Cattelino A, Albertinazzi C, Bossi M, Critchley D R, de Curtis I
Cell Adhesion Unit, Department for Biological and Technological Research, San Raffaele Scientific Institute, 20132 Milan, Italy.
Mol Biol Cell. 1999 Feb;10(2):373-91. doi: 10.1091/mbc.10.2.373.
Assembly and modulation of focal adhesions during dynamic adhesive processes are poorly understood. We describe here the use of ventral plasma membranes from adherent fibroblasts to explore mechanisms regulating integrin distribution and function in a system that preserves the integration of these receptors into the plasma membrane. We find that partial disruption of the cellular organization responsible for the maintenance of organized adhesive sites allows modulation of integrin distribution by divalent cations. High Ca2+ concentrations induce quasi-reversible diffusion of beta1 integrins out of focal adhesions, whereas low Ca2+ concentrations induce irreversible recruitment of beta1 receptors along extracellular matrix fibrils, as shown by immunofluorescence and electron microscopy. Both effects are independent from the presence of actin stress fibers in this system. Experiments with cells expressing truncated beta1 receptors show that the cytoplasmic portion of beta1 is required for low Ca2+-induced recruitment of the receptors to matrix fibrils. Analysis with function-modulating antibodies indicates that divalent cation-mediated receptor distribution within the membrane correlates with changes in the functional state of the receptors. Moreover, reconstitution experiments show that purified alpha-actinin colocalizes and redistributes with beta1 receptors on ventral plasma membranes depleted of actin, implicating binding of alpha-actinin to the receptors. Finally, we found that recruitment of exogenous actin is specifically restricted to focal adhesions under conditions in which new actin polymerization is inhibited. Our data show that the described system can be exploited to investigate the mechanisms of integrin function in an experimental setup that permits receptor redistribution. The possibility to uncouple, under cell-free conditions, events involved in focal adhesion and actin cytoskeleton assembly should facilitate the comprehension of the underlying molecular mechanisms.
在动态黏附过程中，黏着斑的组装和调节机制尚不清楚。我们在此描述了使用贴壁成纤维细胞的腹侧质膜来探索调节整合素分布和功能的机制，该系统保留了这些受体整合到质膜中的特性。我们发现，负责维持有组织黏附位点的细胞组织的部分破坏允许二价阳离子调节整合素分布。高钙浓度诱导β1整合素从黏着斑中发生准可逆扩散，而低钙浓度则诱导β1受体沿细胞外基质纤维不可逆募集，免疫荧光和电子显微镜观察结果表明了这一点。在该系统中，这两种效应均与肌动蛋白应力纤维的存在无关。对表达截短β1受体的细胞进行的实验表明，低钙诱导受体募集到基质纤维上需要β1的细胞质部分。用功能调节抗体进行的分析表明，膜内二价阳离子介导的受体分布与受体功能状态的变化相关。此外，重组实验表明，纯化的α-辅肌动蛋白与β1受体在缺乏肌动蛋白的腹侧质膜上共定位并重新分布，这表明α-辅肌动蛋白与受体结合。最后，我们发现，在新肌动蛋白聚合受到抑制的条件下，外源性肌动蛋白的募集特异性地局限于黏着斑。我们的数据表明，所述系统可用于在允许受体重新分布的实验设置中研究整合素功能的机制。在无细胞条件下解开黏着斑和肌动蛋白细胞骨架组装相关事件的可能性，应有助于理解其潜在的分子机制。
