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Substrate recognition by osteoclast precursors induces C-src/microtubule association.

Abu-Amer Y, Ross FP, Schlesinger P, Tondravi MM, Teitelbaum SL - J. Cell Biol. (1997)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.

ABSTRACT
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-/- osteopetrotic mice to form ruffled membranes indicates pp60(c-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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C-src associates with microtubules generated in vitro. Isolated microtubules were generated from cell lysates and isolated.  The microtubules were fixed and stained with both antitubulin and anti–c-src antibodies followed by fluorescent-labeled secondary antibodies, Texas red (for c-src) and FITC (for tubulin), and examined by confocal microscopy. b represents excitation of FITC (tubulin), c  represents Texas red (c-src), and d represents both fluorochromes and thus, colocalization of the antibodies. a represents incubation  with irrelevant primary murine and rabbit antibodies and FITC and Texas red and excitation of both fluorochromes. Bar, 10 μm.
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Figure 10: C-src associates with microtubules generated in vitro. Isolated microtubules were generated from cell lysates and isolated. The microtubules were fixed and stained with both antitubulin and anti–c-src antibodies followed by fluorescent-labeled secondary antibodies, Texas red (for c-src) and FITC (for tubulin), and examined by confocal microscopy. b represents excitation of FITC (tubulin), c represents Texas red (c-src), and d represents both fluorochromes and thus, colocalization of the antibodies. a represents incubation with irrelevant primary murine and rabbit antibodies and FITC and Texas red and excitation of both fluorochromes. Bar, 10 μm.

Mentions: Although our lysis conditions are known to preserve cytoskeletal architecture (51), the possibility exists that microtubules depolymerize in vitro. To assure, as in vivo, c-src associates in vitro with microtubules, we reconstituted these structures from cell lysates, separated them on a sucrose gradient from “free” c-src, and examined them using double antibody immunofluorescent confocal microscopy. As seen in Fig. 10, c-src decorates reconstituted microtubules.


Substrate recognition by osteoclast precursors induces C-src/microtubule association.

Abu-Amer Y, Ross FP, Schlesinger P, Tondravi MM, Teitelbaum SL - J. Cell Biol. (1997)

C-src associates with microtubules generated in vitro. Isolated microtubules were generated from cell lysates and isolated.  The microtubules were fixed and stained with both antitubulin and anti–c-src antibodies followed by fluorescent-labeled secondary antibodies, Texas red (for c-src) and FITC (for tubulin), and examined by confocal microscopy. b represents excitation of FITC (tubulin), c  represents Texas red (c-src), and d represents both fluorochromes and thus, colocalization of the antibodies. a represents incubation  with irrelevant primary murine and rabbit antibodies and FITC and Texas red and excitation of both fluorochromes. Bar, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2139850&req=5

Figure 10: C-src associates with microtubules generated in vitro. Isolated microtubules were generated from cell lysates and isolated. The microtubules were fixed and stained with both antitubulin and anti–c-src antibodies followed by fluorescent-labeled secondary antibodies, Texas red (for c-src) and FITC (for tubulin), and examined by confocal microscopy. b represents excitation of FITC (tubulin), c represents Texas red (c-src), and d represents both fluorochromes and thus, colocalization of the antibodies. a represents incubation with irrelevant primary murine and rabbit antibodies and FITC and Texas red and excitation of both fluorochromes. Bar, 10 μm.
Mentions: Although our lysis conditions are known to preserve cytoskeletal architecture (51), the possibility exists that microtubules depolymerize in vitro. To assure, as in vivo, c-src associates in vitro with microtubules, we reconstituted these structures from cell lysates, separated them on a sucrose gradient from “free” c-src, and examined them using double antibody immunofluorescent confocal microscopy. As seen in Fig. 10, c-src decorates reconstituted microtubules.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.

ABSTRACT
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-/- osteopetrotic mice to form ruffled membranes indicates pp60(c-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.

Show MeSH
Related in: MedlinePlus