Centre for Human Development, Stem Cells and Regeneration, Human Development and Health, Institute of Developmental Sciences, University of Southampton, Southampton, United Kingdom.
Centre for Human Development, Stem Cells and Regeneration, Human Development and Health, Institute of Developmental Sciences, University of Southampton, Southampton, United Kingdom; Mechanical Engineering, University of Southampton, Southampton, United Kingdom.
Biophys J. 2018 Jun 5;114(11):2743-2755. doi: 10.1016/j.bpj.2018.03.037.
Extracellular matrix stiffness has a profound effect on the behavior 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 hypothesized 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 extracellular-matrix-functionalized 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-labeled 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 Rho-associated protein kinase-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.
细胞外基质的硬度对许多细胞类型的行为有深远的影响。贴壁细胞对其附着的材料施加收缩力，并感知材料对变形的阻力——即其硬度。这取决于材料的弹性模量和厚度，因此单个细胞能够通过厚度低（<10μm）的软水凝胶材料感知下面的硬材料。在这里，我们假设细胞凝聚体施加的力更大，并且比单细胞产生更多的水凝胶变形，因此它们能够比单细胞更深入地感受下面的材料。为了验证这一点，我们调制了软（1kPa）弹性细胞外基质功能化聚丙烯酰胺水凝胶的厚度，使其附着在玻璃基底上，并允许 MG63 细胞凝聚体在其表面形成。通过延时荧光显微镜成像定量测量细胞形态和荧光标记的水凝胶的变形。单细胞的扩展随水凝胶厚度的减小而增加，数据拟合为半最大响应厚度为 3.2μm 的指数模型。通过定量分析具有定义面积的凝聚体中细胞的面积，我们同样发现，凝聚体中细胞的扩展随水凝胶厚度的减小而增加，但半最大响应厚度为 54μm。深度感应取决于 Rho 相关蛋白激酶介导的细胞收缩性。与薄水凝胶相比，厚水凝胶表面的水凝胶变形显著更大。此外，与薄水凝胶相比，厚水凝胶上的变形从凝聚体的外围延伸出更远的距离。我们的数据表明，通过集体作用，细胞在弹性水凝胶下的刚性材料中比单个细胞更深地进行机械感应。这提出了一种可能性，即凝聚体或薄片中细胞的集体作用可能允许细胞在相对较大的距离上感知具有不同材料特性的结构。
