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MBC in Press, published online ahead of print January 4, 2006
Mol. Biol. Cell 10.1091/mbc.E05-08-0802

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Submitted on August 26, 2005
Revised on December 13, 2005
Accepted on December 27, 2005

The V260I Mutation in Fission Yeast {alpha}-Tubulin Atb2 Affects Microtubule Dynamics and EB1-Mal3 Localization and Activates the Bub1 Branch of the Spindle Checkpoint

Kazuhide Asakawa,*{dagger}{ddagger} Kazunori Kume,{dagger}{sect} Muneyoshi Kanai,{sect} Tetsuya Goshima,{sect} Kohji Miyahara,{sect} Susheela Dhut,* Wee W. Tee,* Dai Hirata,{sect} and Takashi Toda*

*Laboratory of Cell Regulation, Cancer Research UK, London Research Institute, Lincoln’s Inn Fields Laboratories, London WC2A 3PX, United Kingdom; {sect}Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima University, Higashi-Hiroshima 739-8530, Japan

Monitoring Editor: J. Richard McIntosh

We have identified a novel temperature-sensitive mutant of fission yeast {alpha}-tubulin Atb2 (atb2-983) that contains a single amino acid substitution (V260I). Atb2-983 is incorporated into the microtubules and their overall structures are not altered noticeably, but microtubule dynamics is compromised during interphase. atb2-983 displays a high rate of chromosome mis-segregation and is synthetically lethal with deletions in a subset of spindle checkpoint genes including bub1, bub3 and mph1, but not with mad1, mad2 and mad3. During early mitosis in this mutant, Bub1, but not Mad2, remains for a prolonged period in the kinetochores that are situated in close proximity to one of the two SPBs (spindle pole bodies). High dosage mal3+, encoding EB1 homolog, rescues atb2-983, suggesting that Mal3 function is compromised. Consistently, Mal3 localization and a binding between Mal3 and Atb2-983 are impaired significantly and a mal3 single mutant, like atb2-983, displays prolonged Bub1 kinetochore localization. Furthermore in atb2-983 back-and-forth centromere oscillation during prometaphase is abolished. Intriguingly this oscillation still occurs in the mal3 mutant, indicating that there is another defect independent of Mal3. These results show that microtubule dynamics is important for coordinated execution of mitotic events, in which Mal3 plays a vital role.

{dagger}These authors contributed equally to this work.

{ddagger}Present address: Division of Molecular and Developmental Biology, Department of Developmental Genetics, National Institute of Genetics, Yata 1111, Mishima, Shizuoka 411-8540, Japan.

Address correspondence to: Takashi Toda (toda{at}cancer.org.uk)




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