Migration frequently involves Rac-mediated protrusion of lamellipodia, formed by Arp2/3 complex-dependent branching thought to be crucial for force generation and stability of these networks. The formins FMNL2 and FMNL3 are Cdc42 effectors targeting to the lamellipodium tip and shown here to nucleate and elongate actin filaments with complementary activities in vitro. In migrating B16-F1 melanoma cells, both formins contribute to the velocity of lamellipodium protrusion. Loss of FMNL2/3 function in melanoma cells and fibroblasts reduces lamellipodial width, actin filament density and -bundling, without changing patterns of Arp2/3 complex incorporation. Strikingly, in melanoma cells, FMNL2/3 gene inactivation almost completely abolishes protrusion forces exerted by lamellipodia and modifies their ultrastructural organization. Consistently, CRISPR/Cas-mediated depletion of FMNL2/3 in fibroblasts reduces both migration and capability of cells to move against viscous media. Together, we conclude that force generation in lamellipodia strongly depends on FMNL formin activity, operating in addition to Arp2/3 complex-dependent filament branching.

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Endocytosis of extracellular or plasma membrane material is a fundamental process. A variety of endocytic pathways exist, several of which are barely understood in terms of mechanistic execution and biological function. Importantly, some mechanisms have been identified and characterized by following virus internalization into cells. This includes a novel endocytic pathway exploited by human papillomavirus type 16 (HPV16). However, its cellular role and mechanism of endocytic vacuole formation remain unclear. Here, HPV16 was used as a tool to examine the mechanistic execution of vesicle formation by combining systematic perturbation of cellular processes with electron and video microscopy. Our results indicate cargo uptake by uncoated, inward-budding pits facilitated by the membrane bending retromer protein SNX2. Actin polymerization-driven vesicle scission is promoted by WASH, an actin regulator typically not found at the plasma membrane. Uncovering a novel role of WASH in endocytosis, we propose to term the new pathway WASH-mediated endocytosis (WASH-ME).

Salmonella enterica serovar Typhimurium manipulates cellular Rho GTPases for host cell invasion by effector protein translocation via the Type III Secretion System (T3SS). The two Guanine nucleotide exchange (GEF) mimicking factors SopE and -E2 and the inositol phosphate phosphatase (PiPase) SopB activate the Rho GTPases Rac1, Cdc42 and RhoA, thereby mediating bacterial invasion. S. Typhimurium lacking these three effector proteins are largely invasion-defective. Type III secretion is crucial for both early and later phases of the intracellular life of S. Typhimurium. Here we investigated whether and how the small GTPase RhoB, known to localize on endomembrane vesicles and at the invasion site of S. Typhimurium, contributes to bacterial invasion and to subsequent steps relevant for S. Typhimurium lifestyle. We show that RhoB is significantly upregulated within hours of Salmonella infection. This effect depends on the presence of the bacterial effector SopB, but does not require its phosphatase activity. Our data reveal that SopB and RhoB bind to each other, and that RhoB localizes on early phagosomes of intracellular S. Typhimurium. Whereas both SopB and RhoB promote intracellular survival of Salmonella, RhoB is specifically required for Salmonella-induced upregulation of autophagy. Finally, in the absence of RhoB, vacuolar escape and cytosolic hyper-replication of S. Typhimurium is diminished. Our findings thus uncover a role for RhoB in Salmonella-induced autophagy, which supports intracellular survival of the bacterium and is promoted through a positive feedback loop by the Salmonella effector SopB.