Dynamics of epithelial monolayers has recently been interpreted in terms of a jamming or rigidity transition. How cells control such phase transitions is, however, unknown. Here we show that RAB5A, a key endocytic protein, is sufficient to induce large-scale, coordinated motility over tens of cells and ballistic motion in otherwise kinetically-arrested monolayers. This is linked to increased traction forces and to the extension of cell protrusions, which align with local velocity. Molecularly, impairing endocytosis, macropinocytosis or increasing fluid efflux abrogates RAB5A-induced collective motility. A simple model based on mechanical junctional tension and an active cell reorientation mechanism for the velocity of self-propelled cells identifies regimes of monolayer dynamics that explain endocytic reawakening of locomotion in terms of a combination of large-scale directed migration and local unjamming. These changes in multicellular dynamics enable collectives to migrate under physical constraints and may be exploited by tumors for interstitial dissemination.

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...

Cells constantly sense and respond to mechanical signals by reorganizing their actin cytoskeleton. Although a number of studies have explored the effects of mechanical stimuli on actin dynamics, the immediate response of actin after force application has not been studied. We designed a method to monitor the spatiotemporal reorganization of actin after cell stimulation by local force application. We found that force could induce transient actin accumulation in the perinuclear region within ∼2 min. This actin reorganization was triggered by an intracellular Ca 2+ burst induced by force application. Treatment with the calcium ionophore A23187 recapitulated the force-induced perinuclear actin remodeling. Blocking of actin polymerization abolished this process. Overexpression of Klarsicht, ANC-1, Syne Homology (KASH) domain to displace nesprins from the nuclear envelope did not abolish Ca 2+ -dependent perinuclear actin assembly. However, the endoplasmic reticulum-and nuclear membrane-associated inverted formin-2 (INF2), a potent actin polymerization activator (mutations of which are associated with several genetic diseases), was found to be important for perinuclear actin assembly. The perinuclear actin rim structure colocalized with INF2 on stimulation, and INF2 depletion resulted in attenuation of the rim formation. Our study suggests that cells can respond rapidly to external force by remodeling perinuclear actin in a unique Ca 2+ -and INF2-dependent manner.force | mechanotransduction | calcium | formin | perinuclear actin rim C ells can sense and adapt to their physical microenvironment through specific mechanosensing mechanisms. These properties are often mediated by the actin cytoskeleton, which can be modulated by a wide range of forces. Fluid shear stress, for example, induces actin stress fiber assembly and realignment along the direction of flow (1-4), whereas the cyclic stretch of an elastic substrate induces a reorientation of stress fibers under some angle to the direction of stretch (5-8). Applying mechanical force to cells by a microneedle results in focal adhesion growth and activation of formin-type actin nucleators (9, 10). Similarly, local application of force through fibronectin or collagen-coated beads trapped by optical or magnetic tweezers leads to the local reorganization of the actin cytoskeleton. This response is associated with reinforcement of bead attachment (11), recruitment of additional actin-associated proteins (12), and activation of a variety of signaling pathways (13)(14)(15)(16)(17). Most studies to date have explored the effects of force on actin structures directly associated with the sites of force application, such as focal adhesions and stress fibers. However, the immediate effect of force on the assembly of actin structures distal from the sites of force application has not been assessed. Such process is despite distal effects having potential implications in the transduction of local forces from the cell periphery to nuclear events (18).In this study, we used a local mecha...

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Phagocytosis of invading pathogens or cellular debris requires a dramatic change in cell shape driven by actin polymerization. For antibody-covered targets, phagocytosis is thought to proceed through the sequential engagement of Fc-receptors on the phagocyte with antibodies on the target surface, leading to the extension and closure of the phagocytic cup around the target. We find that two actin-dependent molecular motors, class 1 myosins myosin 1e and myosin 1f, are specifically localized to Fc-receptor adhesions and required for efficient phagocytosis of antibody-opsonized targets. Using primary macrophages lacking both myosin 1e and myosin 1f, we find that without the actin-membrane linkage mediated by these myosins, the organization of individual adhesions is compromised, leading to excessive actin polymerization, slower adhesion turnover, and deficient phagocytic internalization. This work identifies a role for class 1 myosins in coordinated adhesion turnover during phagocytosis and supports a mechanism involving membrane-cytoskeletal crosstalk for phagocytic cup closure.