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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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Defects in CBF3 assembly, but not outer kinetochore proteins, cause septin organization defects. (A) Representative examples of wild-type or mutant strains grown for 3 h at nonpermissive temperature, fixed, and processed to stain septins (red), tubulin (green), and chromosomes (blue). The right panels represent a fourfold zoom of the regions in the insets. (B) Quantification of the percentage of cells with defective septins after growth at nonpermissive temperature. (C) The indicated mutants were grown at nonpermissive temperature for 3 h and stained with Calcofluor white to visualize bud scars; the position of bud scars were classified as axial, middle, or polar. (D) Images were recorded using DIC and fluorescent optics. (E) Cells with more than one bud scar were scored and the percentage of total cells with scars in the middle region of the mother (blue) or at the polar side of the mother (red) was measured. Bars, 1 μm.
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fig5: Defects in CBF3 assembly, but not outer kinetochore proteins, cause septin organization defects. (A) Representative examples of wild-type or mutant strains grown for 3 h at nonpermissive temperature, fixed, and processed to stain septins (red), tubulin (green), and chromosomes (blue). The right panels represent a fourfold zoom of the regions in the insets. (B) Quantification of the percentage of cells with defective septins after growth at nonpermissive temperature. (C) The indicated mutants were grown at nonpermissive temperature for 3 h and stained with Calcofluor white to visualize bud scars; the position of bud scars were classified as axial, middle, or polar. (D) Images were recorded using DIC and fluorescent optics. (E) Cells with more than one bud scar were scored and the percentage of total cells with scars in the middle region of the mother (blue) or at the polar side of the mother (red) was measured. Bars, 1 μm.

Mentions: Although kinetochores remain intact and anaphase chromosome segregation occurs normally after the depletion of CBF3 (Rodrigo-Brenni et al., 2004), it is possible that there are subtle changes in outer kinetochore complexes that affect septin organization. To test this possibility, we examined septins in a variety of mutants that compromise either CBF3 or outer kinetochore complexes. We observed little change in septin organization in mutants that effect outer kinetochore complexes (e.g., ndc80-1; Fig. 5, A and B). Although some outer kinetochore alleles arrest in metaphase because of the spindle checkpoint, we and others have observed that some alleles are checkpoint defective and continue past metaphase (e.g., ctf19-26, ctf19-58, ndc80-1, and dam1-1; Hyland et al., 1999; Jones et al., 2001; Wigge and Kilmartin, 2001), making it unlikely that a pre-anaphase arrest masks a septin defect. In contrast, alleles that affect CBF3 assembly exhibit a high percentage of cells with disorganized septins (Fig. 5 A and Fig. S2, available at http://www.jcb.org/cgi/content/full/jcb.200507017/DC1). As was the case for the GAL1-CTF13 strain, we observed partially formed or misoriented septin rings, especially in anaphase cells. Interestingly, the most dramatic affects on septins were observed in SKP1 and SGT1 alleles that we have previously shown compromise the CBF3 cycle (Fig. 5 B; Lingelbach and Kaplan, 2004; Rodrigo-Brenni et al., 2004). Specifically, the skp1-4 allele, which prevents CBF3 assembly by blocking the interaction between Skp1p and Sgt1p, gives rise to defects in septin organization. The skp1-3 allele, which stabilizes the Skp1p–Sgt1p interaction and prevents CBF3 turnover (Rodrigo-Brenni et al., 2004), results in an even higher percentage of cells with disorganized septins. SKP1 and SGT1 have also been implicated in the function of the Skp1-cullin-F-box (SCF)–E3 ubiquitin ligase complex. To rule out a role for SCF in regulating septin organization, we examined cdc34-2, which is a conditional allele encoding the ubiquitin-ligase subunit of SCF. We observed no effect on septin organization despite the high percentage of cells with elongated buds (Fig. 5, A and B). In contrast to the more subtle effects of SKP1 and SGT1 alleles, the ndc10-1 allele entirely eliminates kinetochore formation on CEN DNA and cells inappropriately proceed through the cell cycle (Gardner et al., 2001); the resulting multibudded ndc10-1 cells frequently exhibit a complete failure to form septins rings (38% for ndc10-1 and 5% for the wild-type W303 strain) and are therefore not included in our analysis of “disorganized” septins. This phenotype is similar to that recently shown by Bouck and Bloom (2005). However, we note that this extreme phenotype is not observed when ndc10-1 is backcrossed to the S288C background used in all of the other data in our studies, instead we observe a pattern of septin disorganization more typical of CBF3 mutants (Fig. 5 B and Fig. S2; see Discussion). Thus, even the most severe kinetochore mutant available produces the same septin defect observed in mutants, which perturbs the CBF3 cycle but not kinetochore formation. Together, these results strongly argue that CBF3 functions independently from its role at the kinetochore to regulate septins during anaphase.


