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Cytokinesis-based constraints on polarized cell growth in fission yeast.

Bohnert KA, Gould KL - PLoS Genet. (2012)

Bottom Line: Intriguingly, such cells elongated constitutively at new ends unless cytokinesis was perturbed.We posit that such constraints facilitate invasive fungal growth, as cytokinesis mutants displaying bipolar growth defects formed numerous pseudohyphae.Collectively, these data highlight a role for previous cell cycles in defining a cell's capacity to polarize at specific sites, and they additionally provide insight into how a unicellular yeast can transition into a quasi-multicellular state.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute and Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, Tennessee, USA.

ABSTRACT
The rod-shaped fission yeast Schizosaccharomyces pombe, which undergoes cycles of monopolar-to-bipolar tip growth, is an attractive organism for studying cell-cycle regulation of polarity establishment. While previous research has described factors mediating this process from interphase cell tips, we found that division site signaling also impacts the re-establishment of bipolar cell growth in the ensuing cell cycle. Complete loss or targeted disruption of the non-essential cytokinesis protein Fic1 at the division site, but not at interphase cell tips, resulted in many cells failing to grow at new ends created by cell division. This appeared due to faulty disassembly and abnormal persistence of the cell division machinery at new ends of fic1Δ cells. Moreover, additional mutants defective in the final stages of cytokinesis exhibited analogous growth polarity defects, supporting that robust completion of cell division contributes to new end-growth competency. To test this model, we genetically manipulated S. pombe cells to undergo new end take-off immediately after cell division. Intriguingly, such cells elongated constitutively at new ends unless cytokinesis was perturbed. Thus, cell division imposes constraints that partially override positive controls on growth. We posit that such constraints facilitate invasive fungal growth, as cytokinesis mutants displaying bipolar growth defects formed numerous pseudohyphae. Collectively, these data highlight a role for previous cell cycles in defining a cell's capacity to polarize at specific sites, and they additionally provide insight into how a unicellular yeast can transition into a quasi-multicellular state.

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Fic1 participates in protein–protein interactions at the CR that guide subsequent growth polarity.(A) Live-cell images of calcofluor-stained cdc15ΔSH3, fic1-P257A, imp2Δ, and cyk3Δ cells. Arrowheads indicate monopolar cells. (B) Quantification of (A), with three trials per genotype and n>300 for each trial. Data are presented as mean ± SEM for each category. (C) Quantification of septated cells in (A) and (B), with three trials per genotype and n>300 for each trial. Data are presented as mean ± SEM for each category. (D) Anti-Cdc15 and anti-FLAG immunoprecipitates from cells of the indicated genotypes were blotted with anti-Cdc15 and/or anti-FLAG antibodies. cdc25-22 cells and nda3-KM311 cells were arrested in G2 and prometaphase, respectively, prior to pelleting and lysis. (E) Anti-FLAG and anti-V5 immunoprecipitates from prometaphase-arrested cells of the indicated genotypes were blotted with anti-FLAG and/or anti-V5 antibodies. (F) Live-cell GFP movie of a cyk3-GFP sid4-GFP cell, with images every 3 min (Bars = 5 µm).
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pgen-1003004-g004: Fic1 participates in protein–protein interactions at the CR that guide subsequent growth polarity.(A) Live-cell images of calcofluor-stained cdc15ΔSH3, fic1-P257A, imp2Δ, and cyk3Δ cells. Arrowheads indicate monopolar cells. (B) Quantification of (A), with three trials per genotype and n>300 for each trial. Data are presented as mean ± SEM for each category. (C) Quantification of septated cells in (A) and (B), with three trials per genotype and n>300 for each trial. Data are presented as mean ± SEM for each category. (D) Anti-Cdc15 and anti-FLAG immunoprecipitates from cells of the indicated genotypes were blotted with anti-Cdc15 and/or anti-FLAG antibodies. cdc25-22 cells and nda3-KM311 cells were arrested in G2 and prometaphase, respectively, prior to pelleting and lysis. (E) Anti-FLAG and anti-V5 immunoprecipitates from prometaphase-arrested cells of the indicated genotypes were blotted with anti-FLAG and/or anti-V5 antibodies. (F) Live-cell GFP movie of a cyk3-GFP sid4-GFP cell, with images every 3 min (Bars = 5 µm).

Mentions: Because Fic1's C terminus was necessary and sufficient for proper growth polarity, we examined whether protein-protein interactions at the CR mediated by Fic1's C-terminal PxxP motifs, which bind SH3 domains, govern Fic1's polarity function. Fic1 was originally identified based on its interaction with Cdc15's SH3 domain [28]. As would be expected if association of Cdc15 with Fic1's C terminus is important in establishing the timing of NETO, calcofluor-stained cdc15ΔSH3 cells, which are viable but lack Fic1-Cdc15 interaction [28], exhibited growth polarity defects (Figure 4A–4C and Figure S3A–S3B).


