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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 requires CBF3 assembly during anaphase. (A) The indicated strains were grown as outlined in the flow chart. (B and C) Stages where cells were removed for analysis are indicated by colors (blue, S phase; red, metaphase; and green, postanaphase cells). (B) DIC and fluorescent images of Cdc11-GFP were collected as indicated in A. (C) The total percentage of cells with disorganized septins was calculated. Bar, 1 μm.
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fig2: Septin organization requires CBF3 assembly during anaphase. (A) The indicated strains were grown as outlined in the flow chart. (B and C) Stages where cells were removed for analysis are indicated by colors (blue, S phase; red, metaphase; and green, postanaphase cells). (B) DIC and fluorescent images of Cdc11-GFP were collected as indicated in A. (C) The total percentage of cells with disorganized septins was calculated. Bar, 1 μm.

Mentions: Disorganized septins may reflect a failure to properly regulate their dynamics during the cell cycle. In addition, we observed the most severe septin defects in anaphase cells (Fig. S1 D). To examine the requirement for CBF3 assembly during anaphase more directly, we arrested strains that contain Cdc11-GFP and either CTF13 or GAL1-CTF13 in S phase, using hydroxyurea. Cells were transferred to media containing raffinose for 2 h in the presence of hydroxyurea (Fig. 2 A). We observed no change in septin organization after the hydroxyurea arrest in either control or GAL1-CTF13 strains (Fig. 2 B, +raffinose HU arrest). Cells were released from S phase by washing out the hydroxyurea and returning them to medium containing raffinose with or without nocodazole, a microtubule poison that arrests cells in metaphase. In cells arrested in metaphase, inhibition of CBF3 assembly had no effect on the Cdc11-GFP pattern (Fig. 2 B, +raffinose, HU release, +nocodazole). Finally, we released cells from the hydroxyurea-arrest into medium containing raffinose and allowed them to progress past metaphase; fixing and staining cells indicated that >90% of large budded cells were in anaphase under these conditions (unpublished data). In control cells, we observed the normal separation of septin rings marking the beginning of anaphase. In contrast, a high percentage of cells inhibited for CBF3 assembly exhibited disorganized septins during this same time period (Fig. 2, B [+raffinose, HU release] and C). From these data, we concluded that CBF3 assembly is critical for septin organization after the movement of chromosomes to the poles in anaphase, at a time when septins are separating to form the cytokinetic furrow.


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 requires CBF3 assembly during anaphase. (A) The indicated strains were grown as outlined in the flow chart. (B and C) Stages where cells were removed for analysis are indicated by colors (blue, S phase; red, metaphase; and green, postanaphase cells). (B) DIC and fluorescent images of Cdc11-GFP were collected as indicated in A. (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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fig2: Septin organization requires CBF3 assembly during anaphase. (A) The indicated strains were grown as outlined in the flow chart. (B and C) Stages where cells were removed for analysis are indicated by colors (blue, S phase; red, metaphase; and green, postanaphase cells). (B) DIC and fluorescent images of Cdc11-GFP were collected as indicated in A. (C) The total percentage of cells with disorganized septins was calculated. Bar, 1 μm.
Mentions: Disorganized septins may reflect a failure to properly regulate their dynamics during the cell cycle. In addition, we observed the most severe septin defects in anaphase cells (Fig. S1 D). To examine the requirement for CBF3 assembly during anaphase more directly, we arrested strains that contain Cdc11-GFP and either CTF13 or GAL1-CTF13 in S phase, using hydroxyurea. Cells were transferred to media containing raffinose for 2 h in the presence of hydroxyurea (Fig. 2 A). We observed no change in septin organization after the hydroxyurea arrest in either control or GAL1-CTF13 strains (Fig. 2 B, +raffinose HU arrest). Cells were released from S phase by washing out the hydroxyurea and returning them to medium containing raffinose with or without nocodazole, a microtubule poison that arrests cells in metaphase. In cells arrested in metaphase, inhibition of CBF3 assembly had no effect on the Cdc11-GFP pattern (Fig. 2 B, +raffinose, HU release, +nocodazole). Finally, we released cells from the hydroxyurea-arrest into medium containing raffinose and allowed them to progress past metaphase; fixing and staining cells indicated that >90% of large budded cells were in anaphase under these conditions (unpublished data). In control cells, we observed the normal separation of septin rings marking the beginning of anaphase. In contrast, a high percentage of cells inhibited for CBF3 assembly exhibited disorganized septins during this same time period (Fig. 2, B [+raffinose, HU release] and C). From these data, we concluded that CBF3 assembly is critical for septin organization after the movement of chromosomes to the poles in anaphase, at a time when septins are separating to form the cytokinetic furrow.

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