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.
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Research Article|
July 26 2005
Overexpression of truncated γ-tubulins disrupts mitotic aster formation in Xenopus oocyte extracts
Tomoya Kotani
;
Tomoya Kotani
1
1
Laboratory of Molecular and Cellular Interactions, Division of Biological Sciences, Graduate School of Science, Hokkaido University, Sapporo 060-0810, Japan1
To 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 tkotani@lab.nig.ac.jp).
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Masakane Yamashita
Masakane Yamashita
1
Laboratory of Molecular and Cellular Interactions, Division of Biological Sciences, Graduate School of Science, Hokkaido University, Sapporo 060-0810, Japan
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Biochem J (2005) 389 (3): 611-617.
Article history
Received:
February 08 2005
Revision Received:
April 04 2005
Accepted:
April 08 2005
Accepted Manuscript online:
April 08 2005
Citation
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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