Cytokinesis-based constraints on polarized cell growth in fission yeast.

Bohnert KA, Gould KL - PLoS Genet. (2012)

Fic1 participates in protein–protein interactions at the CR that guide subsequent growth polarity.(A) Live-cell images of calcofluor-stained cdc15ΔSH3, fic1-P257A, imp2Δ, and cyk3Δ cells. Arrowheads indicate monopolar cells. (B) Quantification of (A), with three trials per genotype and n>300 for each trial. Data are presented as mean ± SEM for each category. (C) Quantification of septated cells in (A) and (B), with three trials per genotype and n>300 for each trial. Data are presented as mean ± SEM for each category. (D) Anti-Cdc15 and anti-FLAG immunoprecipitates from cells of the indicated genotypes were blotted with anti-Cdc15 and/or anti-FLAG antibodies. cdc25-22 cells and nda3-KM311 cells were arrested in G2 and prometaphase, respectively, prior to pelleting and lysis. (E) Anti-FLAG and anti-V5 immunoprecipitates from prometaphase-arrested cells of the indicated genotypes were blotted with anti-FLAG and/or anti-V5 antibodies. (F) Live-cell GFP movie of a cyk3-GFP sid4-GFP cell, with images every 3 min (Bars = 5 µm).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3475658&req=5

pgen-1003004-g004: Fic1 participates in protein–protein interactions at the CR that guide subsequent growth polarity.(A) Live-cell images of calcofluor-stained cdc15ΔSH3, fic1-P257A, imp2Δ, and cyk3Δ cells. Arrowheads indicate monopolar cells. (B) Quantification of (A), with three trials per genotype and n>300 for each trial. Data are presented as mean ± SEM for each category. (C) Quantification of septated cells in (A) and (B), with three trials per genotype and n>300 for each trial. Data are presented as mean ± SEM for each category. (D) Anti-Cdc15 and anti-FLAG immunoprecipitates from cells of the indicated genotypes were blotted with anti-Cdc15 and/or anti-FLAG antibodies. cdc25-22 cells and nda3-KM311 cells were arrested in G2 and prometaphase, respectively, prior to pelleting and lysis. (E) Anti-FLAG and anti-V5 immunoprecipitates from prometaphase-arrested cells of the indicated genotypes were blotted with anti-FLAG and/or anti-V5 antibodies. (F) Live-cell GFP movie of a cyk3-GFP sid4-GFP cell, with images every 3 min (Bars = 5 µm).
Mentions: Because Fic1's C terminus was necessary and sufficient for proper growth polarity, we examined whether protein-protein interactions at the CR mediated by Fic1's C-terminal PxxP motifs, which bind SH3 domains, govern Fic1's polarity function. Fic1 was originally identified based on its interaction with Cdc15's SH3 domain [28]. As would be expected if association of Cdc15 with Fic1's C terminus is important in establishing the timing of NETO, calcofluor-stained cdc15ΔSH3 cells, which are viable but lack Fic1-Cdc15 interaction [28], exhibited growth polarity defects (Figure 4A–4C and Figure S3A–S3B).

Bottom Line: Intriguingly, such cells elongated constitutively at new ends unless cytokinesis was perturbed.We posit that such constraints facilitate invasive fungal growth, as cytokinesis mutants displaying bipolar growth defects formed numerous pseudohyphae.Collectively, these data highlight a role for previous cell cycles in defining a cell's capacity to polarize at specific sites, and they additionally provide insight into how a unicellular yeast can transition into a quasi-multicellular state.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute and Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, Tennessee, USA.

ABSTRACT
The rod-shaped fission yeast Schizosaccharomyces pombe, which undergoes cycles of monopolar-to-bipolar tip growth, is an attractive organism for studying cell-cycle regulation of polarity establishment. While previous research has described factors mediating this process from interphase cell tips, we found that division site signaling also impacts the re-establishment of bipolar cell growth in the ensuing cell cycle. Complete loss or targeted disruption of the non-essential cytokinesis protein Fic1 at the division site, but not at interphase cell tips, resulted in many cells failing to grow at new ends created by cell division. This appeared due to faulty disassembly and abnormal persistence of the cell division machinery at new ends of fic1Δ cells. Moreover, additional mutants defective in the final stages of cytokinesis exhibited analogous growth polarity defects, supporting that robust completion of cell division contributes to new end-growth competency. To test this model, we genetically manipulated S. pombe cells to undergo new end take-off immediately after cell division. Intriguingly, such cells elongated constitutively at new ends unless cytokinesis was perturbed. Thus, cell division imposes constraints that partially override positive controls on growth. We posit that such constraints facilitate invasive fungal growth, as cytokinesis mutants displaying bipolar growth defects formed numerous pseudohyphae. Collectively, these data highlight a role for previous cell cycles in defining a cell's capacity to polarize at specific sites, and they additionally provide insight into how a unicellular yeast can transition into a quasi-multicellular state.

Show MeSH
Related in: MedlinePlus