the localization of ARF6 when overexpressed in NRK, or HeLa, cells. Treatments which pertu rb cortical actin in NRK cells, such as replating of cells after tryps inization or treatment with phorbol ester, resulted in the recruitment of endogenous ARF6 to the regions of cortical actin rearrangement. AR F6 activation and subsequent membrane recycling was required for cell spreading activity since expression of the dominant-negative, GTP-bind ing defective mutant of ARF6, T27N, previously shown to inhibit ARF6-r egulated membrane recycling, inhibited cell attachment and spreading i n HeLa cells. Furthermore, phorbol ester treatment enhanced the cell s preading activities in NRK cells, and in HeLa cells, but was not obser ved in cells expressing T27N, Taken together, these observations suppo rt a role for endogenous ARF6 in modeling the plasma membrane and cort ical actin cytoskeleton.