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)

Defects in CBF3 assembly, but not outer kinetochore proteins, cause septin organization defects. (A) Representative examples of wild-type or mutant strains grown for 3 h at nonpermissive temperature, fixed, and processed to stain septins (red), tubulin (green), and chromosomes (blue). The right panels represent a fourfold zoom of the regions in the insets. (B) Quantification of the percentage of cells with defective septins after growth at nonpermissive temperature. (C) The indicated mutants were grown at nonpermissive temperature for 3 h and stained with Calcofluor white to visualize bud scars; the position of bud scars were classified as axial, middle, or polar. (D) Images were recorded using DIC and fluorescent optics. (E) Cells with more than one bud scar were scored and the percentage of total cells with scars in the middle region of the mother (blue) or at the polar side of the mother (red) was measured. Bars, 1 μm.
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Related In: Results  -  Collection

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

fig5: Defects in CBF3 assembly, but not outer kinetochore proteins, cause septin organization defects. (A) Representative examples of wild-type or mutant strains grown for 3 h at nonpermissive temperature, fixed, and processed to stain septins (red), tubulin (green), and chromosomes (blue). The right panels represent a fourfold zoom of the regions in the insets. (B) Quantification of the percentage of cells with defective septins after growth at nonpermissive temperature. (C) The indicated mutants were grown at nonpermissive temperature for 3 h and stained with Calcofluor white to visualize bud scars; the position of bud scars were classified as axial, middle, or polar. (D) Images were recorded using DIC and fluorescent optics. (E) Cells with more than one bud scar were scored and the percentage of total cells with scars in the middle region of the mother (blue) or at the polar side of the mother (red) was measured. Bars, 1 μm.
Mentions: Although kinetochores remain intact and anaphase chromosome segregation occurs normally after the depletion of CBF3 (Rodrigo-Brenni et al., 2004), it is possible that there are subtle changes in outer kinetochore complexes that affect septin organization. To test this possibility, we examined septins in a variety of mutants that compromise either CBF3 or outer kinetochore complexes. We observed little change in septin organization in mutants that effect outer kinetochore complexes (e.g., ndc80-1; Fig. 5, A and B). Although some outer kinetochore alleles arrest in metaphase because of the spindle checkpoint, we and others have observed that some alleles are checkpoint defective and continue past metaphase (e.g., ctf19-26, ctf19-58, ndc80-1, and dam1-1; Hyland et al., 1999; Jones et al., 2001; Wigge and Kilmartin, 2001), making it unlikely that a pre-anaphase arrest masks a septin defect. In contrast, alleles that affect CBF3 assembly exhibit a high percentage of cells with disorganized septins (Fig. 5 A and Fig. S2, available at http://www.jcb.org/cgi/content/full/jcb.200507017/DC1). As was the case for the GAL1-CTF13 strain, we observed partially formed or misoriented septin rings, especially in anaphase cells. Interestingly, the most dramatic affects on septins were observed in SKP1 and SGT1 alleles that we have previously shown compromise the CBF3 cycle (Fig. 5 B; Lingelbach and Kaplan, 2004; Rodrigo-Brenni et al., 2004). Specifically, the skp1-4 allele, which prevents CBF3 assembly by blocking the interaction between Skp1p and Sgt1p, gives rise to defects in septin organization. The skp1-3 allele, which stabilizes the Skp1p–Sgt1p interaction and prevents CBF3 turnover (Rodrigo-Brenni et al., 2004), results in an even higher percentage of cells with disorganized septins. SKP1 and SGT1 have also been implicated in the function of the Skp1-cullin-F-box (SCF)–E3 ubiquitin ligase complex. To rule out a role for SCF in regulating septin organization, we examined cdc34-2, which is a conditional allele encoding the ubiquitin-ligase subunit of SCF. We observed no effect on septin organization despite the high percentage of cells with elongated buds (Fig. 5, A and B). In contrast to the more subtle effects of SKP1 and SGT1 alleles, the ndc10-1 allele entirely eliminates kinetochore formation on CEN DNA and cells inappropriately proceed through the cell cycle (Gardner et al., 2001); the resulting multibudded ndc10-1 cells frequently exhibit a complete failure to form septins rings (38% for ndc10-1 and 5% for the wild-type W303 strain) and are therefore not included in our analysis of “disorganized” septins. This phenotype is similar to that recently shown by Bouck and Bloom (2005). However, we note that this extreme phenotype is not observed when ndc10-1 is backcrossed to the S288C background used in all of the other data in our studies, instead we observe a pattern of septin disorganization more typical of CBF3 mutants (Fig. 5 B and Fig. S2; see Discussion). Thus, even the most severe kinetochore mutant available produces the same septin defect observed in mutants, which perturbs the CBF3 cycle but not kinetochore formation. Together, these results strongly argue that CBF3 functions independently from its role at the kinetochore to regulate septins during anaphase.

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