The dynamic interrelationships of actin and vinculin in cultured cells

The dynamic state of cytoskeletal proteins actin and vinculin was studied in living cells using microinjection of fluorescently-labeled proteins combined with fluorescence photobleaching recovery (FPR). It is shown that both proteins maintain a dynamic equilibrium between their diffusible pools in the cytoplasms and their "organized" cytoskeletal fraction. These interrelationships could be simulated in model systems consisting of isolated substrate attached membranes. It was demonstrated that fluorophore bound vinculin was incorporated into the exposed focal contacts and that this binding was largely actin independent. These results are in line with the hypothesis that local contacts induce binding of vinculin to the endofacial surface of the membranes and that this region serves as a nucleation center for the assembly of actin bundles.