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A novel role for the CBF3 kinetochore-scaffold complex in regulating septin dynamics and cytokinesis.

Gillis AN, Thomas S, Hansen SD, Kaplan KB - J. Cell Biol. (2005)

Bottom Line: Biol.These results demonstrate a novel role for CBF3 in regulating cytokinesis, a role that is reminiscent of passenger proteins.Mutants in Bir1p similarly affect septin organization, leading us to propose that CBF3 and Bir1p act as passenger proteins to coordinate chromosome segregation with cytokinesis.

View Article: PubMed Central - PubMed

Affiliation: The Section of Molecular and Cellular Biology, University of California, Davis, Davis, CA 95616, USA.

ABSTRACT
In budding yeast, the kinetochore scaffold complex centromere binding factor 3 (CBF3) is required to form kinetochores on centromere DNA and to allow proper chromosome segregation. We have previously shown that SKP1 and SGT1 balance the assembly and turnover of CBF3 complexes, a cycle that we suggest is independent of its role in chromosome segregation (Rodrigo-Brenni, M.C., S. Thomas, D.C. Bouck, and K.B. Kaplan. 2004. Mol. Biol. Cell. 15:3366-3378). We provide evidence that this cycle contributes to a second, kinetochore-independent function of CBF3. In this study, we show that inhibiting the assembly of CBF3 causes disorganized septins and defects in cell polarity that give rise to cytokinesis failures. Specifically, we show that septin ring separation and disassembly is delayed in anaphase, suggesting that CBF3 regulates septin dynamics. Only mutations that affect the CBF3 cycle, and not mutants in outer kinetochore subunits, cause defects in septins. These results demonstrate a novel role for CBF3 in regulating cytokinesis, a role that is reminiscent of passenger proteins. Consistent with this possibility, we find that CBF3 interacts with Bir1p, the homologue of the passenger protein Survivin. Mutants in Bir1p similarly affect septin organization, leading us to propose that CBF3 and Bir1p act as passenger proteins to coordinate chromosome segregation with cytokinesis.

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Bir1p localizes to interpolar microtubules and interacts with Ndc10p. (A) Bir1-GFP was imaged in premetaphase cells (top, arrow) and in anaphase cells (bottom, arrow); fluorescence and DIC images were recorded. (B) The indicated bait and prey yeast two-hybrid vectors were plated on HIS selection; growth indicates an interaction. (C) Ndc10p was translated in vitro in the presence of 35S-methionine. The translated protein was incubated with GST-Bir1p or GST and the fraction of the Ndc10p bound to the beads was calculated.
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fig7: Bir1p localizes to interpolar microtubules and interacts with Ndc10p. (A) Bir1-GFP was imaged in premetaphase cells (top, arrow) and in anaphase cells (bottom, arrow); fluorescence and DIC images were recorded. (B) The indicated bait and prey yeast two-hybrid vectors were plated on HIS selection; growth indicates an interaction. (C) Ndc10p was translated in vitro in the presence of 35S-methionine. The translated protein was incubated with GST-Bir1p or GST and the fraction of the Ndc10p bound to the beads was calculated.

Mentions: The requirement of CBF3 to regulate septins during anaphase is reminiscent of the role ascribed to passenger proteins in higher eukaryotes. Interestingly, we and others have observed that CBF3 subunits are found both at kinetochores and on interpolar microtubules during anaphase (Fig. S3, Ndc10-GFP), a localization change that is similar to passenger protein behavior in animal cells (Buvelot et al., 2003; Pereira and Schiebel, 2003). Furthermore, in yeast BIR1 (Survivin), IPL1 (Aurora B) and SLI15 (INCENP) encode likely homologues of known passenger proteins, and both Ipl1p and Sli15p have been shown to localize to interpolar microtubules late in anaphase (Buvelot et al., 2003; Pereira and Schiebel, 2003). We have confirmed these localizations (not depicted), and we now show that Bir1-GFP also localizes to kinetochores early in the cell cycle and is found on interpolar microtubules in anaphase (Fig. 7 A). Previous work identified that the CBF3 subunit, Ndc10p, interacts with Bir1p via yeast two hybrid (Yoon and Carbon, 1999). We confirmed this result and showed that neither Ipl1p nor Sli15p interact with Ndc10p by yeast two hybrid (Fig. 7 B). To show that the interaction between Ndc10p and Bir1p is direct, we translated Ndc10p in vitro and incubated it with GST or a GST-Bir1p fusion. The enrichment of translated Bir1p with GST-Bir1p but not GST is consistent with the direct interaction between these two proteins (Fig. 7 C), indicating that CBF3 and Bir1p may function together.


