Figure 7.

Requirement of microtubules, but not microfilaments, for the formation of extensions. (A) After replating of 388-Cbl–expressing cells for 30 min in the presence of cytochalasin D, nocodazole, or taxol, phase contrast images were collected at 0, 60, 180, and 360 min of addition of Y-27632 to the culture media. Bar, 50 μm. (B) 388-Cbl–expressing cells were replated for 30 min in the presence of cytochalasin D (1 and 2), nocodazole (3 and 4), or taxol (5 and 6). Confocal immunofluorescence microscopy images of phalloidin (red), anti-tubulin antibody (green), and Hoescht (blue) staining of 388-Cbl were then collected after a 4-h incubation with DMSO (1, 3, and 5) or Y-27632 (2, 4, and 6). Bar, 50 μm. (C) 388-Cbl–expressing cells were replated in the absence (no addition) or presence of Y-27632, Y-27632 plus cytochalasin D, and Y-27632 plus taxol. Median maximal distances from the cell boundary to the nucleus were obtained from separate triplicate anti-tubulin immunofluorescence images as described in MATERIALS AND METHODS. Average values and standard deviations are indicated for samples taken at 0, 1, 3, 6, and 12 h of adhesion to the culture substrate after replating. (D) 388-Cbl–expressing cells were replated for 6 h either with no drug treatment (lanes 1 and 5), Y-27632 (lane 2), taxol (lane 3), or nocodazole (for the final 60 min, lane 4), and microtubule cytoskeleton fractions (lanes 1–4) or total cell lysates (lane 5) were probed with anti-tubulin antibody after SDS-PAGE and Western blot.