Department of Biology, Stanford University, Stanford, CA 94305.
Department of Bioengineering, Stanford University, Stanford, CA 94305.
Proc Natl Acad Sci U S A. 2017 Jul 18;114(29):E5835-E5844. doi: 10.1073/pnas.1618676114. Epub 2017 Jul 3.
Mechanical cues are sensed and transduced by cell adhesion complexes to regulate diverse cell behaviors. Extracellular matrix (ECM) rigidity sensing by integrin adhesions has been well studied, but rigidity sensing by cadherins during cell adhesion is largely unexplored. Using mechanically tunable polyacrylamide (PA) gels functionalized with the extracellular domain of E-cadherin (Ecad-Fc), we showed that E-cadherin-dependent epithelial cell adhesion was sensitive to changes in PA gel elastic modulus that produced striking differences in cell morphology, actin organization, and membrane dynamics. Traction force microscopy (TFM) revealed that cells produced the greatest tractions at the cell periphery, where distinct types of actin-based membrane protrusions formed. Cells responded to substrate rigidity by reorganizing the distribution and size of high-traction-stress regions at the cell periphery. Differences in adhesion and protrusion dynamics were mediated by balancing the activities of specific signaling molecules. Cell adhesion to a 30-kPa Ecad-Fc PA gel required Cdc42- and formin-dependent filopodia formation, whereas adhesion to a 60-kPa Ecad-Fc PA gel induced Arp2/3-dependent lamellipodial protrusions. A quantitative 3D cell-cell adhesion assay and live cell imaging of cell-cell contact formation revealed that inhibition of Cdc42, formin, and Arp2/3 activities blocked the initiation, but not the maintenance of established cell-cell adhesions. These results indicate that the same signaling molecules activated by E-cadherin rigidity sensing on PA gels contribute to actin organization and membrane dynamics during cell-cell adhesion. We hypothesize that a transition in the stiffness of E-cadherin homotypic interactions regulates actin and membrane dynamics during initial stages of cell-cell adhesion.
机械线索通过细胞黏附复合物被感知和转导，从而调节多种细胞行为。整合素黏附对细胞外基质（ECM）硬度的感知已得到充分研究，但细胞黏附过程中钙黏蛋白对硬度的感知在很大程度上仍未被探索。我们使用机械可调聚丙酰胺（PA）凝胶，该凝胶被 E-钙黏蛋白（Ecad-Fc）的细胞外结构域功能化，结果表明，E-钙黏蛋白依赖性上皮细胞黏附对 PA 凝胶弹性模量的变化敏感，这种变化会导致细胞形态、肌动蛋白组织和膜动力学的显著差异。牵引力显微镜（TFM）显示，细胞在细胞边缘产生最大的牵引力，在那里形成了不同类型的基于肌动蛋白的膜突起。细胞通过重新组织细胞边缘的高牵引力-压力区域的分布和大小来响应基质硬度。黏附和突起动力学的差异是通过平衡特定信号分子的活性来介导的。细胞黏附到 30kPa 的 Ecad-Fc PA 凝胶需要 Cdc42 和形成蛋白依赖性丝状伪足形成，而黏附到 60kPa 的 Ecad-Fc PA 凝胶则诱导 Arp2/3 依赖性片状伪足的形成。定量的 3D 细胞-细胞黏附测定和活细胞成像显示，抑制 Cdc42、形成蛋白和 Arp2/3 的活性阻断了细胞-细胞黏附的起始，但不阻断已建立的细胞-细胞黏附的维持。这些结果表明，PA 凝胶上 E-钙黏蛋白硬度感知激活的相同信号分子有助于细胞-细胞黏附过程中的肌动蛋白组织和膜动力学。我们假设，E-钙黏蛋白同质相互作用的刚度转变调节细胞-细胞黏附初始阶段的肌动蛋白和膜动力学。
