The small GTPases, Rab5 and Rac, are essential for endocytosis and actin remodeling, respectively. Coordination of these processes is critical to achieve spatial restriction of intracellular signaling, which is essential for a variety of polarized functions. Here, we show that clathrin- and Rab5-mediated endocytosis are required for the activation of Rac induced by motogenic stimuli. Rac activation occurs on early endosomes, where the RacGEF Tiam1 is also recruited. Subsequent recycling of Rac to the plasma membrane ensures localized signaling, leading to the formation of actin-based migratory protrusions. Thus, membrane trafficking of Rac is required for the spatial resolution of Rac-dependent motogenic signals. We further demonstrate that a Rab5-to-Rac circuitry controls the morphology of motile mammalian tumor cells and primordial germinal cells during zebrafish development, suggesting that this circuitry is relevant for the regulation of migratory programs in various cells, in both in vitro settings and whole organisms.

Rab5 is a small GTPase involved in the control of intracellular trafficking, both at the level of receptor endocytosis and endosomal dynamics. The finding that Rab5 can be activated by receptor tyrosine kinases (RTK) raised the question of whether it also participates in effector pathways emanating from these receptors. Here we show that Rab5 is indispensable for a form of RTK-induced actin remodelling, called circular ruffling. Three independent signals, originating from Rab5, phosphatidylinositol-3-OH kinase and Rac, respectively, are simultaneously required for the induction of circular ruffles. Rab5 signals to the actin cytoskeleton through RN-tre, a previously identified Rab5-specific GTPase-activating protein (GAP). Here we demonstrate that RN-tre has the dual function of Rab5-GAP and Rab5 effector. We also show that RN-tre is critical for macropinocytosis, a process previously connected to the formation of circular ruffles. Finally, RN-tre interacts with both F-actin and actinin-4, an F-actin bundling protein. We propose that RN-tre establishes a three-pronged connection with Rab5, F-actin and actinin-4. This may aid crosslinking of actin fibres into actin networks at the plasma membrane. Thus, we have shown that Rab5 is a signalling GTPase and have elucidated the major molecular elements of its downstream pathway.

Filopodia explore the environment, sensing soluble and mechanical cues during directional motility and tissue morphogenesis. How filopodia are initiated and spatially restricted to specific sites on the plasma membrane is still unclear. Here, we show that the membrane deforming and curvature sensing IRSp53 (Insulin Receptor Substrate of 53 kDa) protein slows down actin filament barbed end growth. This inhibition is relieved by CDC42 and counteracted by VASP, which also binds to IRSp53. The VASP:IRSp53 interaction is regulated by activated CDC42 and promotes high-density clustering of VASP, which is required for processive actin filament elongation. The interaction also mediates VASP recruitment to liposomes. In cells, IRSp53 and VASP accumulate at discrete foci at the leading edge, where filopodia are initiated. Genetic removal of IRSp53 impairs the formation of VASP foci, filopodia and chemotactic motility, while IRSp53 null mice display defective wound healing. Thus, IRSp53 dampens barbed end growth. CDC42 activation inhibits this activity and promotes IRSp53-dependent recruitment and clustering of VASP to drive actin assembly. These events result in spatial restriction of VASP filament elongation for initiation of filopodia during cell migration, invasion, and tissue repair.

145-words) 31 32During wound repair, branching morphogenesis and carcinoma dissemination, cellular 33 rearrangements are fostered by a solid-to-liquid transition, known as unjamming. The biomolecular 34 machinery behind unjamming and its pathophysiological relevance remain, however, unclear. Here, 35 we study unjamming in a variety of normal and tumorigenic epithelial 2D and 3D collectives. 36 Biologically, the increased level of the small GTPase RAB5A sparks unjamming by promoting non-37 clathrin-dependent internalization of epidermal growth factor receptor that leads to hyper-activation 38 of the kinase ERK1/2 and phosphorylation of the actin nucleator WAVE2. This cascade triggers 39 collective motility effects with striking biophysical consequences. Specifically, unjamming in tumor 40 spheroids is accompanied by persistent and coordinated rotations that progressively remodel the 41 extracellular matrix, while simultaneously fluidizing cells at the periphery. This concurrent action 42 results in collective invasion, supporting the concept that the endo-ERK1/2 pathway is a 43 physicochemical switch to initiate collective invasion and dissemination of otherwise jammed 44 carcinoma. 45 46 47 48 among each other and with their environment 1, 2 . During tissue growth cells are free to move, as in a 49 fluid, but their motion becomes constrained as density increases. At a critical density -depending on 50 a variety of biophysical parameters, such as intercellular adhesion, cortical tension, single cell 51 motility, and cell shape variance, motility ceases and collectives rigidify undergoing jamming 52 transition 3-7 . This transition ensures proper development of barrier properties in epithelial tissues, but 53 also to act as a tumour suppressive mechanism 3, 8 . The reverse solid-to-liquid (unjamming) transition 54 might, instead, represent a complementary gateway to epithelial cell migration, enabling mature 55 tissues to flow 3, 8, 9 . However, how cells control the jamming/unjamming transition is unclear. 56Consistently with the emerging role of membrane trafficking in regulating cell migration plasticity 57 and the mechanics of cell-cell interactions 10, 11 , we recently found that RAB5A, a master regulator of 58 early endosomes necessary to promote a mesenchymal program of individual cancer invasion 12, 13 , 59 impacts on the mechanics and dynamics of multicellular, normal and tumorigenic cell assemblies 14 . 60RAB5A overexpression re-awakens the motility of otherwise kinetically-arrested epithelial 61 monolayers, promoting millimetre-scale, multicellular, ballistic cell locomotion and a flocking-fluid 62 motility pattern through large-scale coordinated migration and local cell rearrangements 14-16 . 63 Concurrently, monolayer stiffness, cell-cell surface contact and junctional tension increase, as well 64 as the turnover of junctional E-cadherin and the extension of RAC1-driven protrusions 14 . 65Molecularly, impairing endocytosis, macropinocytosis or increasing fluid efflux abrogated RAB5A-66 indu...

RAB5A and RAB4 promote breast tumor cell dissemination by controlling the trafficking of proteins necessary for localized invadosome formation.