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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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Loss of Fic1 impairs CR disassembly and leads to persistence of division site factors.(A) Fixed-cell DIC and DAPI/methyl blue images of asynchronous and G2-arrested cells of the indicated genotypes. Arrowheads indicate cells that are still joined following ingression. (B) Quantification of (A), with four trials per genotype and n>300 for each trial. Percentages are presented as mean ± SEM. (C) Live-cell GFP movies of rlc1-GFP sid4-GFP and fic1Δ rlc1-GFP sid4-GFP cells. Images were acquired every 2 min, and representative images are given for 10 min intervals. (D) Quantification of times from spindle pole body (SPB) separation to the completion of CR constriction in (C). n>20 for each genotype. 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. (E) Quantification of times from septum closure to disappearance of the CR at the division site for GFP-cps1 rlc1-mCherry3 and fic1Δ GFP-cps1 rlc1-mCherry3 cells. n>30 for each genotype. 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. (F) Live-cell GFP (colored green) and mCherry (mCh) (colored magenta) movies of GFP-cps1 rlc1-mCherry3 and fic1Δ GFP-cps1 rlc1-mCherry3 cells, with time intervals indicated and GFP/mCherry images merged. White arrows in GFP images mark the septa's leading edges. The time point with only one arrow drawn marks septum closure. In the mCh images, “C” marks the point of CR closure, and arrowheads denote CR remnants persisting after this point. (G) Fixed-cell images of actin stained with Alexa Fluor 488 Phalloidin. Single z planes as well as maximum projections of multiple z planes are given. Red arrows indicate division planes, whereas yellow arrows indicate unusual actin masses lining the division plane (Bars = 5 µm).
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pgen-1003004-g005: Loss of Fic1 impairs CR disassembly and leads to persistence of division site factors.(A) Fixed-cell DIC and DAPI/methyl blue images of asynchronous and G2-arrested cells of the indicated genotypes. Arrowheads indicate cells that are still joined following ingression. (B) Quantification of (A), with four trials per genotype and n>300 for each trial. Percentages are presented as mean ± SEM. (C) Live-cell GFP movies of rlc1-GFP sid4-GFP and fic1Δ rlc1-GFP sid4-GFP cells. Images were acquired every 2 min, and representative images are given for 10 min intervals. (D) Quantification of times from spindle pole body (SPB) separation to the completion of CR constriction in (C). n>20 for each genotype. 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. (E) Quantification of times from septum closure to disappearance of the CR at the division site for GFP-cps1 rlc1-mCherry3 and fic1Δ GFP-cps1 rlc1-mCherry3 cells. n>30 for each genotype. 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. (F) Live-cell GFP (colored green) and mCherry (mCh) (colored magenta) movies of GFP-cps1 rlc1-mCherry3 and fic1Δ GFP-cps1 rlc1-mCherry3 cells, with time intervals indicated and GFP/mCherry images merged. White arrows in GFP images mark the septa's leading edges. The time point with only one arrow drawn marks septum closure. In the mCh images, “C” marks the point of CR closure, and arrowheads denote CR remnants persisting after this point. (G) Fixed-cell images of actin stained with Alexa Fluor 488 Phalloidin. Single z planes as well as maximum projections of multiple z planes are given. Red arrows indicate division planes, whereas yellow arrows indicate unusual actin masses lining the division plane (Bars = 5 µm).

Mentions: To discern how loss of Fic1 scaffold function during cytokinesis impacts subsequent new end growth, we next defined what aspects of cytokinesis are perturbed in fic1Δ cells. Previous data demonstrated that fic1Δ was synthetically lethal with sid2-250 [28], a temperature-sensitive allele of the SIN kinase Sid2. Consistent with Fic1 and associated factors working in parallel to the SIN, we found that fic1Δ and cyk3Δ suppressed the hyperactive SIN allele cdc16-116 (Figure S4A), and that fic1Δ and cyk3Δ were synthetically sick or lethal with a variety of SIN alleles conferring loss of SIN function (Figure S4A–S4B). These genetic data implied that Fic1 most likely functions during late stages of cytokinesis. In line with this idea, the percentage of fic1Δ cells that had undergone ingression but were still joined at their division sites was more than four times that of wild-type cells (Figure 5A–5B). When cells were arrested in G2 using the cdc25-22 allele, this difference increased, with the percentage of joined cells roughly 15 times greater in the absence of Fic1 (Figure 5A–5B). Similar to S. cerevisiae inn1Δ cells [29] and S. pombe cdc15ΔSH3 cells [28], many G2-arrested fic1Δ daughter cells that were still joined at division sites exhibited membranous bridges (Figure S4C). These findings verified that the completion of cell division is perturbed in the absence of Fic1.


