Overexpression of truncated gamma-tubulins disrupts mitotic aster formation in Xenopus oocyte extracts.

Mechanisms of spindle pole formation rely on minus-end-directed motor proteins. gamma-Tubulin is present at the centre of poles, but its function during pole formation is completely unknown. To address the role of gamma-tubulin in spindle pole formation, we overexpressed GFP (green fluorescent protein)-fused gamma-tubulin (gamma-Tu-GFP) in Xenopus oocytes and produced self-assembled mitotic asters in the oocyte extracts. gamma-Tu-GFP associated with endogenous alpha-, beta- and gamma-tubulin, suggesting that it acts in the same manner as that of endogenous gamma-tubulin. During the process of aster formation, gamma-Tu-GFP aggregated as dots on microtubules, and then the dots were translocated to the centre of the aster along microtubules in a manner dependent on cytoplasmic dynein activity. Inhibition of the function of gamma-tubulin by an anti-gamma-tubulin antibody resulted in failure of microtubule organization into asters. This defect was restored by overexpression of gamma-Tu-GFP, confirming the necessity of gamma-tubulin in microtubule recruitment for aster formation. We also examined the effects of truncated gamma-tubulin mutants, which are difficult to solubly express in other systems, on aster formation. The middle part of gamma-tubulin caused abnormal organization of microtubules in which minus ends of microtubules were not tethered, but dispersed. An N-terminus-deleted mutant prevented recruitment of microtubules into asters, similar to the effect of the anti-gamma-tubulin antibody. The results indicate possible roles of gamma-tubulin in spindle pole formation and show that the system developed in the present study could be useful for analysing roles of many proteins that are difficult to solubly express.