Interaction between vacuolar H+-ATPase and microfilaments during osteoclast activation

Vacuolar H+-ATPases (V-ATPases) are multisubunit enzymes that acidify compa rtments of the vacuolar system of all eukaryotic cells. In osteoclasts, the cells that degrade bone, V-ATPases, are recruited from intracellular membr ane compartments to the ruffled membrane, a specialized domain of the plasm a membrane, where they are maintained at high densities, serving to acidify the resorption bay at the osteoclast attachment site on bone (Blair, H. C, , Teitelbaum, S. L,, Ghiselli, R., and Gluck, S. L. (1989) Science 249, 855 -857). Here, we describe a new mechanism involved in controlling the activi ty of the bone-resorptive cell. V-ATPase in osteoclasts cultured in vitro w as found to form a detergent-insoluble complex with actin and myosin II thr ough direct binding of V-ATPase to actin filaments. Plating bone marrow cel ls onto dentine slices, a physiologic stimulus that activates osteoclast re sorption, produced a profound change in the association of the V-ATPase wit h actin, assayed by coimmunoprecipitation and immunocytochemical colocaliza tion of actin filaments and V-ATPase in osteoclasts. Mouse marrow and bovin e kidney V-ATPase bound rabbit muscle F-actin directly with a maximum stoic hiometry of 1 mol of V-ATPase per 8 mol of F-actin and an apparent affinity of 0.05 mu M. Electron microscopy of negatively stained samples confirmed the binding interaction. These findings link transport of V-ATPase to reorg anization of the actin cytoskeleton during osteoclast activation.