Mechanisms of spindle pole formation rely on minus-end-directed motor proteins. γ-Tubulin is present at the centre of poles, but its function during pole formation is completely unknown. To address the role of γ-tubulin in spindle pole formation, we overexpressed GFP (green fluorescent protein)-fused γ-tubulin (γ-Tu-GFP) in Xenopus oocytes and produced self-assembled mitotic asters in the oocyte extracts. γ-Tu-GFP associated with endogenous α-, β- and γ-tubulin, suggesting that it acts in the same manner as that of endogenous γ-tubulin. During the process of aster formation, γ-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 γ-tubulin by an anti-γ-tubulin antibody resulted in failure of microtubule organization into asters. This defect was restored by overexpression of γ-Tu-GFP, confirming the necessity of γ-tubulin in microtubule recruitment for aster formation. We also examined the effects of truncated γ-tubulin mutants, which are difficult to solubly express in other systems, on aster formation. The middle part of γ-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-γ-tubulin antibody. The results indicate possible roles of γ-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.
Research Article| July 26 2005
Overexpression of truncated γ-tubulins disrupts mitotic aster formation in Xenopus oocyte extracts
Tomoya Kotani 1
1Laboratory of Molecular and Cellular Interactions, Division of Biological Sciences, Graduate School of Science, Hokkaido University, Sapporo 060-0810, Japan
1To whom correspondence should be addressed at the present address: Division of Molecular and Developmental Biology, National Institute of Genetics, Yata, Mishima, Shizuoka 411-8540, Japan (email email@example.com).
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Tomoya Kotani, Masakane Yamashita; Overexpression of truncated γ-tubulins disrupts mitotic aster formation in Xenopus oocyte extracts. Biochem J 1 August 2005; 389 (3): 611–617. doi: https://doi.org/10.1042/BJ20050243
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