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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 is required for septin organization. (A) The indicated mutants expressing Cdc11-GFP were grown at permissive (25°C) and nonpermissive temperature (37°C) for 3 h; fluorescence and DIC images were recorded. The boxed regions indicate the areas on the right. (B) The percentages of cells with disorganized septins were calculated for mutants and isogenic wild-type parents grown at permissive (white bars) or nonpermissive (gray bars) temperatures. (C) The indicated mutants were grown at nonpermissive temperatures, stained with Calcofluor white to visualize bud scars and the position of the bud scars were classified as in Fig. 3 C. (D) Images were recorded and the percentage of cells with nonaxial bud scars were determined as in Fig. 3 D. Bar, 1 μm.
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fig8: Bir1p is required for septin organization. (A) The indicated mutants expressing Cdc11-GFP were grown at permissive (25°C) and nonpermissive temperature (37°C) for 3 h; fluorescence and DIC images were recorded. The boxed regions indicate the areas on the right. (B) The percentages of cells with disorganized septins were calculated for mutants and isogenic wild-type parents grown at permissive (white bars) or nonpermissive (gray bars) temperatures. (C) The indicated mutants were grown at nonpermissive temperatures, stained with Calcofluor white to visualize bud scars and the position of the bud scars were classified as in Fig. 3 C. (D) Images were recorded and the percentage of cells with nonaxial bud scars were determined as in Fig. 3 D. Bar, 1 μm.

Mentions: We next asked whether yeast passenger protein homologues also have a role in regulating septins. To address this question, we used preexisting alleles of IPL1 or SLI15 and created conditional alleles of BIR1. Mutant strains were engineered to express Cdc11-GFP, and septins were analyzed after growth of cells at permissive (25°C) or nonpermissive (37°C) temperatures. At 25°C, all strains exhibited normally organized septins; when shifted to 37°C, ipl1-321, ipl1-182, sli15-3, and sli15-1 all exhibited only a small increase in cells with disorganized septins, equivalent to isogenic wild-type control strains (Fig. 8, A and B; and Fig. S4). In contrast, growth of bir1-33 at nonpermissive temperature gave rise to a dramatic defect in septin organization in a significant percentage of cells. Septins appeared in disorganized clusters and rings were often fragmented or partially formed (Fig. 8 B and Fig. S4). This phenotype is remarkably similar to that observed for mutants in CBF3 assembly and shows that Bir1p and CBF3 function together to regulate septins.


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 is required for septin organization. (A) The indicated mutants expressing Cdc11-GFP were grown at permissive (25°C) and nonpermissive temperature (37°C) for 3 h; fluorescence and DIC images were recorded. The boxed regions indicate the areas on the right. (B) The percentages of cells with disorganized septins were calculated for mutants and isogenic wild-type parents grown at permissive (white bars) or nonpermissive (gray bars) temperatures. (C) The indicated mutants were grown at nonpermissive temperatures, stained with Calcofluor white to visualize bud scars and the position of the bud scars were classified as in Fig. 3 C. (D) Images were recorded and the percentage of cells with nonaxial bud scars were determined as in Fig. 3 D. Bar, 1 μm.
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fig8: Bir1p is required for septin organization. (A) The indicated mutants expressing Cdc11-GFP were grown at permissive (25°C) and nonpermissive temperature (37°C) for 3 h; fluorescence and DIC images were recorded. The boxed regions indicate the areas on the right. (B) The percentages of cells with disorganized septins were calculated for mutants and isogenic wild-type parents grown at permissive (white bars) or nonpermissive (gray bars) temperatures. (C) The indicated mutants were grown at nonpermissive temperatures, stained with Calcofluor white to visualize bud scars and the position of the bud scars were classified as in Fig. 3 C. (D) Images were recorded and the percentage of cells with nonaxial bud scars were determined as in Fig. 3 D. Bar, 1 μm.
Mentions: We next asked whether yeast passenger protein homologues also have a role in regulating septins. To address this question, we used preexisting alleles of IPL1 or SLI15 and created conditional alleles of BIR1. Mutant strains were engineered to express Cdc11-GFP, and septins were analyzed after growth of cells at permissive (25°C) or nonpermissive (37°C) temperatures. At 25°C, all strains exhibited normally organized septins; when shifted to 37°C, ipl1-321, ipl1-182, sli15-3, and sli15-1 all exhibited only a small increase in cells with disorganized septins, equivalent to isogenic wild-type control strains (Fig. 8, A and B; and Fig. S4). In contrast, growth of bir1-33 at nonpermissive temperature gave rise to a dramatic defect in septin organization in a significant percentage of cells. Septins appeared in disorganized clusters and rings were often fragmented or partially formed (Fig. 8 B and Fig. S4). This phenotype is remarkably similar to that observed for mutants in CBF3 assembly and shows that Bir1p and CBF3 function together to regulate septins.

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