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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 septin ring separation and disassembly. After growth in raffinose for 120 min, large-budded cells expressing Cdc11-GFP and containing CTF13 (A) or GAL1-CTF13 (B) were selected for recording. Panels depict the major transitions in septin behavior for CTF13 or GAL1-CTF13 strains (see Videos 1–3 for the complete sequence, available at http://www.jcb.org/cgi/content/full/jcb.200507017/DC1). (C) Time 0 represents the first appearance of distinct septin rings; the transitions are defined as follows and the length of time for each transition is plotted for multiple cells. The violet column indicates the time between SI and SII or when septin ring separation is complete. The magenta column represents the time between SII and the disassembly of the septin ring in the mother cell (D). The yellow column represents time between disassembly (D) and reformation of a new septin ring in the mother cell (R).
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fig3: Inhibition of CBF3 assembly causes defects in septin ring separation and disassembly. After growth in raffinose for 120 min, large-budded cells expressing Cdc11-GFP and containing CTF13 (A) or GAL1-CTF13 (B) were selected for recording. Panels depict the major transitions in septin behavior for CTF13 or GAL1-CTF13 strains (see Videos 1–3 for the complete sequence, available at http://www.jcb.org/cgi/content/full/jcb.200507017/DC1). (C) Time 0 represents the first appearance of distinct septin rings; the transitions are defined as follows and the length of time for each transition is plotted for multiple cells. The violet column indicates the time between SI and SII or when septin ring separation is complete. The magenta column represents the time between SII and the disassembly of the septin ring in the mother cell (D). The yellow column represents time between disassembly (D) and reformation of a new septin ring in the mother cell (R).

Mentions: To more directly address how CBF3 assembly regulates anaphase septins, we filmed the behavior of Cdc11-GFP in mitotic cells. In wild-type cells, septins switch from a filamentous structure in G2 to distinct rings as mitosis begins (compare Fig. 3 [G2] with Fig. S1; also compare HU arrest with HU release in Fig. 2 B). Septin rings undergo further separation 15–45 min after they become distinct during anaphase (Fig. 3 A, separation II; shortly [2–6 min] after septins are maximally separated, the septin ring in the mother cell undergoes disassembly [Fig. 3 A, disassembly]); cells finish cytokinesis and rapidly return to G1 as indicated by the reformation of the septin ring in the mother cell (7–13 min after disassembly; Fig. 3 A, reformation). The general timing of anaphase as judged by septin behavior is remarkably consistent between cells with very similar transitions between each septin stage (Fig. 3 C, CTF13; and Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200507017/DC1). In cells where CBF3 assembly is inhibited (Fig. 3 B, GAL1-CTF13), septin rings become distinct in large budded cells but show more variable kinetics during the transition from SI to SII (wild type = 32 ± 11 min; GAL1-CTF13 = 48 ± 42 min; Fig. 3 C). More dramatically, separation of rings occurs asymmetrically in the majority of cells inhibited for CBF3 assembly (92% in CBF3-inhibited cells compared with 0.2% in wild-type cells); in these cells, one portion of the ring in the mother cell remains in close proximity to the daughter cell ring (Fig. 3 B, 34-min time point). Finally, after this aberrant ring separation we observed that the mother cell ring is dramatically delayed for disassembly (Fig. 3, B and C; and Video 2). In most cases, the ring fails to disassemble during the time course of the experiment (mean >70 min) in contrast to the rapid disassembly observed in wild-type cells (mean = 10 min). Remarkably, we also observed several unbudded (G1) cells where the septin ring disassembled and then reformed in a nonaxial position, suggesting that the timing of septin dynamics is misregulated in cells inhibited for CBF3 assembly (Video 3). Together, these data argue that the CBF3 cycle regulates the timing of septin dynamics during the anaphase and G1, possibly through changes in their posttranslation modifications.


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 septin ring separation and disassembly. After growth in raffinose for 120 min, large-budded cells expressing Cdc11-GFP and containing CTF13 (A) or GAL1-CTF13 (B) were selected for recording. Panels depict the major transitions in septin behavior for CTF13 or GAL1-CTF13 strains (see Videos 1–3 for the complete sequence, available at http://www.jcb.org/cgi/content/full/jcb.200507017/DC1). (C) Time 0 represents the first appearance of distinct septin rings; the transitions are defined as follows and the length of time for each transition is plotted for multiple cells. The violet column indicates the time between SI and SII or when septin ring separation is complete. The magenta column represents the time between SII and the disassembly of the septin ring in the mother cell (D). The yellow column represents time between disassembly (D) and reformation of a new septin ring in the mother cell (R).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171293&req=5

fig3: Inhibition of CBF3 assembly causes defects in septin ring separation and disassembly. After growth in raffinose for 120 min, large-budded cells expressing Cdc11-GFP and containing CTF13 (A) or GAL1-CTF13 (B) were selected for recording. Panels depict the major transitions in septin behavior for CTF13 or GAL1-CTF13 strains (see Videos 1–3 for the complete sequence, available at http://www.jcb.org/cgi/content/full/jcb.200507017/DC1). (C) Time 0 represents the first appearance of distinct septin rings; the transitions are defined as follows and the length of time for each transition is plotted for multiple cells. The violet column indicates the time between SI and SII or when septin ring separation is complete. The magenta column represents the time between SII and the disassembly of the septin ring in the mother cell (D). The yellow column represents time between disassembly (D) and reformation of a new septin ring in the mother cell (R).
Mentions: To more directly address how CBF3 assembly regulates anaphase septins, we filmed the behavior of Cdc11-GFP in mitotic cells. In wild-type cells, septins switch from a filamentous structure in G2 to distinct rings as mitosis begins (compare Fig. 3 [G2] with Fig. S1; also compare HU arrest with HU release in Fig. 2 B). Septin rings undergo further separation 15–45 min after they become distinct during anaphase (Fig. 3 A, separation II; shortly [2–6 min] after septins are maximally separated, the septin ring in the mother cell undergoes disassembly [Fig. 3 A, disassembly]); cells finish cytokinesis and rapidly return to G1 as indicated by the reformation of the septin ring in the mother cell (7–13 min after disassembly; Fig. 3 A, reformation). The general timing of anaphase as judged by septin behavior is remarkably consistent between cells with very similar transitions between each septin stage (Fig. 3 C, CTF13; and Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200507017/DC1). In cells where CBF3 assembly is inhibited (Fig. 3 B, GAL1-CTF13), septin rings become distinct in large budded cells but show more variable kinetics during the transition from SI to SII (wild type = 32 ± 11 min; GAL1-CTF13 = 48 ± 42 min; Fig. 3 C). More dramatically, separation of rings occurs asymmetrically in the majority of cells inhibited for CBF3 assembly (92% in CBF3-inhibited cells compared with 0.2% in wild-type cells); in these cells, one portion of the ring in the mother cell remains in close proximity to the daughter cell ring (Fig. 3 B, 34-min time point). Finally, after this aberrant ring separation we observed that the mother cell ring is dramatically delayed for disassembly (Fig. 3, B and C; and Video 2). In most cases, the ring fails to disassemble during the time course of the experiment (mean >70 min) in contrast to the rapid disassembly observed in wild-type cells (mean = 10 min). Remarkably, we also observed several unbudded (G1) cells where the septin ring disassembled and then reformed in a nonaxial position, suggesting that the timing of septin dynamics is misregulated in cells inhibited for CBF3 assembly (Video 3). Together, these data argue that the CBF3 cycle regulates the timing of septin dynamics during the anaphase and G1, possibly through changes in their posttranslation modifications.

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