A novel role for the CBF3 kinetochore-scaffold complex in regulating septin dynamics and cytokinesis.

Gillis AN, Thomas S, Hansen SD, Kaplan KB - J. Cell Biol. (2005)

Bir1p localizes to interpolar microtubules and interacts with Ndc10p. (A) Bir1-GFP was imaged in premetaphase cells (top, arrow) and in anaphase cells (bottom, arrow); fluorescence and DIC images were recorded. (B) The indicated bait and prey yeast two-hybrid vectors were plated on HIS selection; growth indicates an interaction. (C) Ndc10p was translated in vitro in the presence of 35S-methionine. The translated protein was incubated with GST-Bir1p or GST and the fraction of the Ndc10p bound to the beads was calculated.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171293&req=5

fig7: Bir1p localizes to interpolar microtubules and interacts with Ndc10p. (A) Bir1-GFP was imaged in premetaphase cells (top, arrow) and in anaphase cells (bottom, arrow); fluorescence and DIC images were recorded. (B) The indicated bait and prey yeast two-hybrid vectors were plated on HIS selection; growth indicates an interaction. (C) Ndc10p was translated in vitro in the presence of 35S-methionine. The translated protein was incubated with GST-Bir1p or GST and the fraction of the Ndc10p bound to the beads was calculated.
Mentions: The requirement of CBF3 to regulate septins during anaphase is reminiscent of the role ascribed to passenger proteins in higher eukaryotes. Interestingly, we and others have observed that CBF3 subunits are found both at kinetochores and on interpolar microtubules during anaphase (Fig. S3, Ndc10-GFP), a localization change that is similar to passenger protein behavior in animal cells (Buvelot et al., 2003; Pereira and Schiebel, 2003). Furthermore, in yeast BIR1 (Survivin), IPL1 (Aurora B) and SLI15 (INCENP) encode likely homologues of known passenger proteins, and both Ipl1p and Sli15p have been shown to localize to interpolar microtubules late in anaphase (Buvelot et al., 2003; Pereira and Schiebel, 2003). We have confirmed these localizations (not depicted), and we now show that Bir1-GFP also localizes to kinetochores early in the cell cycle and is found on interpolar microtubules in anaphase (Fig. 7 A). Previous work identified that the CBF3 subunit, Ndc10p, interacts with Bir1p via yeast two hybrid (Yoon and Carbon, 1999). We confirmed this result and showed that neither Ipl1p nor Sli15p interact with Ndc10p by yeast two hybrid (Fig. 7 B). To show that the interaction between Ndc10p and Bir1p is direct, we translated Ndc10p in vitro and incubated it with GST or a GST-Bir1p fusion. The enrichment of translated Bir1p with GST-Bir1p but not GST is consistent with the direct interaction between these two proteins (Fig. 7 C), indicating that CBF3 and Bir1p may function together.

Bottom Line: Biol.These results demonstrate a novel role for CBF3 in regulating cytokinesis, a role that is reminiscent of passenger proteins.Mutants in Bir1p similarly affect septin organization, leading us to propose that CBF3 and Bir1p act as passenger proteins to coordinate chromosome segregation with cytokinesis.

View Article: PubMed Central - PubMed

Affiliation: The Section of Molecular and Cellular Biology, University of California, Davis, Davis, CA 95616, USA.

ABSTRACT
In budding yeast, the kinetochore scaffold complex centromere binding factor 3 (CBF3) is required to form kinetochores on centromere DNA and to allow proper chromosome segregation. We have previously shown that SKP1 and SGT1 balance the assembly and turnover of CBF3 complexes, a cycle that we suggest is independent of its role in chromosome segregation (Rodrigo-Brenni, M.C., S. Thomas, D.C. Bouck, and K.B. Kaplan. 2004. Mol. Biol. Cell. 15:3366-3378). We provide evidence that this cycle contributes to a second, kinetochore-independent function of CBF3. In this study, we show that inhibiting the assembly of CBF3 causes disorganized septins and defects in cell polarity that give rise to cytokinesis failures. Specifically, we show that septin ring separation and disassembly is delayed in anaphase, suggesting that CBF3 regulates septin dynamics. Only mutations that affect the CBF3 cycle, and not mutants in outer kinetochore subunits, cause defects in septins. These results demonstrate a novel role for CBF3 in regulating cytokinesis, a role that is reminiscent of passenger proteins. Consistent with this possibility, we find that CBF3 interacts with Bir1p, the homologue of the passenger protein Survivin. Mutants in Bir1p similarly affect septin organization, leading us to propose that CBF3 and Bir1p act as passenger proteins to coordinate chromosome segregation with cytokinesis.

Show MeSH
Related in: MedlinePlus