Arf6 coordinates actin assembly through the WAVE complex,a mechanism usurped by Salmonella to invade host cells

ADP ribosylation factor (Arf) 6 anchors to the plasma membrane, where it coordinates membrane trafficking and cytoskeleton remodelling, but how it assembles actin filaments is unknown. By reconstituting membrane-associated actin assembly mediated by the WASP family veroprolin homolog (WAVE) regulatory complex (WRC), we recapitulated an Arf6-driven actin polymerization pathway. We show that Arf6 is divergent from other Arf members, as it was incapable of directly recruiting WRC. We demonstrate that Arf6 triggers actin assembly at the membrane indirectly by recruiting the Arf guanine nucleotide exchange factor (GEF) ARNO that activates Arf1 to enable WRC-dependent actin assembly. The pathogen Salmonella usurped Arf6 for host cell invasion by recruiting its canonical GEFs EFA6 and BRAG2. Arf6 and its GEFs facilitated membrane ruffling and pathogen invasion via ARNO, and triggered actin assembly by generating an Arf1–WRC signaling hub at the membrane in vitro and in cells. This study reconstitutes Arf6-dependent actin assembly to reveal a mechanism by which related Arf GTPases orchestrate distinct steps in the WRC cytoskeleton remodelling pathway.ADP ribosylation factor (Arf) GTPases are best known for their roles in vesicle and organelle trafficking (1). Class I and II Arfs (Arf1, Arf3, Arf4, and Arf5) are found predominantly in and around the Golgi apparatus. In contrast, the more divergent Class III Arf (Arf6) operates almost exclusively at the plasma membrane (2). Consistent with its localization, Arf6 has been heavily implicated in trafficking events at the cell surface, including the regulation of endocytosis and exocytosis (1). In particular, Arf6 and its guanine nucleotide exchange factors (GEFs) are believed to be pivotal to the recycling of endosomes and receptors to and from the plasma membrane (3, 4). Arf6 also has a clear role in cortical cytoskeleton rearrangement (5). This is strongly supported by evidence of Arf6 and Rac1 (Ras-related C3 botulinum toxin substrate) interplay (6), exemplified by Arf6 recruitment of the Rac1 GEF Kalirin, Arf6 promotion of Rac1 activation, and lamellipodia formation (7–9).Rac1 is required for generation of lamellipodia that lead to membrane ruffles and macropinocytosis (10). Rac1 is thought to achieve this by activating the WRC, which comprises WAVE (WASP family veroprolin homolog), Abi (abl-interactor 1), Cyfip (cytoplasmic FMR1 interacting protein), Nap1 (NCK-associated protein 1), HSPC300 (heat shock protein C300), or their homologs (10–12). We recently established that Rac1 was not sufficient for WRC recruitment to the membrane and its activation, which instead requires direct binding by Rac1 and an Arf GTPase (13). This in vitro Arf activity could be supplied by multiple isoforms including Arf1 and Arf5, but only Arf1 facilitated WRC-dependent lamellipodia formation and macropinocytosis of the bacterial pathogen Salmonella into human host cells (13–15). Intriguingly, Arf6 also promoted Salmonella invasion (14), and, given the capability of Arfs to modulate WRC, it seems likely that Arf6 also recruits and activates the WRC at the plasma membrane. However, despite mounting evidence that Arf6 remodels the cytoskeleton, a molecular mechanism by which Arf6 drives Arp2/3-dependent actin assembly has not been resolved.