IMMUNODETECTION OF ALPHA-ACTININ IN FOCAL ADHESIONS IS LIMITED BY ANTIBODY INACCESSIBILITY

In this study we demonstrate that alpha-actinin is a prominent compone nt of the focal adhesions of nonmuscle cells but that the alpha-actini n in focal adhesions is largely inaccessible to staining with antibodi es against alpha-actinin, Our results explain a controversy that has e xisted in the literature. Investigators who microinject alpha-actinin into nonmuscle cells have routinely observed significant incorporation of alpha-actinin into focal adhesions as well as stress fibers, Immun ofluorescence and immunoelectron microscopy have, however, indicated t hat alpha-actinin is located farther from the membrane than either tal in or vinculin, Immunofluorescence studies of smooth muscle dense plaq ues and myotendinous junctions have also yielded conflicting results r egarding the presence or absence of alpha-actinin at these sites, Here , we confirm that cu-actinin immunofluorescence of fibroblasts yields weak or absent staining of focal adhesions, We also demonstrate that m icroinjected alpha-actinin readily incorporates into focal adhesions, However, various antisera against either the cell's endogenous alpha-a ctinin or against the microinjected chicken gizzard alpha-actinin fail to stain focal adhesions despite the presence of microinjected alpha- actinin at these sites, Furthermore, disassembly of stress fibers indu ced by dibutyrl cAMP demonstrates that alpha-actinin persists in focal adhesions in the absence of associated stress fibers, suggesting that alpha-actinin's association with focal adhesions is independent of st ress fibers. (C) 1995 Academic Press, Inc.