Figure 4: Microtubule (MT) morphology and dynamics are dictated by integrin activation state.

(a) Enrichment of talin and three +TIPs, EB1, ACF7 and CKAP5, in complexes associated with active β1 integrin shown by western blotting (see Supplementary Fig. 10 for original blots). (b) HFFs spread on FN, stimulatory and inhibitory anti-β1 integrin mAbs stained for actin (red) and α-tubulin (green), with corresponding high-power images highlighting the difference in the location of MTs at the cell periphery in cells spread on the inhibitory mAb. MT density was calculated by counting the number of MTs within a 5 × 2 μm region of the cell periphery. Results are mean±s.d. (n=9, 10 and 8 cells for FN, stimulatory and inhibitory, respectively). (c) HFFs spread on FN, stimulatory and inhibitory mAbs for 1 h before treatment with 10 μM nocodazole for 45 min and subsequent washout for a further 45 min to examine MT regrowth. Cells were stained for tubulin; dotted line in bottom-right image indicates cell periphery. MT density was measured as in b. Results are mean±s.d. (n=3, 3 and 4 cells for FN, stimulatory and inhibitory, respectively). (d) HFFs spread on stimulatory and inhibitory mAbs for 1 h before addition of 20 μM cytochalasin D or dimethylsulphoxide (DMSO) vehicle control for a further 1 h. Cells were stained for actin (red) and α-tubulin (green); dotted line in bottom-right image indicates cell periphery. MT density was measured as in b. Results are mean±s.d. (n=5 and 5 DMSO-treated cells and 5 and 7 cytochalasin D-treated cells for stimulatory and inhibitory, respectively). Scale bars, 10 μm. ***P<0.001, ****P<0.0001; one-way analysis of variance with Tukey’s post hoc correction in b, two-way analysis of variance with Tukey’s post hoc correction in c and d (see Supplementary Table 4 for statistics source data). Inhib., inhibitory; MW, molecular weight; NS, nonsignificant; Stim., stimulatory.