Glogauer M, Arora P, Yao G, Sokholov I, Ferrier J, McCulloch C A
MRC Group in Periodontal Physiology, University of Toronto, Ontario, Canada.
J Cell Sci. 1997 Jan;110 ( Pt 1):11-21. doi: 10.1242/jcs.110.1.11.
The actin-dependent sensory and response elements of stromal cells that are involved in mechanical signal transduction are poorly understood. To study mechanotransduction we have described previously a collagen-magnetic bead model in which application of well-defined forces to integrins induces an immediate (< 1 second) calcium influx. In this report we used the model to determine the role of calcium ions and tyrosine-phosphorylation in the regulation of force-mediated actin assembly and the resulting change in membrane rigidity. Collagen-beads were bound to cells through the focal adhesion-associated proteins talin, vinculin, alpha 2-integrin and beta-actin, indicating that force application was mediated through cytoskeletal elements. When force (2 N/m2) was applied to collagen beads, confocal microscopy showed a marked vertical extension of the cell which was counteracted by an actin-mediated retraction. Immunoblotting showed that force application induced F-actin accumulation at the bead-membrane complex but vinculin, talin and alpha 2-integrin remained unchanged. Atomic force microscopy showed that membrane rigidity increased 6-fold in the vicinity of beads which had been exposed to force. Force also induced tyrosine phosphorylation of several cytoplasmic proteins including paxillin. The force-induced actin accumulation was blocked in cells loaded with BAPTA/AM or in cells preincubated with genistein, an inhibitor of tyrosine phosphorylation. Repeated force application progressively inhibited the amplitude of force-induced calcium ion flux. As force-induced actin reorganization was dependent on calcium and tyrosine phosphorylation, and as progressive increases of filamentous actin in the submembrane cortex were correlated with increased membrane rigidity and dampened calcium influx, we suggest that cortical actin regulates stretch-activated cation permeable channel activity and provides a desensitization mechanism for cells exposed to repeated long-term mechanical stimuli. The actin response may be cytoprotective since it counteracts the initial force-mediated membrane extension and potentially strengthens cytoskeletal integrity at force-transfer points.
基质细胞中参与机械信号转导的肌动蛋白依赖性感觉和反应元件目前还知之甚少。为了研究机械转导，我们之前描述了一种胶原蛋白 - 磁珠模型，在该模型中，对整合素施加明确的力会立即（<1秒）引起钙离子内流。在本报告中，我们使用该模型来确定钙离子和酪氨酸磷酸化在力介导的肌动蛋白组装调节以及由此导致的膜刚性变化中的作用。胶原蛋白珠通过与粘着斑相关的蛋白踝蛋白、纽蛋白、α2整合素和β - 肌动蛋白与细胞结合，表明力的施加是通过细胞骨架元件介导的。当对胶原蛋白珠施加力（2 N/m2）时，共聚焦显微镜显示细胞有明显的垂直延伸，这被肌动蛋白介导的回缩所抵消。免疫印迹显示，施加力会诱导F - 肌动蛋白在珠 - 膜复合物处积累，但纽蛋白、踝蛋白和α2整合素保持不变。原子力显微镜显示，在受到力作用的珠附近，膜刚性增加了6倍。力还诱导了包括桩蛋白在内的几种细胞质蛋白的酪氨酸磷酸化。在加载了BAPTA/AM的细胞或用酪氨酸磷酸化抑制剂染料木黄酮预孵育的细胞中，力诱导的肌动蛋白积累被阻断。重复施加力会逐渐抑制力诱导的钙离子内流幅度。由于力诱导的肌动蛋白重组依赖于钙离子和酪氨酸磷酸化，并且由于膜下皮质中丝状肌动蛋白的逐渐增加与膜刚性增加和钙离子内流减弱相关，我们认为皮质肌动蛋白调节拉伸激活的阳离子渗透通道活性，并为暴露于重复长期机械刺激的细胞提供脱敏机制。肌动蛋白反应可能具有细胞保护作用，因为它抵消了最初力介导的膜延伸，并可能在力传递点加强细胞骨架的完整性。
