Laboratory of Functional Molecular Imaging, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892, USA.
Proc Natl Acad Sci U S A. 2013 Jun 25;110(26):E2352-61. doi: 10.1073/pnas.1221637110. Epub 2013 Jun 10.
Cells are mechanosensitive to extracellular matrix (ECM) deformation, which can be caused by muscle contraction or changes in hydrostatic pressure. Focal adhesions (FAs) mediate the linkage between the cell and the ECM and initiate mechanically stimulated signaling events. We developed a stretching apparatus in which cells grown on fibronectin-coated elastic substrates can be stretched and imaged live to study how FAs dynamically respond to ECM deformation. Human bone osteosarcoma epithelial cell line U2OS was transfected with GFP-paxillin as an FA marker and subjected to sustained uniaxial stretching. Two responses at different timescales were observed: rapid FA growth within seconds after stretching, and delayed FA disassembly and loss of cell polarity that occurred over tens of minutes. Rapid FA growth occurred in all cells; however, delayed responses to stretch occurred in an orientation-specific manner, specifically in cells with their long axes perpendicular to the stretching direction, but not in cells with their long axes parallel to stretch. Pharmacological treatments demonstrated that FA kinase (FAK) promotes but Src inhibits rapid FA growth, whereas FAK, Src, and calpain 2 all contribute to delayed FA disassembly and loss of polarity in cells perpendicular to stretching. Immunostaining for phospho-FAK after stretching revealed that FAK activation was maximal at 5 s after stretching, specifically in FAs oriented perpendicular to stretch. We hypothesize that orientation-specific activation of strain/stress-sensitive proteins in FAs upstream to FAK and Src promote orientation-specific responses in FA growth and disassembly that mediate polarity rearrangement in response to sustained stretch.
细胞对细胞外基质（ECM）的变形具有机械敏感性，这种变形可以由肌肉收缩或静水压力的变化引起。黏附斑（FA）介导细胞与 ECM 之间的连接，并启动机械刺激信号事件。我们开发了一种拉伸装置，在该装置中，可以拉伸生长在纤维连接蛋白涂覆的弹性基底上的细胞，并对其进行实时成像，以研究 FA 如何动态响应 ECM 的变形。人骨肉瘤上皮细胞系 U2OS 转染 GFP-桩蛋白作为 FA 标记物，并进行持续的单轴拉伸。观察到两种不同时间尺度的反应：拉伸后几秒钟内快速 FA 生长，以及数十分钟内延迟的 FA 解体和细胞极性丧失。快速 FA 生长发生在所有细胞中；然而，对拉伸的延迟反应以定向特异性的方式发生，特别是在其长轴垂直于拉伸方向的细胞中，但在其长轴平行于拉伸的细胞中不发生。药理处理表明，FA 激酶（FAK）促进但Src 抑制快速 FA 生长，而 FAK、Src 和钙蛋白酶 2 都有助于垂直于拉伸的细胞中 FA 的解体和极性丧失。拉伸后 FA 中磷酸化 FAK 的免疫染色显示，FAK 的激活在拉伸后 5 秒达到最大值，特别是在垂直于拉伸的 FA 中。我们假设 FAK 和 Src 上游的应变/应激敏感蛋白在 FA 中的定向特异性激活促进了 FA 生长和解体的定向特异性反应，从而介导对持续拉伸的极性重排。
