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...

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There is growing evidence that tyrosine phosphatases display an intrinsic enzymatic preference for the sequence context flanking the target phosphotyrosines. On the other hand, substrate selection in vivo is decisively guided by the enzyme-substrate connectivity in the protein interaction network. We describe here a system wide strategy to infer physiological substrates of protein-tyrosine phosphatases. Here we integrate, by a Bayesian model, proteome wide evidence about in vitro substrate preference, as determined by a novel high-density peptide chip technology, and “closeness” in the protein interaction network. This allows to rank candidate substrates of the human PTP1B phosphatase. Ultimately a variety of in vitro and in vivo approaches were used to verify the prediction that the tyrosine phosphorylation levels of five high-ranking substrates, PLC-γ1, Gab1, SHP2, EGFR, and SHP1, are indeed specifically modulated by PTP1B. In addition, we demonstrate that the PTP1B-mediated dephosphorylation of Gab1 negatively affects its EGF-induced association with the phosphatase SHP2. The dissociation of this signaling complex is accompanied by a decrease of ERK MAP kinase phosphorylation and activation.

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The ability of cells to alter their phenotypic and morphological characteristics, known as cellular plasticity, is critical in normal embryonic development and adult tissue repair and contributes to the pathogenesis of diseases, such as organ fibrosis and cancer. The epithelial-to-mesenchymal transition (EMT) is a type of cellular plasticity. This transition involves genetic and epigenetic changes as well as alterations in protein expression and post-translational modifications. These changes result in reduced cell-cell adhesion, enhanced cell adhesion to the extracellular matrix, and altered organization of the cytoskeleton and of cell polarity. Among these modifications, loss of cell polarity represents the nearly invariable, distinguishing feature of EMT that frequently precedes the other traits or might even occur in their absence. EMT transforms cell morphology and physiology, and hence cell identity, from one typical of cells that form a tight barrier, like epithelial and endothelial cells, to one characterized by a highly motile mesenchymal phenotype. Time-resolved proteomic and phosphoproteomic analyses of cells undergoing EMT recently identified thousands of changes in proteins involved in many cellular processes, including cell proliferation and motility, DNA repair, and – unexpectedly – membrane trafficking (1). These results have highlighted a picture of great complexity. First, the EMT transition is not an all-or-none response but rather a gradual process that develops over time. Second, EMT events are highly dynamic and frequently reversible, involving both cell-autonomous and non-autonomous mechanisms. The net results is that EMT generates populations of mixed cells, with partial or full phenotypes, possibly accounting (at least in part) for the physiological as well as pathological cellular heterogeneity of some tissues. Endocytic circuitries have emerged as complex connectivity infrastructures for numerous cellular networks required for the execution of different biological processes, with a primary role in the control of polarized functions. Thus, they may be relevant for controlling EMT or certain aspects of it. Here, by discussing a few paradigmatic cases, we will outline how endocytosis may be harnessed by the EMT process to promote dynamic changes in cellular identity, and to increase cellular flexibility and adaptation to micro-environmental cues, ultimately impacting on physiological and pathological processes, first and foremost cancer progression.

Abstract. Epithelial cells cultured in a monolayer are very motile in isolation but reach a near-jammed state when mitotic division increases their number above a critical threshold. We have recently shown that a monolayer can be reawakened by over-expression of a single protein, RAB5A, a master regulator of endocytosis. This reawakening of motility was explained in term of a flocking transition that promotes the emergence of a large-scale collective migratory pattern. Here we focus on the impact of this reawakening on the structural properties of the monolayer. We find that the unjammed monolayer is characterised by a fluidisation at the single cell level and by enhanced non-equilibrium large-scale number fluctuations at a larger length scale. Also with the help of numerical simulations, we trace back the origin of these fluctuations to the selfpropelled active nature of the constituents and to the existence of a local alignment mechanism, leading to the spontaneous breaking of the orientational symmetry.

How cells move chemotactically remains a major unmet challenge in cell biology. Emerging evidence indicates that for interpreting noisy, shallow gradients of soluble cues a system must behave as an excitable process. Here, through an RNAi-based, high-content screening approach, we identify RAB35 as necessary for the formation of growth factors (GFs)-induced waves of circular dorsal ruffles (CDRs), apically restricted actin-rich migratory protrusions. RAB35 is sufficient to induce recurrent and polarized CDRs that travel as propagating waves, thus behaving as an excitable system that can be biased to control cell steering. Consistently, RAB35 is essential for promoting directed chemotactic migration and chemoinvasion of various cells in response to gradients of motogenic GFs. Molecularly, RAB35 does so by directly regulating the activity of p85/PI3K polarity axis. We propose that RAB35 is a molecular determinant for the control of an excitable, oscillatory system that acts as a steering wheel for GF-mediated chemotaxis and chemoinvasion.