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

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

Loss of Fic1 impairs CR disassembly and leads to persistence of division site factors.(A) Fixed-cell DIC and DAPI/methyl blue images of asynchronous and G2-arrested cells of the indicated genotypes. Arrowheads indicate cells that are still joined following ingression. (B) Quantification of (A), with four trials per genotype and n>300 for each trial. Percentages are presented as mean ± SEM. (C) Live-cell GFP movies of rlc1-GFP sid4-GFP and fic1Δ rlc1-GFP sid4-GFP cells. Images were acquired every 2 min, and representative images are given for 10 min intervals. (D) Quantification of times from spindle pole body (SPB) separation to the completion of CR constriction in (C). n>20 for each genotype. 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. (E) Quantification of times from septum closure to disappearance of the CR at the division site for GFP-cps1 rlc1-mCherry3 and fic1Δ GFP-cps1 rlc1-mCherry3 cells. n>30 for each genotype. 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. (F) Live-cell GFP (colored green) and mCherry (mCh) (colored magenta) movies of GFP-cps1 rlc1-mCherry3 and fic1Δ GFP-cps1 rlc1-mCherry3 cells, with time intervals indicated and GFP/mCherry images merged. White arrows in GFP images mark the septa's leading edges. The time point with only one arrow drawn marks septum closure. In the mCh images, “C” marks the point of CR closure, and arrowheads denote CR remnants persisting after this point. (G) Fixed-cell images of actin stained with Alexa Fluor 488 Phalloidin. Single z planes as well as maximum projections of multiple z planes are given. Red arrows indicate division planes, whereas yellow arrows indicate unusual actin masses lining the division plane (Bars = 5 µm).
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pgen-1003004-g005: Loss of Fic1 impairs CR disassembly and leads to persistence of division site factors.(A) Fixed-cell DIC and DAPI/methyl blue images of asynchronous and G2-arrested cells of the indicated genotypes. Arrowheads indicate cells that are still joined following ingression. (B) Quantification of (A), with four trials per genotype and n>300 for each trial. Percentages are presented as mean ± SEM. (C) Live-cell GFP movies of rlc1-GFP sid4-GFP and fic1Δ rlc1-GFP sid4-GFP cells. Images were acquired every 2 min, and representative images are given for 10 min intervals. (D) Quantification of times from spindle pole body (SPB) separation to the completion of CR constriction in (C). n>20 for each genotype. 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. (E) Quantification of times from septum closure to disappearance of the CR at the division site for GFP-cps1 rlc1-mCherry3 and fic1Δ GFP-cps1 rlc1-mCherry3 cells. n>30 for each genotype. 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. (F) Live-cell GFP (colored green) and mCherry (mCh) (colored magenta) movies of GFP-cps1 rlc1-mCherry3 and fic1Δ GFP-cps1 rlc1-mCherry3 cells, with time intervals indicated and GFP/mCherry images merged. White arrows in GFP images mark the septa's leading edges. The time point with only one arrow drawn marks septum closure. In the mCh images, “C” marks the point of CR closure, and arrowheads denote CR remnants persisting after this point. (G) Fixed-cell images of actin stained with Alexa Fluor 488 Phalloidin. Single z planes as well as maximum projections of multiple z planes are given. Red arrows indicate division planes, whereas yellow arrows indicate unusual actin masses lining the division plane (Bars = 5 µm).
Mentions: To discern how loss of Fic1 scaffold function during cytokinesis impacts subsequent new end growth, we next defined what aspects of cytokinesis are perturbed in fic1Δ cells. Previous data demonstrated that fic1Δ was synthetically lethal with sid2-250 [28], a temperature-sensitive allele of the SIN kinase Sid2. Consistent with Fic1 and associated factors working in parallel to the SIN, we found that fic1Δ and cyk3Δ suppressed the hyperactive SIN allele cdc16-116 (Figure S4A), and that fic1Δ and cyk3Δ were synthetically sick or lethal with a variety of SIN alleles conferring loss of SIN function (Figure S4A–S4B). These genetic data implied that Fic1 most likely functions during late stages of cytokinesis. In line with this idea, the percentage of fic1Δ cells that had undergone ingression but were still joined at their division sites was more than four times that of wild-type cells (Figure 5A–5B). When cells were arrested in G2 using the cdc25-22 allele, this difference increased, with the percentage of joined cells roughly 15 times greater in the absence of Fic1 (Figure 5A–5B). Similar to S. cerevisiae inn1Δ cells [29] and S. pombe cdc15ΔSH3 cells [28], many G2-arrested fic1Δ daughter cells that were still joined at division sites exhibited membranous bridges (Figure S4C). These findings verified that the completion of cell division is perturbed in the absence of Fic1.

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