The Arf6 GTPase regulates membrane traffic between the plasma membrane (PM) and an endosomal compartment and influences the dynamics of the cortical actin cytoskeleton. In many cells this pathway is the route followed by PM proteins that lack clathrin localization sequences and hence are endocytosed into cells independently of clathrin. After internalization, a portion of this membrane recycles back to the PM upon Arf6 activation. The movement of membrane through this Arf6-regulated pathway is required for the dynamic, cortical actin rearrangements observed during cell spreading, protrusion formation, and Rac-mediated ruffling. To understand how Arf6 carrries out these dual functions and to identify potential Arf6 mediators, we have been using a model transfected-cell system that allows us to follow membrane dynamics and cell shape alterations in live and fixed cells. We find that Arf6 functions by regulating the trafficking and activity of phosphatidylinositol 4-phosphate 5-kinase (PIP 5-kinase), an enzyme that generates phosphatidylinositol 4,5-bisphosphate (PIP2). We localized PIP2 in cells with a chimera of a plextrin homology domain (PH) that specifically recognizes membrane-bound PIP2 and green fluorescent protein (GFP). PIP2 was found associated with the PM and also on the Arf6-associated endosomal membranes. Activation of Arf6 achieved by expression of a GTP exchange factor for Arf6, EFA6, resulted in the formation of protrusions, the uptake of membrane into PIP2-enriched macropinosomes and the recycling of this internalized membrane back to the PM. These """"""""activated"""""""" cells continue to protrude and cycle membrane since endogenous Arf GTPase activating proteins (GAPs) can still inactivate Arf6-GTP allowing continual activation and inactivation of Arf6. By contrast, expression of constitutively active Arf6, Q67L, resulted in the internalization of PIP2-enriched membrane into the cell, the fusion of these membranes generating larger vacuoles and a block in recycling of that membrane back to the PM. Overexpression of human PIP 5-kinase alpha mimicked the effects seen with Arf6Q67L demonstrating that PIP 5-kinase and PIP2 turnover regulated by Arf6 are critical for trafficking through the Arf6 endosomal system.
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