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* Department of Pathology, The osteoclast is distinguished from other
macrophage polykaryons by its polarization, a feature
induced by substrate recognition. The most striking
component of the polarized osteoclast is its ruffled
membrane, probably reflecting insertion of intracellular vesicles into the bone apposed plasmalemma. The
failure of osteoclasts in c-src Department of Cell Biology, Washington University School of Medicine, St. Louis, Missouri 63110
/ osteopetrotic mice to
form ruffled membranes indicates pp60c-src (c-src) is essential to osteoclast polarization. Interestingly, c-src itself is a vesicular protein that targets the ruffled membrane. This being the case, we hypothesized that matrix
recognition by osteoclasts, and their precursors, induces c-src to associate with microtubules that traffic
proteins to the cell surface. We find abundant c-src associates with tubulin immunoprecipitated from avian
marrow macrophages (osteoclast precursors) maintained in the adherent, but not nonadherent, state.
Since the two proteins colocalize only within adherent
avian osteoclast-like cells examined by double antibody
immunoconfocal microscopy, c-src/tubulin association reflects an authentic intracellular event. C-src/tubulin
association is evident within 90 min of cell-substrate
recognition, and the event does not reflect increased
expression of either protein. In vitro kinase assay demonstrates tubulin-associated c-src is enzymatically active, phosphorylating itself as well as exogenous substrate. The increase in microtubule-associated kinase
activity attending adhesion mirrors tubulin-bound c-src
and does not reflect enhanced specific activity. The fact
that microtubule-dissociating drugs, as well as cold,
prevent adherence-induced c-src/tubulin association indicates the protooncogene complexes primarily, if not
exclusively, with polymerized tubulin. Association of
the two proteins does not depend upon protein tyrosine
phosphorylation and is substrate specific, as it is induced by vitronectin and fibronectin but not type 1 collagen. Finally, consistent with cotransport of c-src and
the osteoclast vacuolar proton pump to the polarized
plasmalemma, the H+-ATPase decorates microtubules
in a manner similar to the protooncogene, specifically
coimmunoprecipitates with c-src from the osteoclast light Golgi membrane fraction, and is present, with c-src,
in preparations enriched with acidifying vesicles reconstituted from the osteoclast ruffled membrane.
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