The cytoplasm is the largest part of the cell by volume and hence its rheology sets the rate at which cellular shape changes can occur. Recent experimental evidence suggests that cytoplasmic rheology can be described by a poroelastic model, in which the cytoplasm is treated as a biphasic material consisting of a porous elastic solid meshwork (cytoskeleton, organelles, macromolecules) bathed in an interstitial fluid (cytosol). In this picture, the rate of cellular deformation is limited by the rate at which intracellular water can redistribute within the cytoplasm. However, direct supporting evidence for the model is lacking. Here we directly validate the poroelastic model to explain cellular rheology at physiologically relevant timescales using microindentation tests in conjunction with mechanical, chemical and genetic treatments. Our results show that water redistribution through the solid phase of the cytoplasm (cytoskeleton and macromolecular crowders) plays a fundamental role in setting cellular rheology.

Abstract:The ability of cells to follow gradients of extracellular matrix stiffnessdurotaxis-has been implicated in development, fibrosis and cancer. Durotaxis is established as a single cell phenomenon but whether it can direct the motion of cell collectives is unknown. Here we found that multicellular clusters exhibited durotaxis even if isolated constituent cells did not. This emergent mode of directed collective cell migration applied to a variety of epithelial cell types, and required the action of myosin motors and the integrity of cell-cell junctions. By extending traction microscopy to extracellular matrices of arbitrary stiffness profiles we showed that collective durotaxis originated from supracellular transmission of contractile physical forces. To explain the observed phenomenology, we developed a generalized clutch model in which local stickslip dynamics of cell-matrix adhesions is integrated to the tissue level through cell-cell junctions. Collective durotaxis is far more efficient than single cell durotaxis; it thus emerges as a robust mechanism to direct cell migration during development, wound healing, and collective cancer cell invasion. One Sentence Summary: Mechanical cooperation between cells enables an emergent mode of collective movement -3- Main Text:The ability of living cells to migrate following environmental gradients underlies a broad range of phenomena in development, homeostasis, and disease (1, 2). The best understood mode of directed cell migration is chemotaxis, the well-established ability of cells to follow gradients of soluble chemical cues (1). Some cell types are also able to follow gradients in the stiffness of their extracellular matrix (ECM), a process known as durotaxis (3-10).Durotaxis has been implicated in development (11), fibrosis (12) and cancer (13), but its underlying mechanisms remain unclear.Most of our understanding of directed cell migration has been obtained in single isolated cells. However, fundamental processes during development, wound healing, tissue regeneration, and some forms of cancer cell invasion are driven by directed migration of cell groups (14-16). Cell-cell interactions within these groups provide cooperative mechanisms of cell guidance that are altogether inaccessible to single cells (14-20). Here we investigated whether cell groups undergo collective durotaxis, and the cooperative nature of underlying mechanisms.Using stencils of magnetic PDMS, we micropatterned rectangular clusters (500 µm width) of human mammary epithelial cells (MCF-10A) on fibronectin-coated polyacrylamide gel substrates exhibiting uniform stiffness or a stiffness gradient (51 ± 17 kPa/mm, Fig. S1) (21). Upon removal of the PDMS stencil, clusters migrating on uniform gels displayed symmetric expansion (Fig. 1A,C,E,G, Fig. S2, Movie S1), whereas clusters migrating on stiffness gradients displayed a robust asymmetry characterized by faster, more persistent expansion towards the stiff edge (Fig. 1B-D-F-H, Fig. S2, Movie S1). This result was also -4-observed in clusters of...

Fundamental biological processes are carried out by curved epithelial sheets enclosing a pressurized lumen. How these sheets develop and withstand three-dimensional deformations has remained unclear. By combining measurements of epithelial tension and shape with theoretical modeling, here we show that epithelial sheets are active superelastic materials. We produce arrays of epithelial domes with controlled geometry. Quantification of luminal pressure and epithelial tension reveals a tensional plateau over several-fold areal strains. These extreme tissue strains are accommodated by highly heterogeneous cellular strains, in seeming contradiction with the measured tensional uniformity. This phenomenology is reminiscent of superelasticity, a behavior generally attributed to microscopic material instabilities in metal alloys. We show that this instability is triggered in epithelial cells by a stretch-induced dilution of the actin cortex and rescued by the intermediate filament network. Our study unveils a new type of mechanical behavior -active superelasticity- that enables epithelial sheets to sustain extreme stretching under constant tension.

