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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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fic1Δ cells fail at new end growth independently of known cell cycle controls on NETO.(A–B) Live-cell DIC movies of wild-type or fic1Δ cells. Solid arrows denote old end growth, whereas dashed arrows indicate new end growth. Birth scars are marked by asterisks. Time points are noted. (C) Quantification of growth patterns for cells imaged in (A) and (B), with sample size (n) provided. (D) Quantification of growth patterns for tea1Δ and tea1Δ fic1Δ cells, with sample size (n) provided. (E) Live-cell DIC movie of a tea1Δ fic1Δ cell that gives rise to a T-shaped daughter cell. The solid arrow denotes old end growth, whereas the dashed arrow indicates non-tip growth. Birth scars are marked by asterisks. Time points are noted. (F) Quantification of T-shaped cells in tea1Δ and tea1Δ fic1Δ strains grown at 25°C, with three trials per genotype and n>300 for each trial. Data are presented as mean ± SEM for each genotype. (G) Live-cell images of calcofluor-stained tea1Δ and tea1Δ fic1Δ cells grown at 25°C. Arrows indicate T-shaped cells. (H) Quantification of times from septum splitting to initiation of new end growth in cells that undergo NETO prior to the next septation in (A–C). Data are presented in box-and-whisker plots showing the median (line in the box), 25th–75th percentiles (box), and 5th–95th percentiles (whiskers) for each genotype. (I) Live-cell images of calcofluor-stained cdc25-22 and fic1Δ cdc25-22 cells that had been arrested in G2. Arrowheads indicate monopolar cells. (J) Quantification of (I), with three trials per genotype and n>300 for each trial. Data are presented as mean ± SEM for each category. (K) 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. The dashed line represents the minimum length required for NETO [5] (Bars = 5 µm).
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pgen-1003004-g002: fic1Δ cells fail at new end growth independently of known cell cycle controls on NETO.(A–B) Live-cell DIC movies of wild-type or fic1Δ cells. Solid arrows denote old end growth, whereas dashed arrows indicate new end growth. Birth scars are marked by asterisks. Time points are noted. (C) Quantification of growth patterns for cells imaged in (A) and (B), with sample size (n) provided. (D) Quantification of growth patterns for tea1Δ and tea1Δ fic1Δ cells, with sample size (n) provided. (E) Live-cell DIC movie of a tea1Δ fic1Δ cell that gives rise to a T-shaped daughter cell. The solid arrow denotes old end growth, whereas the dashed arrow indicates non-tip growth. Birth scars are marked by asterisks. Time points are noted. (F) Quantification of T-shaped cells in tea1Δ and tea1Δ fic1Δ strains grown at 25°C, with three trials per genotype and n>300 for each trial. Data are presented as mean ± SEM for each genotype. (G) Live-cell images of calcofluor-stained tea1Δ and tea1Δ fic1Δ cells grown at 25°C. Arrows indicate T-shaped cells. (H) Quantification of times from septum splitting to initiation of new end growth in cells that undergo NETO prior to the next septation in (A–C). Data are presented in box-and-whisker plots showing the median (line in the box), 25th–75th percentiles (box), and 5th–95th percentiles (whiskers) for each genotype. (I) Live-cell images of calcofluor-stained cdc25-22 and fic1Δ cdc25-22 cells that had been arrested in G2. Arrowheads indicate monopolar cells. (J) Quantification of (I), with three trials per genotype and n>300 for each trial. Data are presented as mean ± SEM for each category. (K) 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. The dashed line represents the minimum length required for NETO [5] (Bars = 5 µm).

Mentions: To discern whether new and/or old ends were defective in resuming growth following cell division in fic1Δ cells, we performed time-lapse DIC imaging to trace birth scars in live cells. As expected, nearly all wild-type cells underwent NETO prior to subsequent septation (Figure 2A and 2C). However, following roughly two-thirds of fic1Δ cell divisions, either one or both daughter cells failed to initiate new end growth prior to the next septation (Figure 2B–2C). The most predominant growth pattern in fic1Δ cells was that in which one daughter cell underwent NETO while the other did not (Figure 2B–2C), with nearly 70% of those daughter cells that did not exhibit NETO being the younger daughter cell. Unlike tea1Δ and tea4Δ cells, in which one daughter cell commonly fails at its new end and the other daughter cell fails at its old end (Figure 2D) [8], [22], [23], fic1Δ cells were specifically defective in the re-establishment of growth at new ends following cell division (Figure 2B–2C). Intriguingly, tea1Δ fic1Δ double mutants grew mainly in a tea1Δ pattern, though nearly one-fifth of cell divisions produced a T-shaped daughter cell (Figure 2D–2E). Consistent with this, roughly 10% of tea1Δ fic1Δ cells were T-shaped at 25°C, while T-shaped tea1Δ cells were almost never observed at this temperature (Figure 2F). T-shapes always arose in cells that the tea1Δ growth pattern dictated should grow at their new ends (Figure 2D–2E) but that actually grew at neither (Figure 2E and 2G), suggesting these cells polarize at sites other than their tips because growth is inhibited at both ends. These data confirmed that the polarity defect caused by loss of Fic1 stochastically impacts new end growth in a variety of genetic backgrounds. Importantly, fic1Δ new ends that failed to extend in one cell cycle initiated growth as an old end in the next cell cycle, suggesting the defect in growth polarity caused by loss of Fic1 was not permanent. Consistent with a delay but not a block in growth, new ends that initiated growth prior to the next septation did so much later on average in fic1Δ cells than in wild-type cells (120 min versus 75 min) (Figure 2H).


