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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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Inhibition of CBF3 assembly causes defects in polarized cell growth and septin organization. CTF13 or GAL1-CTF13 strains were grown in the presence of galactose and switched to fresh medium containing either galactose or raffinose for 300 min. Samples were collected, fixed, and stained with phalloidin coupled to Texas red to visualize actin (red) and with DAPI to visualize chromosomes (blue). GAL1-CTF13 strains with either SWE1 or SWE1Δ, a single mass of chromosomes, and elongated buds (A) were recorded and the percentage of cells with elongated buds was calculated for each condition (B). (C) Anaphase cells from the aforementioned strains were recorded, and the percentage of anaphase cells that failed to concentrate actin at the mother bud neck was calculated (D). Arrows indicate aberrantly localized actin patches in anaphase cells. (E) CTF13 or GAL1-CTF13 strains were grown in the indicated medium for 120 min, fixed, and processed for immunofluorescence with antibodies to Cdc11p (red). Chromosomes were stained with DAPI (blue). Inset represents a 1.5-fold zoom of the septin staining. The indicated strains expressing Cdc11-GFP from its endogenous promoter (Table I) were grown in raffinose for 120 min and photographed in DIC and fluorescent channels. Bar, 1 μm.
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fig1: Inhibition of CBF3 assembly causes defects in polarized cell growth and septin organization. CTF13 or GAL1-CTF13 strains were grown in the presence of galactose and switched to fresh medium containing either galactose or raffinose for 300 min. Samples were collected, fixed, and stained with phalloidin coupled to Texas red to visualize actin (red) and with DAPI to visualize chromosomes (blue). GAL1-CTF13 strains with either SWE1 or SWE1Δ, a single mass of chromosomes, and elongated buds (A) were recorded and the percentage of cells with elongated buds was calculated for each condition (B). (C) Anaphase cells from the aforementioned strains were recorded, and the percentage of anaphase cells that failed to concentrate actin at the mother bud neck was calculated (D). Arrows indicate aberrantly localized actin patches in anaphase cells. (E) CTF13 or GAL1-CTF13 strains were grown in the indicated medium for 120 min, fixed, and processed for immunofluorescence with antibodies to Cdc11p (red). Chromosomes were stained with DAPI (blue). Inset represents a 1.5-fold zoom of the septin staining. The indicated strains expressing Cdc11-GFP from its endogenous promoter (Table I) were grown in raffinose for 120 min and photographed in DIC and fluorescent channels. Bar, 1 μm.

Mentions: To investigate whether the cycle of CBF3 assembly and turnover plays a role in mitotic progression independent of chromosome segregation, we examined the consequences of inhibiting CBF3 assembly in dividing yeast cells. We used a haploid strain of yeast containing a GAL1 promoter that was integrated in place of the normal CTF13 promoter (GAL1-CTF13). Ctf13p is the core of the CBF3 complex and is targeted for ubiquitin-mediated degradation (Kaplan and Sorger, 1997). Growth of this strain in the presence of dextrose or raffinose results in the rapid depletion of Ctf13p (t1/2 = 10 min) and CBF3 complexes (t1/2 = 25 min) as measured in cell extracts (Rodrigo-Brenni et al., 2004). As previously reported, inhibition of CBF3 assembly does not compromise the integrity of kinetochores as judged by GFP-kinetochore markers and chromatin immunoprecipitation experiments, supporting the idea that CEN-associated CBF3 is not rapidly turned over (Rodrigo-Brenni et al., 2004). Consistent with this observation, cells inhibited for CBF3 assembly only transiently arrest in metaphase and segregate their chromosomes (unpublished data). After the growth of these cells in raffinose to inhibit CBF3 assembly, we observed that 30–60% of cells had elongated buds (Fig. 1, A and B; and Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200507017/DC1) with concentrated patches of actin at their bud tips, which is consistent with failure to switch from axial to isotropic bud growth (Fig. 1 A). In contrast, the parent strain containing the wild-type CTF13 promoter did not show a significant number of cells with elongated buds and exhibited a normal pattern of actin staining (Fig. S1 B and not depicted). Overexpression of Ctf13p (galactose) also increases the percentage of cells with elongated buds, although to a lesser extent than the inhibition of CBF3 assembly (10–14% compared with 2% in wild-type cells; Figs. 1 B and S1 B). Deletion of SWE1, a negative regulator of CDK–cyclin complexes, favors early isotropic growth and frequently rescues elongated buds in polarization mutants (Richman et al., 1999). We found that this was also the case in cells inhibited for CBF3 assembly; the deletion of SWE1 eliminates the appearance of elongated buds and restores the isotropic distribution of actin in the bud, even after 5 h of growth in raffinose (Fig. 1, A and B). In addition to the patches of actin at the bud tips, we also observed that actin failed to properly accumulate at the mother bud neck in anaphase cells inhibited for CBF3 assembly; actin was often found in disorganized patches positioned away from the mother bud neck of anaphase cells (Fig. 1 C, arrows). In contrast to the hyperpolarized bud growth, however, deletion of SWE1 did not rescue the mislocalized actin patches in anaphase cells (Fig. 1, C and D). Together, these data suggest that perturbing the CBF3 cycle compromises the ability of wild-type cells to properly position actin, either at bud tips or at the site of cell division.


