Dynamics of Alpha-actinin in Focal Adhesions and Stress Fibers Visualized with Alpha-actinin-green Fluorescent Protein

Motile cells undergo changes in cell adhesion, behavior, and shape that are mediated by small-scale cytoskeletal rearrangements. These rearrangements have proven difficult to follow quantitatively in living cells, without disrupting the very structures and delicate protein balances under study. We have expressed a prominent cytoskeletal protein, alpha-actinin, as a fusion with green fluorescent protein (alpha AGFP), and have followed this construct's movements within transfected mouse Swiss 3T3 and BALB/c fibroblasts. alpha AGFP was expressed at low levels to avoid overexpression artifacts. alpha AGFP localized to cellular structures, including stress fibers, focal adhesions, microspikes, and lamellipodia. High-resolution video-microscopy revealed that the alpha AGFP construct could be seen relocating to focal adhesions early in their formation and shortly thereafter to stress-fiber dense bodies. By Fluorescent Recovery After Photo-bleaching (FRAP) techniques, alpha AGFP was found to have similar exchange rates and protein stability in focal adhesions and stress fibers (despite the known differences in protein composition in these two structures). This raises the possibility that the two structures share common key regulatory factors and may not be as affected by protein-protein binding interactions as previously suggested. Additionally, the exchange rates revealed by video-microscopy and FRAP analysis of alpha AGFP are more rapid than those reported previously, which were obtained using microinjection of large excesses of fluorescently-tagged protein.