Spc24 and Stu2 Promote Spindle Integrity When DNA Replication Is Stalled

Abstract

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. INTRODUCTIONPreserving the integrity of the genome is a fundamental requirement for eukaryotic cell viability. DNA replication must be completed before segregation of the chromosomes to prevent the transmission of partially replicated chromosomes to daughter cells. In the budding yeast Saccharomyces cerevisiae, cells undergo a closed mitosis and the microtubule (MT) organizing centers, or spindle pole bodies (SPBs), are imbedded in the nuclear envelope. SPB duplication begins at the end of anaphase and spindle formation begins during S phase when duplicated SPBs separate from each other (Adams and Kilmartin, 1999;Jaspersen and Winey, 2004). Because chromosomes remain attached to kinetochore MTs throughout the cell cycle, spindle expansion must be restrained until all 16 chromosomes have duplicated and kinetochores on sister chromatids have formed bipolar MT attachments. When DNA replication is stalled by hydroxyurea (HU) treatment, cells arrest with a large bud, an undivided nucleus positioned at the mother-bud neck and a short bipolar spindle (Allen et al., 1994). Maintaining a short spindle is crucial for cell survival during HU-induced arrest, which activates the DNA replication checkpoint effectors Mec1 and Rad53 (Kolodner et al., 2002). When mec1 and rad53 mutants are treated with HU, the DNA replication checkpoint is not activated, and, as a result, replication forks are not stabilized, the spindle expands, and unequal division of incompletely replicated nuclear material occurs-all of these events contribute to cell lethality (Allen et al., 1994;Weinert et al., 1994;Lopes et al., 2001).In human cells, stalled replication forks activate the ataxia telangiectasia mutat...

A TOG Protein Confers Tension Sensitivity to Kinetochore-Microtubule Attachments

“…Kinetochore particles were purified from cells carrying temperature sensitive alleles of a component of the Dam1 ( dad1-1 ), Ndc80 ( ndc80-1 ) or KNL1 Spc105 ( spc105-15 ) complexes. The Stu2 kinetochore-association was disrupted only in cells carrying an ndc80-1 allele (Figure 1B), in agreement with previously reported chromatin immunoprecipitation data (He et al, 2001; Ma et al, 2007). We further confirmed this by isolating kinetochores from cells carrying an ndc80-AID (auxin inducible degron; Nishimura et al, 2009) allele indicating there is a kinetochore-bound pool of Stu2 that requires the Ndc80 complex for its association (Figure S1A).…”

Stu1 inversely regulates kinetochore capture and spindle stability

“…Stu1 fulfilled these criteria (Yin et al 2002;Ma et al 2007). We confirmed and extended the current knowledge on Stu1 localization in wildtype cells.…”

Laterally attached kinetochores recruit the checkpoint protein Bub1, but satisfy the spindle checkpoint

“…Stu2 can be found both colocalized with kinetochores and along the spindle in DAM1-765 (Figure 1B). This result is consistent with the observed localization in wild-type cells and with previous reports that Stu2 binds the kinetochore independently of microtubules 26, 40. Cin8 localizes along the DAM1-765 spindle, and in some cells also colocalizes with kinetochores, as it does in wild-type cells (Figure 1C).…”

“…In wild-type cells, the plus-end binding proteins (Bik1, Bim1, and Stu2) and plus-end directed motors (Cin8 and Kip3) each localize into two peaks of fluorescence that overlap with the two peaks of kinetochore fluorescence (Figure 1B, C). These localizations are consistent with an enrichment of these proteins at microtubule plus ends, where the kinetochores attach 30, 37-40…”