Unjamming overcomes kinetic and proliferation arrest in terminally differentiated cells and promotes collective motility of carcinoma

Abstract

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...

Membrane tension mediated mechanotransduction drives fate choice in embryonic stem cells

“…3g, h), suggesting that high membrane tension antagonizes endocytosis, consistent with previous findings in other systems(23) (24). To test whether enhanced endocytosis could overcome the block in differentiation imposed by EZR_CA, we transfected iEZR_CA cells with Rab5a, a master regulator of early endosomes (26). Elevation of Rab5a levels has been shown to promote clathrin-independent endocytosis (26) (27).…”

“…Rab5a and FRET plasmids were provided by Giorgio Scita's lab (26) Ezr_GFP and EZR_CA_GFP were provided by Guillaume Charras (15) Primers Dusp6: 5' -GCTGATACCTGCCAAGCAAT-3' 5' -TCCTATCTGGATCACTGGAG -3' Spry4: 5' -TGCACCATGACTGAGCTG -3' 5' -AGCGATGCTTGTGACTCTG -3' Spry2: 5' -CATCAGGTCTTGGCAGTGT-3' 5' -CATCGCTGGAAGAAGAGGAT-3' Egr1: 5' -GATAACTCGTCTCCACCATCG -3' 5' -AGCGCCTTCAATCCTCAAG -3'…”

“…We next asked if a specific signalling pathway related to regulation of naive pluripotency was affected by the changes in cell mechanics and endocytosis we observed. The most obvious candidate was FGF/ERK, as it is key for exit from naive pluripotenc (28) , and has been shown to be regulated by endocytosis in cancer cells (22) (26). We thus quantified pERK levels during exit from naive pluripotency, and observed that at all time points ERK activity was highly attenuated in the iEZR_CA cells ( Fig.…”

“…We then specifically probed pERK activity at endosomes using a recently described FRET sensor reporting on ERK activity at early endosomes ( Fig. 4e) (26). Using live imaging, we found that pERK at endosomes sharply increased as cells spread (Supp Fig.…”

Emergence of single cell mechanical behavior and polarity within epithelial monolayers drives collective cell migration

“…Our experimental and numerical approaches show that the maintenance of coordinated migration requires the acquisition of a front-back polarity within each single cell but does not require the maintenance of cell-cell junctions. Taken together, these unexpected findings demonstrate that collective cell dynamics in closed environments as observed in multiple in vitro and in vivo situations 5,6,8,9 can arise from single cell behavior through a sustained memory of cell polarity. 3…”