Spc24 and Stu2 promote spindle integrity when DNA replication is stalled.
The kinetochore, a protein complex that links chromosomes to microtubules (MTs), is required to prevent spindle expansion during S phase in budding yeast, but the mechanism of how the kinetochore maintains integrity of the bipolar spindle before mitosis is not well understood. Here, we demonstrate that a mutation of Spc24, a component of the conserved Ndc80 kinetochore complex, causes lethality when cells are exposed to the DNA replication inhibitor hydroxyurea (HU) due to premature spindle expansion and segregation of incompletely replicated DNA. Overexpression of Stu1, a CLASP-related MT-associated protein or a truncated form of the XMAP215 orthologue Stu2 rescues spc24-9 HU lethality and prevents spindle expansion. Truncated Stu2 likely acts in a dominant-negative manner, because overexpression of full-length STU2 does not rescue spc24-9 HU lethality, and spindle expansion in spc24-9 HU-treated cells requires active Stu2. Stu1 and Stu2 localize to the kinetochore early in the cell cycle and Stu2 kinetochore localization depends on Spc24. We propose that mislocalization of Stu2 results in premature spindle expansion in S phase stalled spc24-9 mutants. Identifying factors that restrain spindle expansion upon inhibition of DNA replication is likely applicable to the mechanism by which spindle elongation is regulated during a normal cell cycle.
Ma
Lina
L
Genetics Graduate Program, University of British Columbia, Vancouver, British Columbia, V6T 1Z4 Canada.
McQueen
Jennifer
J
Cuschieri
Lara
L
Vogel
Jackie
J
Measday
Vivien
V
D000705:Anaphase / Q000187:drug effects; D016764:Cell Polarity / Q000187:drug effects; D002868:Chromosomal Proteins, Non-Histone / Q000378:metabolism*; D004261:DNA Replication / Q000187:drug effects; D005800:Genes, Fungal; D006918:Hydroxyurea / Q000494:pharmacology; D018386:Kinetochores / Q000187:drug effects / Q000378:metabolism; D050296:Microbial Viability / Q000187:drug effects; D008869:Microtubule-Associated Proteins / Q000378:metabolism*; D009154:Mutation / Q000235:genetics; D009687:Nuclear Proteins / Q000378:metabolism; D021381:Protein Transport / Q000187:drug effects; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D008941:Spindle Apparatus / Q000187:drug effects / Q000378:metabolism*
D002868:Chromosomal Proteins, Non-Histone; D008869:Microtubule-Associated Proteins; C112472:NDC80 protein, S cerevisiae; D009687:Nuclear Proteins; C109414:STU2 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; C500717:Spc24 protein, S cerevisiae; D006918:Hydroxyurea