Figure 5: Acetylation of microtubules increases surface density of α5β1 integrin and inhibits cell migration.

(a) Representative TIRF images of HFFs expressing different tubulin mutants and immunostained with an mAb11 anti-α5 antibody without permeabilizing cell membranes. (b) Quantification of a; intensities were normalized to the control. A minimum of 30 cells were analysed per condition (n=4). (c) Normalized level of endocytosis or recycling from recycling endosomes of α5β1 integrin after TSA treatment (n>3). (d) Normalized level of endocytosis or recycling from recycling endosomes of α5β1 integrins in cells expressing GFP (control) or GFP-tagged acetyl-mimetic tubulin (HyperAcMT) (n>4). (e) Migration velocity of HFFs treated with TSA and function-blocking antibody to α5β1 integrin (mAb16). The concentration of mAb16 reversing the TSA-induced migration defect is highlighted in red. A minimum of 40 time points were used per measurement from >10 cells per condition (n=3). (f) Migration velocity of HFFs stably expressing the indicated tubulins in the absence or presence of function-blocking antibody to α5β1 integrin (mAb16). A minimum of 40 time points were used per measurement from >29 cells per condition (n>4). (g) Representative confocal images of HFFs expressing the indicated tubulin in the presence or absence of function-blocking mAb16. FN was visualized with an anti-FN antibody (green) and α5β1 integrin with anti-α5 antibody (red). Data shown as mean±s.e.m. *P<0.05. Two-way analysis of variance (ANOVA) followed by Tukey post test was used for b and f, and Student’s t-test was used for c and d. Scale bar, 20 μm.