Fig. 4: Vinexin depletion upregulates YAP/TAZ by countering YAP/TAZ cytosolic sequestration by angiomotins.

a HeLa cells were depleted of vinexin beta using an individual siRNA oligonucleotide against SORBS3 (siSORBS3; oligo 7). Endogenous YAP/TAZ were immunoprecipitated using an antibody raised in mouse (YAP/TAZ IP). Normal mouse IgG was used as a negative control (IgG). Endogenous angiomotin-like protein 1 (AMOTL1), YAP/TAZ and tubulin were examined by western blotting. AMOTL1/YAP IP values are the ratio of AMOTL1 (both bands in doublet) to YAP in the in YAP/TAZ IP calculated after normalising AMOTL1 in YAP/TAZ IP to AMOTL1 in the input and YAP in the YAP/TAZ IP to YAP/TAZ in the input. TAZ is not quantified as obscured by IgG heavy chain in the IP. SE = short exposure; LE = long exposure; molecular weights shown in kDa. b HeLa cells were co-transfected with Flag-YAP and HA-AMOT(p130) for 48 h. Cells were treated with latrunculin A (0.5 μM) or DMSO vehicle control for 6 h. Exogenous Flag-YAP was immunoprecipitated using a mouse antibody against Flag (Flag IP). Normal mouse IgG was used as a negative control (IgG). Flag-YAP and HA-AMOT(p130) were examined by western blotting. Representative blot from three independent experiments is shown. Molecular weights shown in kDa. c Quantification of three independent experiments described in b. Amount of Flag-YAP and HA-AMOT(p130) in Flag IP are expressed relative to Flag-YAP and HA-AMOT(p130) in Input and HA-AMOT(p130) IP normalised to Flag-YAP IP. Independent experiment 1 is illustrated with green data points, experiment 2 with red data points and experiment 3 with blue data points. * = p < 0.05 by two-tailed paired t-test. Error bars indicate SEM. d HeLa cells were depleted of vinexin beta as in a. siCntrl and siSORBS3 treated cells were transfected with pcDNA-HA-AMOT(p130) or empty vector control (pcDNA-empty) for 24 h. Haemagglutinin (HA) and endogenous YAP/TAZ were examined by immunofluorescence. Representative confocal images from three independent experiments are shown. Red = HA (Alexa Fluor 568); green = YAP/TAZ (Alexa Fluor 488); blue = DAPI. Scale bars indicate 20 µm. e Cells with predominantly nuclear YAP/TAZ (N > C), YAP/TAZ equally distributed between nucleus and cytosol (N = C) and predominantly cytosolic YAP/TAZ (C > N) where manually quantified. Quantification of the representative experiment shown in d. ns = p > 0.05; ** = p < 0.01 by two-tailed Student’s t-test. Red asterisks represent p value for N > C; blue asterisks represent N < C p value. n = 56 (pcDNA-empty; siCntrl); 68 (pcDNA-empty; siSORBS3); 70 (pcDNA-HA-AMOT; siCntrl); 45 (pcDNA-HA-AMOT; siSORBS3). Error bars indicate SD. f. HeLa cells stably expressing GFP-LC3 were depleted of vinexin beta. siCntrl and siSORBS3 treated cells were transfected with pcDNA-HA-AMOT(p130) or empty vector control (pcDNA-empty) for 24 h. GFP-LC3 vesicles were counted manually. Quantification of 4 independent experiments is shown. ns = p > 0.05; * = p < 0.05 by two-tailed paired t-test. Error bars indicate SEM. g (top) HeLa cells were transfected with either siCntrl or siSORBS3 and then transfected with pcDNA-HA-AMOT(p130) or empty vector control (pcDNA-empty) for 24 h. Cells were treated with BAF (400 nM) or DMSO vehicle control for 4 h. Haemagglutinin (HA), GAPDH and LC3 protein levels were examined by western blotting. Representative blots from four independent experiments are shown. SE = short exposure; LE = long exposure; molecular weights shown in kDa. (Below). (bottom) Quantification of the four independent experiments is shown below. LC3-II (lower band of LC3 doublet) levels are expressed relative to GAPDH loading control and normalised to LC3-II/GAPDH in control siRNA (siCntrl) treated cells. ns = p > 0.05; * = p < 0.05; *** = p < 0.001 by two-tailed paired t-test. Error bars indicate SEM. h Schematic of proposed mechanism. In cells with reduced SORBS3 expression (↓ SORBS3 expression) relative to baseline (baseline SORBS3 expression) increased F-actin structures compete with YAP/TAZ for binding to angiomotins (AMOTs). Therefore, more YAP/TAZ is free to enter the nucleus and upregulate autophagy via increased YAP/TAZ/TEAD transcriptional activity.