Dissecting the link between stress fibres and focal adhesions by CALI with EGFP fusion proteins
Dissecting the link between stress fibres and focal adhesions by CALI with EGFP fusion proteins
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DOI：
10.1038/ncb772
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发表时间：
2002-04-01
影响因子：
21.3
通讯作者：
Jacobson, K
中科院分区：
文献类型：
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作者：
Rajfur, Z;Roy, P;Jacobson, K
发色团辅助激光灭活(CALI)是一种光介导技术，用于选择性地灭活细胞内的蛋白质。在这里，我们证明了绿色荧光蛋白可以作为一种钙试剂来局部灭活活细胞中的蛋白质。我们发现，聚焦激光照射在瑞士3T3成纤维细胞中表达的EGFP-α-肌动蛋白导致应力纤维从局灶性粘连(FA)中分离，而干涉反射显微镜(IRM)确定的FA的完整性被保留。此外，与粘着斑激酶(FAK)在FA信号而不是FA结构中的作用一致，激光照射EGFP-FAK既没有引起可见的FA损伤，也没有引起应力性纤维脱落，尽管在体外分离的EGFP-FAK降低了它的激酶活性，但不能与Paxlin结合。这些数据表明，特定FA组分的CALI可以用来精确地剖析维持这种细胞骨架结构所需的单个蛋白质的功能意义。体外CALI实验还表明，EGFP-α-肌动蛋白与β(1)整合素亚基的细胞质区域结合减少，但不与肌动蛋白结合。因此，α-肌动蛋白对于微丝与FA中的整合素的结合是必不可少的。钙诱导的α-肌动蛋白的变化导致该连接的断裂和随后应力纤维的回缩。
Chromophore-assisted laser inactivation (CALI) is a light-mediated technique used to selectively inactivate proteins within cells. Here, we demonstrate that GFP can be used as a CALI reagent to locally inactivate proteins in living cells. We show that focused laser irradiation of EGFP-alpha-actinin expressed in Swiss 3T3 fibroblasts results in the detachment of stress fibres from focal adhesions (FAs), whereas the integrity of FAs, as determined by interference reflection microscopy (IRM), is preserved. Moreover, consistent with a function for focal adhesion kinase (FAK) in FA signalling and not FA structure, laser irradiation of EGFP-FAK did not cause either visible FA damage or stress fibre detachment, although in vitro CALI of isolated EGFP-FAK decreased its kinase activity, but not its binding to paxillin. These data indicate that CALI of specific FA components may be used to precisely dissect the functional significance of individual proteins required for the maintenance of this cytoskeletal structure. In vitro CALI experiments also demonstrated a reduction of EGFP-alpha-actinin binding to the cytoplasmic domain of the beta(1) integrin subunit, but not to actin. Thus, alpha-actinin is essential for the binding of microfilaments to integrins in the FA. CALI-induced changes in alpha-actinin result in the breakage of that link and the subsequent retraction of the stress fibre.