Contractile forces are the end effectors of cell migration, division, morphogenesis, wound healing and cancer invasion. Here we report optogenetic tools to upregulate and downregulate such forces with high spatiotemporal accuracy. The technology relies on controlling the subcellular activation of RhoA using the CRY2/CIBN light-gated dimerizer system. We fused the catalytic domain (DHPH domain) of the RhoA activator ARHGEF11 to CRY2-mCherry (optoGEF-RhoA) and engineered its binding partner CIBN to bind either to the plasma membrane or to the mitochondrial membrane. Translocation of optoGEF-RhoA to the plasma membrane causes a rapid and local increase in cellular traction, intercellular tension and tissue compaction. By contrast, translocation of optoGEF-RhoA to mitochondria results in opposite changes in these physical properties. Cellular changes in contractility are paralleled by modifications in the nuclear localization of the transcriptional regulator YAP, thus showing the ability of our approach to control mechanotransductory signalling pathways in time and space.

SummaryThe plasticity of developing tissues relies on the adjustment of cell survival and growth rate to environmental cues. This includes the effect of mechanical cues on cell survival. Accordingly, compaction of an epithelium can lead to cell extrusion and cell death. This process was proposed to contribute to tissue homeostasis but also to facilitate the expansion of pretumoral cells through the compaction and elimination of the neighboring healthy cells. However, we know very little about the pathways that can trigger apoptosis upon tissue deformation, and the contribution of compaction-driven death to clone expansion has never been assessed in vivo. Using the Drosophila pupal notum and a new live sensor of ERK, we show first that tissue compaction induces cell elimination through the downregulation of epidermal growth factor receptor/extracellular signal regulated kinase (EGFR/ERK) pathway and the upregulation of the pro-apoptotic protein Hid. Those results suggest that the sensitivity of EGFR/ERK pathway to mechanics could play a more general role in the fine tuning of cell elimination during morphogenesis and tissue homeostasis. Second, we assessed in vivo the contribution of compaction-driven death to pretumoral cell expansion. We found that the activation of the oncogene Ras in clones can downregulate ERK and activate apoptosis in the neighboring cells through their compaction, which eventually contributes to Ras clone expansion. The mechanical modulation of EGFR/ERK during growth-mediated competition for space may contribute to tumor progression.

Recently developed optogenetic methods promise to revolutionize cell biology by allowing signaling perturbations to be controlled in space and time with light. However, a quantitative analysis of the relationship between a custom-defined illumination pattern and the resulting signaling perturbation is lacking. Here, we characterize the biophysical processes governing the localized recruitment of the Cryptochrome CRY2 to its membrane-anchored CIBN partner. We develop a quantitative framework and present simple procedures that enable predictive manipulation of protein distributions on the plasma membrane with a spatial resolution of 5 μm. We show that protein gradients of desired levels can be established in a few tens of seconds and then steadily maintained. These protein gradients can be entirely relocalized in a few minutes. We apply our approach to the control of the Cdc42 Rho GTPase activity. By inducing strong localized signaling perturbation, we are able to monitor the initiation of cell polarity and migration with a remarkable reproducibility despite cell-to-cell variability.

Cell motility can be initiated without prior cytoskeleton polarity by the stochastic stick-slip dynamics of cell adhesions.

Summary What regulates the spatiotemporal distribution of cell elimination in tissues remains largely unknown. This is particularly relevant for epithelia with high rates of cell elimination where simultaneous death of neighboring cells could impair epithelial sealing. Here, using the Drosophila pupal notum (a single-layer epithelium) and a new optogenetic tool to trigger caspase activation and cell extrusion, we first showed that death of clusters of at least three cells impaired epithelial sealing; yet, such clusters were almost never observed in vivo . Accordingly, statistical analysis and simulations of cell death distribution highlighted a transient and local protective phase occurring near every cell death. This protection is driven by a transient activation of ERK in cells neighboring extruding cells, which inhibits caspase activation and prevents elimination of cells in clusters. This suggests that the robustness of epithelia with high rates of cell elimination is an emerging property of local ERK feedback.