Immunodetection of alpha-actinin in focal adhesions is limited by antibody inaccessibility

In this study we demonstrate that alpha-actinin is a prominent component of the focal adhesions of nonmuscle cells but that the alpha-actinin in focal adhesions is largely inaccessible to staining with antibodies against alpha-actinin. Our results explain a controversy that has existed 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. Immunofluorescence and immunoelectron microscopy have, however, indicated that alpha-actinin is located farther from the membrane than either talin or vinculin. Immunofluorescence studies of smooth muscle dense plaques and myotendinous junctions have also yielded conflicting results regarding the presence or absence of alpha-actinin at these sites. Here, we confirm that alpha-actinin immunofluorescence of fibroblasts yields weak or absent staining of focal adhesions. We also demonstrate that microinjected alpha-actinin readily incorporates into focal adhesions. However, various antisera against either the cell's endogenous alpha-actinin 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 induced 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 stress fibers.