TY - JOUR T1 - Collective cell behaviour in mechanosensing of substrate thickness JF - bioRxiv DO - 10.1101/228478 SP - 228478 AU - CG Tusan AU - YH Man AU - H Zarkoob AU - DA Johnson AU - OG Andriotis AU - PJ Thurner AU - S Yang AU - EA Sander AU - E Gentleman AU - BG Sengers AU - ND Evans Y1 - 2017/01/01 UR - http://biorxiv.org/content/early/2017/12/04/228478.abstract N2 - Extracellular matrix stiffness has a profound effect on the behaviour of many cell types. Adherent cells apply contractile forces to the material on which they adhere, and sense the resistance of the material to deformation - its stiffness. This is dependent on both the elastic modulus and the thickness of the material, with the corollary that single cells are able to sense underlying stiff materials through soft hydrogel materials at low (<10 μm) thicknesses. Here, we hypothesised that cohesive colonies of cells exert more force and create more hydrogel deformation than single cells, therefore enabling them to mechanosense more deeply into underlying materials than single cells. To test this, we modulated the thickness of soft (1 kPa) elastic ECM-functionalised polyacrylamide hydrogels adhered to glass substrates and allowed colonies of MG63 cells to form on their surfaces. Cell morphology and deformations of fluorescent fiducial-marker labelled hydrogels were quantified by time-lapse fluorescence microscopy imaging. Single cell spreading increased with respect to decreasing hydrogel thickness, with data fitting to an exponential model with half-maximal response at a thickness of 3.2 μm. By quantifying cell area within colonies of defined area, we similarly found that colony-cell spreading increased with decreasing hydrogel thickness but with a greater half-maximal response at 54 μm. Depth-sensing was dependent on ROCK-mediated cellular contractility. Surface hydrogel deformations were significantly greater on thick hydrogels compared to thin hydrogels. In addition, deformations extended greater distances from the periphery of colonies on thick hydrogels compared to thin hydrogels. Our data suggest that by acting collectively, cells mechanosense rigid materials beneath elastic hydrogels at greater depths than individual cells. This raises the possibility that the collective action of cells in colonies or sheets may allow cells to sense structures of differing material properties at comparatively large distances. ER -