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

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

fic1Δ cells fail at new end growth independently of known cell cycle controls on NETO.(A–B) Live-cell DIC movies of wild-type or fic1Δ cells. Solid arrows denote old end growth, whereas dashed arrows indicate new end growth. Birth scars are marked by asterisks. Time points are noted. (C) Quantification of growth patterns for cells imaged in (A) and (B), with sample size (n) provided. (D) Quantification of growth patterns for tea1Δ and tea1Δ fic1Δ cells, with sample size (n) provided. (E) Live-cell DIC movie of a tea1Δ fic1Δ cell that gives rise to a T-shaped daughter cell. The solid arrow denotes old end growth, whereas the dashed arrow indicates non-tip growth. Birth scars are marked by asterisks. Time points are noted. (F) Quantification of T-shaped cells in tea1Δ and tea1Δ fic1Δ strains grown at 25°C, with three trials per genotype and n>300 for each trial. Data are presented as mean ± SEM for each genotype. (G) Live-cell images of calcofluor-stained tea1Δ and tea1Δ fic1Δ cells grown at 25°C. Arrows indicate T-shaped cells. (H) Quantification of times from septum splitting to initiation of new end growth in cells that undergo NETO prior to the next septation in (A–C). Data are presented in box-and-whisker plots showing the median (line in the box), 25th–75th percentiles (box), and 5th–95th percentiles (whiskers) for each genotype. (I) Live-cell images of calcofluor-stained cdc25-22 and fic1Δ cdc25-22 cells that had been arrested in G2. Arrowheads indicate monopolar cells. (J) Quantification of (I), with three trials per genotype and n>300 for each trial. Data are presented as mean ± SEM for each category. (K) 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. The dashed line represents the minimum length required for NETO [5] (Bars = 5 µm).
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Related In: Results  -  Collection

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

pgen-1003004-g002: fic1Δ cells fail at new end growth independently of known cell cycle controls on NETO.(A–B) Live-cell DIC movies of wild-type or fic1Δ cells. Solid arrows denote old end growth, whereas dashed arrows indicate new end growth. Birth scars are marked by asterisks. Time points are noted. (C) Quantification of growth patterns for cells imaged in (A) and (B), with sample size (n) provided. (D) Quantification of growth patterns for tea1Δ and tea1Δ fic1Δ cells, with sample size (n) provided. (E) Live-cell DIC movie of a tea1Δ fic1Δ cell that gives rise to a T-shaped daughter cell. The solid arrow denotes old end growth, whereas the dashed arrow indicates non-tip growth. Birth scars are marked by asterisks. Time points are noted. (F) Quantification of T-shaped cells in tea1Δ and tea1Δ fic1Δ strains grown at 25°C, with three trials per genotype and n>300 for each trial. Data are presented as mean ± SEM for each genotype. (G) Live-cell images of calcofluor-stained tea1Δ and tea1Δ fic1Δ cells grown at 25°C. Arrows indicate T-shaped cells. (H) Quantification of times from septum splitting to initiation of new end growth in cells that undergo NETO prior to the next septation in (A–C). Data are presented in box-and-whisker plots showing the median (line in the box), 25th–75th percentiles (box), and 5th–95th percentiles (whiskers) for each genotype. (I) Live-cell images of calcofluor-stained cdc25-22 and fic1Δ cdc25-22 cells that had been arrested in G2. Arrowheads indicate monopolar cells. (J) Quantification of (I), with three trials per genotype and n>300 for each trial. Data are presented as mean ± SEM for each category. (K) 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. The dashed line represents the minimum length required for NETO [5] (Bars = 5 µm).
Mentions: To discern whether new and/or old ends were defective in resuming growth following cell division in fic1Δ cells, we performed time-lapse DIC imaging to trace birth scars in live cells. As expected, nearly all wild-type cells underwent NETO prior to subsequent septation (Figure 2A and 2C). However, following roughly two-thirds of fic1Δ cell divisions, either one or both daughter cells failed to initiate new end growth prior to the next septation (Figure 2B–2C). The most predominant growth pattern in fic1Δ cells was that in which one daughter cell underwent NETO while the other did not (Figure 2B–2C), with nearly 70% of those daughter cells that did not exhibit NETO being the younger daughter cell. Unlike tea1Δ and tea4Δ cells, in which one daughter cell commonly fails at its new end and the other daughter cell fails at its old end (Figure 2D) [8], [22], [23], fic1Δ cells were specifically defective in the re-establishment of growth at new ends following cell division (Figure 2B–2C). Intriguingly, tea1Δ fic1Δ double mutants grew mainly in a tea1Δ pattern, though nearly one-fifth of cell divisions produced a T-shaped daughter cell (Figure 2D–2E). Consistent with this, roughly 10% of tea1Δ fic1Δ cells were T-shaped at 25°C, while T-shaped tea1Δ cells were almost never observed at this temperature (Figure 2F). T-shapes always arose in cells that the tea1Δ growth pattern dictated should grow at their new ends (Figure 2D–2E) but that actually grew at neither (Figure 2E and 2G), suggesting these cells polarize at sites other than their tips because growth is inhibited at both ends. These data confirmed that the polarity defect caused by loss of Fic1 stochastically impacts new end growth in a variety of genetic backgrounds. Importantly, fic1Δ new ends that failed to extend in one cell cycle initiated growth as an old end in the next cell cycle, suggesting the defect in growth polarity caused by loss of Fic1 was not permanent. Consistent with a delay but not a block in growth, new ends that initiated growth prior to the next septation did so much later on average in fic1Δ cells than in wild-type cells (120 min versus 75 min) (Figure 2H).

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