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.
Kotani
Tomoya
T
Laboratory of Molecular and Cellular Interactions, Division of Biological Sciences, Graduate School of Science, Hokkaido University, Sapporo 060-0810, Japan. tkotani@lab.nig.ac.jp
Yamashita
Masakane
M
D000818:Animals; D000906:Antibodies; D004398:Dyneins / Q000502:physiology; D015870:Gene Expression / Q000502:physiology; D008870:Microtubules / Q000502:physiology; D008938:Mitosis / Q000502:physiology*; D009154:Mutation; D009865:Oocytes / Q000502:physiology*; D008941:Spindle Apparatus / Q000502:physiology*; D014404:Tubulin / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D015854:Up-Regulation; D014982:Xenopus laevis
D000906:Antibodies; D014404:Tubulin; D004398:Dyneins