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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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Septin organization in shmoos requires CBF3 assembly. (A) The indicated strains were grown as outlined in the flow chart. (B) Stages where cells were removed for analysis are indicated by colors (green, α factor–induced arrest in G1; blue, S phase arrest in hydroxyurea [HU]). (B) DIC and fluorescent images of Cdc11-GFP were collected as indicated in A. The arrows indicate normal septin organization; arrowheads indicate cells with disorganized septins. (C) The total percentage of cells with disorganized septins was calculated. Bar, 1 μm.
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fig4: Septin organization in shmoos requires CBF3 assembly. (A) The indicated strains were grown as outlined in the flow chart. (B) Stages where cells were removed for analysis are indicated by colors (green, α factor–induced arrest in G1; blue, S phase arrest in hydroxyurea [HU]). (B) DIC and fluorescent images of Cdc11-GFP were collected as indicated in A. The arrows indicate normal septin organization; arrowheads indicate cells with disorganized septins. (C) The total percentage of cells with disorganized septins was calculated. Bar, 1 μm.

Mentions: Our observations that unbudded cells (G1) undergo inappropriate septin ring disassembly and reformation led us to ask if CBF3 can regulate septins independent of chromosome segregation and mitosis. As in anaphase, septins also undergo a dramatic reorganization during the mating process. After α factor treatment, haploid cells reorganize their septins into perpendicular (with respect to the bud neck) arrays during formation of the “shmoo” mating projection (Fig. 4 B, CTF13; Ford and Pringle, 1991; Kim et al., 1991; Longtine et al., 1998). To determine if the CBF3 cycle is important for the reorganization of septins in the shmoo, we treated GAL1-CTF13 or control cells with α factor and examined the distribution of Cdc11-GFP. In the GAL1-CTF13 strain, elevated (galactose) or reduced (raffinose) CBF3 levels resulted in less organized septins (Fig. 4 B, arrowheads) in the shmoo tip compared with the control strain. When α factor is removed, cells reinitiate the budding process and septins form the parallel structures typically observed in cycling cells. We released α factor–arrested cells into hydroxyurea to prevent them from entering mitosis and monitored Cdc11-GFP in the presence or absence of CBF3 assembly (Fig. 4 A). When released into media containing galactose, both GAL1-CTF13 and control cells exhibited well organized parallel septin rings at the mother bud neck (Fig. 4, B [arrows] and C). In contrast, when cells were released from the α factor arrest and CBF3 assembly was inhibited, septins became dramatically disorganized. The shape of the resulting bud also indicated that the morphological transition between the shmoo and bud had been compromised (Fig. 4 B, compare differential interference contrast [DIC] images). These results indicate that CBF3 is required to regulate septins during the G1 stage of the cell cycle, supporting the idea that the function of CBF3 in organizing septins is distinct from its role in segregating chromosomes (see Discussion).


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)

Septin organization in shmoos requires CBF3 assembly. (A) The indicated strains were grown as outlined in the flow chart. (B) Stages where cells were removed for analysis are indicated by colors (green, α factor–induced arrest in G1; blue, S phase arrest in hydroxyurea [HU]). (B) DIC and fluorescent images of Cdc11-GFP were collected as indicated in A. The arrows indicate normal septin organization; arrowheads indicate cells with disorganized septins. (C) The total percentage of cells with disorganized septins was calculated. Bar, 1 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171293&req=5

fig4: Septin organization in shmoos requires CBF3 assembly. (A) The indicated strains were grown as outlined in the flow chart. (B) Stages where cells were removed for analysis are indicated by colors (green, α factor–induced arrest in G1; blue, S phase arrest in hydroxyurea [HU]). (B) DIC and fluorescent images of Cdc11-GFP were collected as indicated in A. The arrows indicate normal septin organization; arrowheads indicate cells with disorganized septins. (C) The total percentage of cells with disorganized septins was calculated. Bar, 1 μm.
Mentions: Our observations that unbudded cells (G1) undergo inappropriate septin ring disassembly and reformation led us to ask if CBF3 can regulate septins independent of chromosome segregation and mitosis. As in anaphase, septins also undergo a dramatic reorganization during the mating process. After α factor treatment, haploid cells reorganize their septins into perpendicular (with respect to the bud neck) arrays during formation of the “shmoo” mating projection (Fig. 4 B, CTF13; Ford and Pringle, 1991; Kim et al., 1991; Longtine et al., 1998). To determine if the CBF3 cycle is important for the reorganization of septins in the shmoo, we treated GAL1-CTF13 or control cells with α factor and examined the distribution of Cdc11-GFP. In the GAL1-CTF13 strain, elevated (galactose) or reduced (raffinose) CBF3 levels resulted in less organized septins (Fig. 4 B, arrowheads) in the shmoo tip compared with the control strain. When α factor is removed, cells reinitiate the budding process and septins form the parallel structures typically observed in cycling cells. We released α factor–arrested cells into hydroxyurea to prevent them from entering mitosis and monitored Cdc11-GFP in the presence or absence of CBF3 assembly (Fig. 4 A). When released into media containing galactose, both GAL1-CTF13 and control cells exhibited well organized parallel septin rings at the mother bud neck (Fig. 4, B [arrows] and C). In contrast, when cells were released from the α factor arrest and CBF3 assembly was inhibited, septins became dramatically disorganized. The shape of the resulting bud also indicated that the morphological transition between the shmoo and bud had been compromised (Fig. 4 B, compare differential interference contrast [DIC] images). These results indicate that CBF3 is required to regulate septins during the G1 stage of the cell cycle, supporting the idea that the function of CBF3 in organizing septins is distinct from its role in segregating chromosomes (see Discussion).

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