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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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Cytokinesis is compromised in cells inhibited for CBF3 assembly. (A) GAL1-CTF13 strains were grown in the indicated medium for 300 min, and the percentage of anaphase cells in the population was calculated after DAPI and tubulin staining (not depicted). (B) A GAL1-CTF13, SWE1Δ strain was grown in medium containing galactose or raffinose, cells were stained for actin (red), and chromosomes were visualized with DAPI. (C) Multinucleated cells were calculated as a percentage of the total anaphase cells in the population with more than two chromosome masses. Bar, 1 μm.
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fig6: Cytokinesis is compromised in cells inhibited for CBF3 assembly. (A) GAL1-CTF13 strains were grown in the indicated medium for 300 min, and the percentage of anaphase cells in the population was calculated after DAPI and tubulin staining (not depicted). (B) A GAL1-CTF13, SWE1Δ strain was grown in medium containing galactose or raffinose, cells were stained for actin (red), and chromosomes were visualized with DAPI. (C) Multinucleated cells were calculated as a percentage of the total anaphase cells in the population with more than two chromosome masses. Bar, 1 μm.

Mentions: Septins are used in yeast throughout the cell cycle to control bud growth as well as cytokinesis. To examine whether the defects in septin organization compromise cytokinesis, we monitored the GAL1-CTF13 strain when grown over longer periods of time in either galactose or raffinose. When CBF3 assembly was inhibited (raffinose), we observed an increase in the number of cells with segregated DNA masses and extended anaphase spindles, which is consistent with a defect in cytokinesis (Fig. 6 A). In previous studies, it has been shown that compromised cytokinesis can give rise to multinucleated cells because of defects in septins when the CDK/cyclin regulator SWE1 is deleted (Sreenivasan and Kellogg, 1999). We examined the appearance of multinucleated anaphase cells in a GAL1-CTF13, SWE1Δ strain. We observed a small percentage of multinucleated cells when CBF3 assembly was inhibited in a wild-type SWE1 strain and a dramatic increase in multinucleated cells when SWE1 was deleted (9–52% of anaphase cells; Fig. 6, B and C). The accumulation of anaphase cells with aberrant septins and the increase in multinucleated cells strongly argue that CBF3 assembly is critical for cytokinesis.


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)

Cytokinesis is compromised in cells inhibited for CBF3 assembly. (A) GAL1-CTF13 strains were grown in the indicated medium for 300 min, and the percentage of anaphase cells in the population was calculated after DAPI and tubulin staining (not depicted). (B) A GAL1-CTF13, SWE1Δ strain was grown in medium containing galactose or raffinose, cells were stained for actin (red), and chromosomes were visualized with DAPI. (C) Multinucleated cells were calculated as a percentage of the total anaphase cells in the population with more than two chromosome masses. Bar, 1 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2171293&req=5

fig6: Cytokinesis is compromised in cells inhibited for CBF3 assembly. (A) GAL1-CTF13 strains were grown in the indicated medium for 300 min, and the percentage of anaphase cells in the population was calculated after DAPI and tubulin staining (not depicted). (B) A GAL1-CTF13, SWE1Δ strain was grown in medium containing galactose or raffinose, cells were stained for actin (red), and chromosomes were visualized with DAPI. (C) Multinucleated cells were calculated as a percentage of the total anaphase cells in the population with more than two chromosome masses. Bar, 1 μm.
Mentions: Septins are used in yeast throughout the cell cycle to control bud growth as well as cytokinesis. To examine whether the defects in septin organization compromise cytokinesis, we monitored the GAL1-CTF13 strain when grown over longer periods of time in either galactose or raffinose. When CBF3 assembly was inhibited (raffinose), we observed an increase in the number of cells with segregated DNA masses and extended anaphase spindles, which is consistent with a defect in cytokinesis (Fig. 6 A). In previous studies, it has been shown that compromised cytokinesis can give rise to multinucleated cells because of defects in septins when the CDK/cyclin regulator SWE1 is deleted (Sreenivasan and Kellogg, 1999). We examined the appearance of multinucleated anaphase cells in a GAL1-CTF13, SWE1Δ strain. We observed a small percentage of multinucleated cells when CBF3 assembly was inhibited in a wild-type SWE1 strain and a dramatic increase in multinucleated cells when SWE1 was deleted (9–52% of anaphase cells; Fig. 6, B and C). The accumulation of anaphase cells with aberrant septins and the increase in multinucleated cells strongly argue that CBF3 assembly is critical for cytokinesis.

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