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)

Inhibition of CBF3 assembly causes defects in polarized cell growth and septin organization. CTF13 or GAL1-CTF13 strains were grown in the presence of galactose and switched to fresh medium containing either galactose or raffinose for 300 min. Samples were collected, fixed, and stained with phalloidin coupled to Texas red to visualize actin (red) and with DAPI to visualize chromosomes (blue). GAL1-CTF13 strains with either SWE1 or SWE1Δ, a single mass of chromosomes, and elongated buds (A) were recorded and the percentage of cells with elongated buds was calculated for each condition (B). (C) Anaphase cells from the aforementioned strains were recorded, and the percentage of anaphase cells that failed to concentrate actin at the mother bud neck was calculated (D). Arrows indicate aberrantly localized actin patches in anaphase cells. (E) CTF13 or GAL1-CTF13 strains were grown in the indicated medium for 120 min, fixed, and processed for immunofluorescence with antibodies to Cdc11p (red). Chromosomes were stained with DAPI (blue). Inset represents a 1.5-fold zoom of the septin staining. The indicated strains expressing Cdc11-GFP from its endogenous promoter (Table I) were grown in raffinose for 120 min and photographed in DIC and fluorescent channels. Bar, 1 μm.
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Related In: Results  -  Collection

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fig1: Inhibition of CBF3 assembly causes defects in polarized cell growth and septin organization. CTF13 or GAL1-CTF13 strains were grown in the presence of galactose and switched to fresh medium containing either galactose or raffinose for 300 min. Samples were collected, fixed, and stained with phalloidin coupled to Texas red to visualize actin (red) and with DAPI to visualize chromosomes (blue). GAL1-CTF13 strains with either SWE1 or SWE1Δ, a single mass of chromosomes, and elongated buds (A) were recorded and the percentage of cells with elongated buds was calculated for each condition (B). (C) Anaphase cells from the aforementioned strains were recorded, and the percentage of anaphase cells that failed to concentrate actin at the mother bud neck was calculated (D). Arrows indicate aberrantly localized actin patches in anaphase cells. (E) CTF13 or GAL1-CTF13 strains were grown in the indicated medium for 120 min, fixed, and processed for immunofluorescence with antibodies to Cdc11p (red). Chromosomes were stained with DAPI (blue). Inset represents a 1.5-fold zoom of the septin staining. The indicated strains expressing Cdc11-GFP from its endogenous promoter (Table I) were grown in raffinose for 120 min and photographed in DIC and fluorescent channels. Bar, 1 μm.
Mentions: To investigate whether the cycle of CBF3 assembly and turnover plays a role in mitotic progression independent of chromosome segregation, we examined the consequences of inhibiting CBF3 assembly in dividing yeast cells. We used a haploid strain of yeast containing a GAL1 promoter that was integrated in place of the normal CTF13 promoter (GAL1-CTF13). Ctf13p is the core of the CBF3 complex and is targeted for ubiquitin-mediated degradation (Kaplan and Sorger, 1997). Growth of this strain in the presence of dextrose or raffinose results in the rapid depletion of Ctf13p (t1/2 = 10 min) and CBF3 complexes (t1/2 = 25 min) as measured in cell extracts (Rodrigo-Brenni et al., 2004). As previously reported, inhibition of CBF3 assembly does not compromise the integrity of kinetochores as judged by GFP-kinetochore markers and chromatin immunoprecipitation experiments, supporting the idea that CEN-associated CBF3 is not rapidly turned over (Rodrigo-Brenni et al., 2004). Consistent with this observation, cells inhibited for CBF3 assembly only transiently arrest in metaphase and segregate their chromosomes (unpublished data). After the growth of these cells in raffinose to inhibit CBF3 assembly, we observed that 30–60% of cells had elongated buds (Fig. 1, A and B; and Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200507017/DC1) with concentrated patches of actin at their bud tips, which is consistent with failure to switch from axial to isotropic bud growth (Fig. 1 A). In contrast, the parent strain containing the wild-type CTF13 promoter did not show a significant number of cells with elongated buds and exhibited a normal pattern of actin staining (Fig. S1 B and not depicted). Overexpression of Ctf13p (galactose) also increases the percentage of cells with elongated buds, although to a lesser extent than the inhibition of CBF3 assembly (10–14% compared with 2% in wild-type cells; Figs. 1 B and S1 B). Deletion of SWE1, a negative regulator of CDK–cyclin complexes, favors early isotropic growth and frequently rescues elongated buds in polarization mutants (Richman et al., 1999). We found that this was also the case in cells inhibited for CBF3 assembly; the deletion of SWE1 eliminates the appearance of elongated buds and restores the isotropic distribution of actin in the bud, even after 5 h of growth in raffinose (Fig. 1, A and B). In addition to the patches of actin at the bud tips, we also observed that actin failed to properly accumulate at the mother bud neck in anaphase cells inhibited for CBF3 assembly; actin was often found in disorganized patches positioned away from the mother bud neck of anaphase cells (Fig. 1 C, arrows). In contrast to the hyperpolarized bud growth, however, deletion of SWE1 did not rescue the mislocalized actin patches in anaphase cells (Fig. 1, C and D). Together, these data suggest that perturbing the CBF3 cycle compromises the ability of wild-type cells to properly position actin, either at bud tips or at the site of cell division.

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