Dissecting the link between stress fibres and focal adhesions by CALI with EGFP fusion proteins

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Abstract
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–α-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–α-actinin binding to the cytoplasmic domain of the β1 integrin subunit, but not to actin. Thus, α-actinin is essential for the binding of microfilaments to integrins in the FA. CALI-induced changes in α-actinin result in the breakage of that link and the subsequent retraction of the stress fibre.