The regulation of cellular force production relies on the complex interplay between a well-conserved set of proteins of the cytoskeleton: actin, myosin, and α-actinin. Despite our deep knowledge of the role of these proteins in force production at the molecular scale, our understanding of the biochemical regulation of the magnitude of traction forces generated at the entire-cell level has been limited, notably by the technical challenge of measuring traction forces and the endogenous biochemical composition in the same cell. In this study, we developed an alternative Traction-Force Microscopy (TFM) assay, which used a combination of hydrogel micropatterning to define cell adhesion and shape and an intermediate fixation/immunolabeling step to characterize strain energies and the endogenous protein contents in single epithelial cells. Our results demonstrated that both the signal intensity and the area of the Focal Adhesion (FA)–associated protein vinculin showed a strong positive correlation with strain energy in mature FAs. Individual contents from actin filament and phospho-myosin displayed broader deviation in their linear relationship to strain energies. Instead, our quantitative analyzes demonstrated that their relative amount exhibited an optimum ratio of phospho-myosin to actin, allowing maximum force production by cells. By contrast, although no correlation was identified between individual α-actinin content and strain energy, the ratio of α-actinin to actin filaments was inversely related to strain energy. Hence, our results suggest that, in the cellular model studied, traction-force magnitude is dictated by the relative numbers of molecular motors and cross-linkers per actin filament, rather than the amounts of an individual component in the cytoskeletal network. This assay offers new perspectives to study in more detail the complex interplay between the endogenous biochemical composition of individual cells and the force they produce.

细胞力产生的调节依赖于一组保存完好的细胞骨架蛋白质之间的复杂相互作用：肌动蛋白、肌球蛋白和 α-肌动蛋白。尽管我们对这些蛋白质在分子尺度上的力产生中的作用有深入的了解，但我们对在整个细胞水平上产生的牵引力大小的生化调节的理解受到限制，尤其是测量牵引力的技术挑战和同一细胞内的内源性生化成分。在这项研究中，我们开发了一种替代的牵引力显微镜 (TFM) 测定法，该测定法结合使用水凝胶微图案来定义细胞粘附和形状，并使用中间固定/免疫标记步骤来表征单个上皮细胞中的应变能和内源性蛋白质含量. 我们的结果表明，信号强度和粘着斑（FA）相关蛋白纽蛋白的面积都与成熟 FA 中的应变能呈强正相关。肌动蛋白丝和磷酸肌球蛋白的单个含量与应变能的线性关系显示出更大的偏差。相反，我们的定量分析表明，它们的相对量表现出磷酸肌球蛋白与肌动蛋白的最佳比例，允许细胞产生最大的力。相比之下，虽然在单个 α-肌动蛋白含量和应变能之间没有发现相关性，但 α-肌动蛋白与肌动蛋白丝的比率与应变能呈负相关。因此，我们的结果表明，在所研究的细胞模型中，牵引力的大小取决于每个肌动蛋白丝的分子马达和交联剂的相对数量，而不是细胞骨架网络中单个成分的数量。该测定为更详细地研究单个细胞的内源性生化成分与其产生的力之间的复杂相互作用提供了新的视角。