Summary

Microtubules nucleated from γ-tubulin ring complexes located at the centrosome regulate the localization of organelles, promote vesicular transport and direct cell migration. Although several signaling mechanisms have been identified that regulate microtubule dynamics during interphase, signaling pathways that promote microtubule nucleation remain elusive. We assayed microtubule regrowth following nocodazole washout in human fibroblasts and CHO-K1 cells adhered to fibronectin in either normal serum-free medium or the serum-free, growth-promoting medium, CCM1, which contains IGF1 and androgen, as well as other nutrients. The results indicate that integrin-mediated adhesion is not sufficient to promote rapid microtubule regrowth in either cell type. The addition of androgen, but not IGF1, for 5 minutes was sufficient to promote rapid regrowth and this occurred by a mechanism requiring the androgen receptor. Since Src is a component of the cytoplasmic androgen-receptor-signaling complex, we examined its role using Src siRNA, the Src kinase inhibitor SU6656, and the expression of a constitutively active Src mutant. The data show that Src signaling is both required and sufficient to promote rapid microtubule regrowth in cells adhered to fibronectin. Measurement of the density of microtubules close to the centrosome and the rates of GFP-EB1-labeled microtubules emanating from the centrosome indicated that Src signaling promotes microtubule nucleation. Furthermore, recovery of GFP–γ-tubulin at the centrosome following photobleaching and measurements of endogenous γ-tubulin levels at the centrosome showed that androgen and Src signaling regulate the levels of centrosomal γ-tubulin. Thus, we propose that androgen and Src signaling regulate microtubule nucleation during interphase by promoting the centrosomal localization of γ-tubulin.