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Spc24 and Stu2 promote spindle integrity when DNA replication is stalled
Lina Ma1, Jennifer McQueen, Lara Cuschieri
1Genetics Graduate Program, University of British Columbia, Vancouver, British Columbia, V6T 1Z4 Canada.
Molecular Biology of the Cell
|May 18, 2007
Summary
A mutation in the Spc24 kinetochore protein causes cell death when DNA replication is blocked, leading to premature spindle expansion. Overexpressing Stu1 or truncated Stu2 prevents this, highlighting their role in maintaining spindle integrity during S phase. Keywords: Spc24, kinetochore, spindle integrity, S phase.
Area of Science:
- Cell Biology
- Molecular Biology
- Genetics
Background:
- The kinetochore complex is crucial for linking chromosomes to microtubules (MTs) and maintaining spindle integrity before mitosis.
- The precise mechanisms by which kinetochores prevent premature spindle expansion during S phase, particularly under replication stress, remain incompletely understood.
Purpose of the Study:
- To investigate the role of the Ndc80 kinetochore complex component, Spc24, in maintaining spindle integrity during S phase.
- To elucidate the mechanism by which a loss-of-function mutation in Spc24 leads to premature spindle expansion and lethality under hydroxyurea (HU) treatment.
Main Methods:
- Utilized budding yeast (Saccharomyces cerevisiae) as a model system.
- Generated and analyzed a temperature-sensitive mutant of Spc24 (spc24-9).
- Assessed cell viability, spindle morphology, and DNA content under HU treatment.
- Investigated the effects of overexpressing MT-associated proteins Stu1 and Stu2 (full-length and truncated forms).
- Performed immunofluorescence microscopy to determine protein localization (Stu1, Stu2) at kinetochores.
Main Results:
- The spc24-9 mutation causes lethality upon HU treatment due to premature spindle expansion and segregation of incompletely replicated DNA.
- Overexpression of Stu1 or a truncated form of Stu2 rescues spc24-9 HU lethality and prevents spindle expansion.
- Active Stu2 is required for spindle expansion in spc24-9 mutants treated with HU.
- Stu1 and Stu2 localize to kinetochores early in the cell cycle, and Stu2 kinetochore localization is dependent on Spc24.
Conclusions:
- Mislocalization of Stu2, resulting from Spc24 dysfunction, leads to premature spindle expansion in S phase-stalled spc24-9 mutants.
- Stu1 and Stu2 play critical roles in restraining spindle expansion during replication stress.
- Understanding these factors provides insight into the regulation of spindle elongation during the normal cell cycle.
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