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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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Matrix-dependent intracellular  association of c-src/tubulin association by  confocal microscopy. Avian marrow macrophages were kept in Teflon-coated beakers or plated on coverslips. Adherent cells  were differentiated into osteoclast-like cells  as previously described (1). Adherent and  nonadherent cells were fixed, Triton permeabilized, incubated with both antitubulin  and anti–c-src antibodies followed by fluorescent-labeled secondary antibodies, Texas  red (for src) and FITC (for tubulin), and examined by confocal microscopy. A–C represent cells plated on vitronectin-coated  plates. D–F represent cells plated on collagen type-I, and G–I represent cells kept  nonadherent. Each group represents excitation of the same cell. A, D, and G represent  excitation of Texas red (c-src), and B, E,  and H represent FITC (tubulin). C, F, and I  represent excitation of both fluorochromes  and thus, colocalization of the antibodies.  Similar results were reproduced when using  anti–c-src polyclonal and antitubulin monoclonal antibodies, followed by the relevant  secondary-labeled antibodies (data not  shown).
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Figure 13: Matrix-dependent intracellular association of c-src/tubulin association by confocal microscopy. Avian marrow macrophages were kept in Teflon-coated beakers or plated on coverslips. Adherent cells were differentiated into osteoclast-like cells as previously described (1). Adherent and nonadherent cells were fixed, Triton permeabilized, incubated with both antitubulin and anti–c-src antibodies followed by fluorescent-labeled secondary antibodies, Texas red (for src) and FITC (for tubulin), and examined by confocal microscopy. A–C represent cells plated on vitronectin-coated plates. D–F represent cells plated on collagen type-I, and G–I represent cells kept nonadherent. Each group represents excitation of the same cell. A, D, and G represent excitation of Texas red (c-src), and B, E, and H represent FITC (tubulin). C, F, and I represent excitation of both fluorochromes and thus, colocalization of the antibodies. Similar results were reproduced when using anti–c-src polyclonal and antitubulin monoclonal antibodies, followed by the relevant secondary-labeled antibodies (data not shown).

Mentions: To determine if the specific matrix induction of c-src/tubulin association, demonstrated, by immunoprecipitation (Fig. 12), is an intracellular event, we once again turned to immunoconfocal microscopy (Fig. 13). Macrophages were permeabilized and incubated with anti–c-src and antitubulin antibodies as well as fluoresceinated secondary antibodies as described in Fig. 4. Whereas c-src/microtubule colocalization is extant in cells plated on vitronectin (Fig. 13, A–C), microtubules are not evident in those on collagen (Fig. 13, D–F), which are indistinguishable from their nonadherent counterparts (Fig. 13, G–I), wherein the two molecules fail to associate.


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)

Matrix-dependent intracellular  association of c-src/tubulin association by  confocal microscopy. Avian marrow macrophages were kept in Teflon-coated beakers or plated on coverslips. Adherent cells  were differentiated into osteoclast-like cells  as previously described (1). Adherent and  nonadherent cells were fixed, Triton permeabilized, incubated with both antitubulin  and anti–c-src antibodies followed by fluorescent-labeled secondary antibodies, Texas  red (for src) and FITC (for tubulin), and examined by confocal microscopy. A–C represent cells plated on vitronectin-coated  plates. D–F represent cells plated on collagen type-I, and G–I represent cells kept  nonadherent. Each group represents excitation of the same cell. A, D, and G represent  excitation of Texas red (c-src), and B, E,  and H represent FITC (tubulin). C, F, and I  represent excitation of both fluorochromes  and thus, colocalization of the antibodies.  Similar results were reproduced when using  anti–c-src polyclonal and antitubulin monoclonal antibodies, followed by the relevant  secondary-labeled antibodies (data not  shown).
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Related In: Results  -  Collection

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

Figure 13: Matrix-dependent intracellular association of c-src/tubulin association by confocal microscopy. Avian marrow macrophages were kept in Teflon-coated beakers or plated on coverslips. Adherent cells were differentiated into osteoclast-like cells as previously described (1). Adherent and nonadherent cells were fixed, Triton permeabilized, incubated with both antitubulin and anti–c-src antibodies followed by fluorescent-labeled secondary antibodies, Texas red (for src) and FITC (for tubulin), and examined by confocal microscopy. A–C represent cells plated on vitronectin-coated plates. D–F represent cells plated on collagen type-I, and G–I represent cells kept nonadherent. Each group represents excitation of the same cell. A, D, and G represent excitation of Texas red (c-src), and B, E, and H represent FITC (tubulin). C, F, and I represent excitation of both fluorochromes and thus, colocalization of the antibodies. Similar results were reproduced when using anti–c-src polyclonal and antitubulin monoclonal antibodies, followed by the relevant secondary-labeled antibodies (data not shown).
Mentions: To determine if the specific matrix induction of c-src/tubulin association, demonstrated, by immunoprecipitation (Fig. 12), is an intracellular event, we once again turned to immunoconfocal microscopy (Fig. 13). Macrophages were permeabilized and incubated with anti–c-src and antitubulin antibodies as well as fluoresceinated secondary antibodies as described in Fig. 4. Whereas c-src/microtubule colocalization is extant in cells plated on vitronectin (Fig. 13, A–C), microtubules are not evident in those on collagen (Fig. 13, D–F), which are indistinguishable from their nonadherent counterparts (Fig. 13, G–I), wherein the two molecules fail to associate.

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