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Substrate recognition by osteoclast precursors induces C-src/microtubule association
Y Abu-Amer1, F P Ross, P Schlesinger
1Department of Pathology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
The Journal of Cell Biology
|April 7, 1997
Summary
Osteoclast polarization requires the proto-oncogene pp60(c-src) (c-src) to associate with microtubules, trafficking proteins to the ruffled membrane. This binding is triggered by cell-substrate recognition and is crucial for osteoclast function.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Osteoclasts, crucial for bone resorption, polarize upon substrate recognition, forming a ruffled membrane.
- The proto-oncogene pp60(c-src) (c-src) is essential for osteoclast polarization and ruffled membrane formation.
- c-src is a vesicular protein that localizes to the ruffled membrane.
Purpose of the Study:
- To investigate the hypothesis that matrix recognition induces c-src to associate with microtubules for transport to the osteoclast cell surface.
- To elucidate the role of c-src/tubulin interaction in osteoclast polarization and function.
Main Methods:
- Immunoprecipitation of c-src and tubulin from avian marrow macrophages (osteoclast precursors).
- Double antibody immunoconfocal microscopy to assess protein colocalization.
- In vitro kinase assays to determine the activity of tubulin-associated c-src.
- Treatment with microtubule-disrupting drugs and cold to assess the role of polymerized tubulin.
Main Results:
- c-src associates with tubulin in adherent osteoclast precursors, indicating an intracellular event.
- This association occurs within 90 minutes of cell-substrate recognition and does not involve increased protein expression.
- Tubulin-associated c-src is enzymatically active, and its activity increases upon cell adhesion.
- The association is dependent on polymerized tubulin and specific substrates like vitronectin and fibronectin.
- The H+-ATPase, like c-src, decorates microtubules and co-localizes with c-src in the Golgi and ruffled membrane vesicles.
Conclusions:
- Cell-substrate recognition induces c-src to associate with microtubules in osteoclast precursors.
- This c-src/tubulin complex is actively involved in trafficking proteins, including the H+-ATPase, to the ruffled membrane.
- The findings highlight a novel mechanism for regulating osteoclast polarization and function via c-src-mediated microtubule transport.