Rajfur Zenon, Roy Partha, Otey Carol, Romer Lewis, Jacobson Ken
Department of Cell and Developmental Biology, University of North Carolina, Chapel Hill, NC, USA.
Nat Cell Biol. 2002 Apr;4(4):286-93. doi: 10.1038/ncb772.
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.
生色团辅助激光灭活（CALI）是一种光介导技术，用于选择性地灭活细胞内的蛋白质。在此，我们证明绿色荧光蛋白（GFP）可作为一种CALI试剂，在活细胞中局部灭活蛋白质。我们发现，对瑞士3T3成纤维细胞中表达的增强型绿色荧光蛋白-α-辅肌动蛋白进行聚焦激光照射，会导致应力纤维从粘着斑（FAs）上脱离，而通过干涉反射显微镜（IRM）测定，粘着斑的完整性得以保留。此外，与粘着斑激酶（FAK）在粘着斑信号传导而非粘着斑结构中的功能一致，对增强型绿色荧光蛋白-FAK进行激光照射，既未造成可见的粘着斑损伤亦未导致应力纤维脱离，尽管对分离的增强型绿色荧光蛋白-FAK进行体外CALI降低了其激酶活性，但未影响其与桩蛋白的结合。这些数据表明，特定粘着斑成分的CALI可用于精确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，增强型绿色荧光蛋白-α-辅肌动蛋白与β1整合素亚基细胞质结构域的结合减少，但与肌动蛋白的结合未减少。因此，α-辅肌动蛋白对于微丝在粘着斑中与整合素的结合至关重要。CALI诱导的α-辅肌动蛋白变化导致该连接断裂，随后应力纤维回缩。
