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Fig. 2. VASP and 
vß3 overlap in stationary cells but distribute separately in motile cells. Cells were incubated for 1 hour with 50 µM Rp-cGMPS to inactivate endogenous cGMP activity (A,C) or for 1 hour with 100 µM 8-pCPT-cGMP to induce maximal motility (B,D). (A,B) When cGMP was blocked (top panels), osteoclasts on bone labeled for VASP (red) and vß3 (green) showed distribution consistent with the annular attachment site; merged images confirmed this (yellow labeling). After cGMP activation (bottom panels), VASP distribution was partially separated from vß3. It is seen in parallel rings (arrows, bottom merged image) on one side of the cell. Under these conditions, the cells are highly motile (see Fig. 1). (C,D) When grown on glass, the cells spread uniformly, facilitating labeling, and an analogous labeling pattern to that on bone was clearly seen. With PKG I suppressed by Rp-cGMPS (top), VASP was associated with the integrin forming a yellow, double-labeled ring in the merged image. With the nonhydrolyzable cGMP analog 8-pCPT-cGMP (bottom panels), vß3 and VASP were disassociated at one edge of the cell, now occurring in discrete rings. This polarization was consistent with cell motility. Bar, 10 µm (A,B); 20 µm (C,D).
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