In Myxococcus xanthus the gliding motility machinery is assembled at the leading cell pole to form focal adhesions, translocated rearward to propel the cell, and disassembled at the lagging pole. We show that MglA, a Ras-like small G-protein, is an integral part of this machinery. In this function, MglA stimulates the assembly of the motility complex by directly connecting it to the MreB actin cytoskeleton. Because the nucleotide state of MglA is regulated spatially and MglA only binds MreB in the guanosine triphosphate–bound form, the motility complexes are assembled at the leading pole and dispersed at the lagging pole where the guanosine triphosphatase activating protein MglB disrupts the MglA–MreB interaction. Thus, MglA acts as a nucleotide-dependent molecular switch to regulate the motility machinery spatially. The function of MreB in motility is independent of its function in peptidoglycan synthesis, representing a coopted function. Our findings highlight a new function for the MreB cytoskeleton and suggest that G-protein–cytoskeleton interactions are a universally conserved feature.

The Arf6-specific exchange factor EFA6 coordinates membrane trafficking with actin cytoskeleton remodeling. It localizes to the plasma membrane where it catalyzes Arf6 activation and induces the formation of actin-based membrane ruffles. We have shown previously that the pleckstrin homology (PH) domain of EFA6 was responsible for its membrane localization. In this study we looked for the partners of the PH domain at the plasma membrane. Mutations of the conserved basic residues suspected to be involved in the binding to phosphoinositides redistribute EFA6-PH to the cytosol. In addition, phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂) breakdown also leads to the solubilization of EFA6-PH. Direct binding measured by surface plasmon resonance gives an apparent affinity of ∼0.5 μm EFA6-PH for PI(4,5)P₂. Moreover, we observed in vitro that the catalytic activity of EFA6 is strongly increased by PI(4,5)P₂. These results indicate that the plasma membrane localization of EFA6-PH is based on its interaction with PI(4,5)P₂, and this interaction is necessary for an optimal catalytic activity of EFA6. Furthermore, we demonstrated by fluorescence recovery after photobleaching and Triton X-100 detergent solubility experiments that in addition to the phophoinositides, EFA6-PH is linked to the actin cytoskeleton. We observed both in vivo and in vitro that EFA6-PH interacts directly with F-actin. Finally, we demonstrated that EFA6 could bind simultaneously filamentous actin and phospholipids vesicles. Our results explain how the exchange factor EFA6 via its PH domain could coordinate at the plasma membrane actin cytoskeleton organization with membrane trafficking.

β2-adrenergic receptor (β2AR), a member of the GPCR (G-Proteins Coupled Receptor) family, is internalized in a ligand- and β-arrestin-dependent manner into early endosomes, and subsequently recycled back to the plasma membrane. Here we report that β-arrestin promotes the activation of the small G protein Arf6, which regulates the recycling and degradation of β2AR. We demonstrate in vitro that the C-terminal region of β-arrestin1 interacted directly and simultaneously with Arf6GDP and its specific exchange factor EFA6, to promote Arf6 activation. Similarly, the ligand-mediated activation of β2AR leads to the formation of Arf6GTP in vivo in a β-arrestin-dependent manner. Expression of either EFA6 or an activated Arf6 mutant caused accumulation of β2AR in the degradation pathway. And this phenotype could be rescued by the expression of an activated mutant of Rab4, suggesting that Arf6 acts upstream of Rab4. We propose a model in which Arf6 plays an essential role for the β2AR desensitization. The ligand-mediated stimulation of β2AR relocates β-arrestin to the plasma membrane, and triggers the activation of Arf6 by EFA6. The activated Arf6 leads to accumulation of β2AR to the degradation pathway, and negatively controls the Rab4-dependent fast recycling to prevent the re-sensitization of β2AR.

Significance: The small G protein ADP-ribosylation factor 6 (Arf6) and the exchange factor for Arf6 (EFA6) are involved in endocytic vesicular transport, but their precise functions remain unclear. The Bin/Amphiphysin/Rvs (BAR) domain containing endophilin is known to couple fission to uncoating of the clathrin-coated vesicles. Here, we identified endophilin as a direct interactor of EFA6. We analyzed in vitro the effect of this interaction on EFA6 guanine nucleotide exchange factor activity, and on endophilin lipid binding and remodeling activities. We then studied in vivo the role of the two proteins in transferrin receptor endocytosis. Our results suggest a model in which EFA6 recruits endophilin on flat areas of endocytic zones of the plasma membrane, where endophilin cooperates with EFA6 to activate Arf6 and regulate clathrin-mediated endocytosis.

The endocytic protein NUMB has been implicated in the control of various polarized cellular processes, including the acquisition of mesenchymal migratory traits through molecular mechanisms that have only been partially defined. Here, we report that NUMB is a negative regulator of a specialized set of understudied, apically restricted, actin-based protrusions, the circular dorsal ruffles (CDRs), induced by either PDGF or HGF stimulation. Through its PTB domain, NUMB binds directly to an N-terminal NPLF motif of the ARF6 guanine nucleotide exchange factor, EFA6B, and promotes its exchange activity in vitro. In cells, a NUMB–EFA6B–ARF6 axis regulates the recycling of the actin regulatory cargo RAC1 and is critical for the formation of CDRs that mark the acquisition of a mesenchymal mode of motility. Consistently, loss of NUMB promotes HGF-induced cell migration and invasion. Thus, NUMB negatively controls membrane protrusions and the acquisition of mesenchymal migratory traits by modulating EFA6B–ARF6 activity.

A key step of epithelial morphogenesis is the creation of the lumen. Luminogenesis by hollowing proceeds through the fusion of apical vesicles at cell–cell contacts. The small nascent lumens grow through extension, coalescence and enlargement, coordinated with cell division, to give rise to a single central lumen. Here, by using MDCK cells grown in 3D-culture, we show that EFA6A (also known as PSD) participates in luminogenesis. EFA6A recruits α-actinin 1 (ACTN1) through direct binding. In polarized cells, ACTN1 was found to be enriched at the tight junction where it acts as a primary effector of EFA6A for normal luminogenesis. Both proteins are essential for the lumen extension and enlargement, where they mediate their effect by regulating the cortical acto-myosin contractility. Finally, ACTN1 was also found to act as an effector for the isoform EFA6B (also known as PSD4) in the human mammary tumoral MCF7 cell line. EFA6B restored the glandular morphology of this tumoral cell line in an ACTN1-dependent manner. Thus, we identified new regulators of cyst luminogenesis essential for the proper maturation of a newly-formed lumen into a single central lumen.