Figure 8: GPCR-mediated activation of GEF-H1 does not require microtubule depolymerization.

(a) Thrombin-induced GEF-H1 dephosphorylation is independent of microtubule depolymerization. Cells were treated with thrombin for different times with or without Taxol pretreatment. Lysates were probed for GEF-H1 and phosphoSer885 GEF-H1 levels by western blot. (b) Taxol suppresses nocodazole but not LPA-stimulated redistribution of GEF-H1 from the microtubule to the cytoplasm. GEF-H1^−/− MEFs were transfected with GEF-H1–eGFP, starved for 3 days and then treated with LPA, thrombin or nocodazole with or without 24-h Taxol pretreatment. Cells were fixed and stained with anti-phalloidin and anti-tubulin antibodies and imaged by confocal microscopy. Scale bar, 20 μm. (c) LPA-induced GEF-H1 relocalization is independent of microtubule depolymerization. Real-time live-cell images of GEF-H1 and microtubules following LPA stimulation. GEF-H1^−/− MEFs were transiently transfected with GEF-H1–eGFP and Cherry tubulin, treated by Taxol then stimulated with LPA. Scale bar, 20 μm. (d) The correlation coefficient (R) measuring co-localization between GFP (GEF-H1) and polymerized microtubules in b was determined in at least 63 cells from three independent experiments. Error bars represent s.d. of at least three independent replicates, and P values derived from pairwise t-tests are indicated. (e) Taxol suppresses nocodazole—but not LPA—or thrombin-induced activation of GEF-H1 exchange activity. RhoA nucleotide exchange rates were measured in the lysates derived from HEK293T cells overexpressing GEF-H1–eGFP or co-expressing GEF-H1–eGFP and Flag-Tctex-1, untreated or treated with LPA, thrombin or 20 μM nocodazole, with or without 24-h pretreatment with Taxol. The s.d. derived from at least three independent replicates are indicated, with P values from pairwise t-tests.