Limits...
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.

Show MeSH

Related in: MedlinePlus

An endogenous Tea1-For3 fusion protein is functional but impinges on the cell division machinery.(A) Schematic of Tea1, For3, and Tea1-For3 protein domains and organization. (B) Anti-V5 immunoprecipitates from asynchronous tea1-V53, for3-V53, and tea1-for3-V53 cells were blotted with anti-V5 antibodies. Arrows indicate full-length proteins. Lysates were blotted with anti-Cdc2 as a loading control. (C) Fixed-cell DAPI/methyl blue images of stained wild-type, tea1Δ, for3Δ, tea1Δ for3Δ, and tea1-for3 cells. (D) Quantification of phenotypes of cells in (C), with three trials per genotype and n>300 for each trial. Data are presented as mean ± SEM for each category. (E) Quantification of cell lengths at cell division, with n>200 for each genotype. Data are presented as box-and-whisker plots showing the median (line in the box), 25th–75th percentiles (box), and 5th–95th percentiles (whiskers) for each genotype (Bar = 5 µm).
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3475658&req=5

pgen-1003004-g007: An endogenous Tea1-For3 fusion protein is functional but impinges on the cell division machinery.(A) Schematic of Tea1, For3, and Tea1-For3 protein domains and organization. (B) Anti-V5 immunoprecipitates from asynchronous tea1-V53, for3-V53, and tea1-for3-V53 cells were blotted with anti-V5 antibodies. Arrows indicate full-length proteins. Lysates were blotted with anti-Cdc2 as a loading control. (C) Fixed-cell DAPI/methyl blue images of stained wild-type, tea1Δ, for3Δ, tea1Δ for3Δ, and tea1-for3 cells. (D) Quantification of phenotypes of cells in (C), with three trials per genotype and n>300 for each trial. Data are presented as mean ± SEM for each category. (E) Quantification of cell lengths at cell division, with n>200 for each genotype. Data are presented as box-and-whisker plots showing the median (line in the box), 25th–75th percentiles (box), and 5th–95th percentiles (whiskers) for each genotype (Bar = 5 µm).

Mentions: If faithful remodeling of the division site is important for growth competency of new ends, then one would expect that prematurely triggering NETO signaling just after cell division should not fully rescue the growth polarity defects of late cytokinesis mutants. To test this, we constructed a mutant that would undergo constitutive NETO. As over-expression of a fusion protein linking cell tip-associated Tea1 with formin For3 induces NETO in G1 [8], we integrated a Tea1-For3 fusion (Figure 7A) into the endogenous tea1+ locus and deleted the single copy of the for3+ gene. We confirmed that the Tea1-For3 fusion protein was produced in vivo (Figure 7B) and verified that this fusion was sufficient to induce NETO in a cdc10-V50 G1 arrest (Figure S6A–S6B). As previously reported [7], double deletion of tea1+ and for3+ resulted in general cell rounding (Figure 7C). However, expression of the Tea1-For3 fusion protein in the absence of Tea1 and For3 individually caused cells to regain their rod-shaped appearance (Figure 7C). Intriguingly, a high percentage of tea1-for3 cells were either septated or exhibited cytokinesis defects (Figure 7C–7D), and tea1-for3 cells were significantly longer at division than wild-type cells (on average, 18.3 µm versus 15.3 µm) (Figure 7C and 7E). Thus, though the endogenous Tea1-For3 fusion protein functioned in prematurely triggering NETO, it also affected cell division.


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

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

An endogenous Tea1-For3 fusion protein is functional but impinges on the cell division machinery.(A) Schematic of Tea1, For3, and Tea1-For3 protein domains and organization. (B) Anti-V5 immunoprecipitates from asynchronous tea1-V53, for3-V53, and tea1-for3-V53 cells were blotted with anti-V5 antibodies. Arrows indicate full-length proteins. Lysates were blotted with anti-Cdc2 as a loading control. (C) Fixed-cell DAPI/methyl blue images of stained wild-type, tea1Δ, for3Δ, tea1Δ for3Δ, and tea1-for3 cells. (D) Quantification of phenotypes of cells in (C), with three trials per genotype and n>300 for each trial. Data are presented as mean ± SEM for each category. (E) Quantification of cell lengths at cell division, with n>200 for each genotype. Data are presented as box-and-whisker plots showing the median (line in the box), 25th–75th percentiles (box), and 5th–95th percentiles (whiskers) for each genotype (Bar = 5 µm).
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1003004-g007: An endogenous Tea1-For3 fusion protein is functional but impinges on the cell division machinery.(A) Schematic of Tea1, For3, and Tea1-For3 protein domains and organization. (B) Anti-V5 immunoprecipitates from asynchronous tea1-V53, for3-V53, and tea1-for3-V53 cells were blotted with anti-V5 antibodies. Arrows indicate full-length proteins. Lysates were blotted with anti-Cdc2 as a loading control. (C) Fixed-cell DAPI/methyl blue images of stained wild-type, tea1Δ, for3Δ, tea1Δ for3Δ, and tea1-for3 cells. (D) Quantification of phenotypes of cells in (C), with three trials per genotype and n>300 for each trial. Data are presented as mean ± SEM for each category. (E) Quantification of cell lengths at cell division, with n>200 for each genotype. Data are presented as box-and-whisker plots showing the median (line in the box), 25th–75th percentiles (box), and 5th–95th percentiles (whiskers) for each genotype (Bar = 5 µm).
Mentions: If faithful remodeling of the division site is important for growth competency of new ends, then one would expect that prematurely triggering NETO signaling just after cell division should not fully rescue the growth polarity defects of late cytokinesis mutants. To test this, we constructed a mutant that would undergo constitutive NETO. As over-expression of a fusion protein linking cell tip-associated Tea1 with formin For3 induces NETO in G1 [8], we integrated a Tea1-For3 fusion (Figure 7A) into the endogenous tea1+ locus and deleted the single copy of the for3+ gene. We confirmed that the Tea1-For3 fusion protein was produced in vivo (Figure 7B) and verified that this fusion was sufficient to induce NETO in a cdc10-V50 G1 arrest (Figure S6A–S6B). As previously reported [7], double deletion of tea1+ and for3+ resulted in general cell rounding (Figure 7C). However, expression of the Tea1-For3 fusion protein in the absence of Tea1 and For3 individually caused cells to regain their rod-shaped appearance (Figure 7C). Intriguingly, a high percentage of tea1-for3 cells were either septated or exhibited cytokinesis defects (Figure 7C–7D), and tea1-for3 cells were significantly longer at division than wild-type cells (on average, 18.3 µm versus 15.3 µm) (Figure 7C and 7E). Thus, though the endogenous Tea1-For3 fusion protein functioned in prematurely triggering NETO, it also affected cell division.

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