Arf6 regulates endocytic trafficking in two examples of polarized cells

ADP-Ribosylation Factor-6 (Arf6) is a small GTPase that regulates membrane trafficking from the recycling endosome to the cell surface and also has effects on endocytosis at the plasma membrane. As Arf6 regulates traffic from the two compartments that form synaptic vesicles, the plasma membrane and endosomes, we investigated whether Arf6 plays a role in synaptic vesicle biogenesis. We used a synaptic vesicle marker protein, p-selectin-HRP, to follow the effects of ARF6 expression on synaptic vesicle biogenesis in PC12 neuroendocrine cells. Expression of a constitutively active ARF6 mutant increased, while expression of a nucleotide-free ARF6 mutant decreased, p-selectin-HRP levels in the synaptic vesicle peak. These results provide the first direct evidence for a role for ARF6 in synaptic vesicle biogenesis.; We then examined the endocytic recycling of integrins, a process that has been suggested to play an important role during cell motility by mediating the redistribution of integrins to the leading edge of migrating cells. A recycling pathway from a distinct subset of the recycling endosome was found to be regulated by external stimulation, and this process resulted in the targeted recycling of integrin β1 to surface membrane ruffles. This recycling was regulated by Arf6 and Rab11 and requires the actin cytoskeleton. Supporting the importance of these features of integrin β1 recycling for cell motility, we found that integrin β1-dependent cell motility requires the activation of Arf6 and its actin effector function.; Finally, we investigated how stimulation might induce integrin β1 recycling. We found that while the phosphorylation state of integrin β1 is not altered by stimulation, ACAP1, an Arf6 GAP and potential component of the transport machinery at the recycling endosome, is phosphorylated. Thus stimulation may alter the phosphorylation state of ACAP1, resulting in the inclusion of integrin β1 into transport vesicles.; In conclusion, we have found that Arf6 affects both the biogenesis of synaptic vesicles and integrin β1 recycling. Although these processes are seemingly disparate, they have the common features of cell polarization, Arf6-governed vesicle formation, and stimulation-regulated transport. Our findings may thus provide insight into issues in other systems of polarized or regulated transport.