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Morphogenesis beyond cytokinetic arrest in Saccharomyces cerevisiae.

Jiménez J, Cid VJ, Cenamor R, Yuste M, Molero G, Nombela C, Sánchez M - J. Cell Biol. (1998)

Bottom Line: This morphogenetic response reflects entry into a new round of the cell cycle: the preference for polarization from the distal pole was lost in bud1 cdc15 double mutants; double cdc15-lyt1 cdc28-4 mutants, defective for START, did not develop apical projections and apical polarization was accompanied by DNA replication.Apical polarization was delayed in cdc15 mutants as compared with budding in control cells and this delay was abolished in a septin mutant.Our results suggest that the delayed M/G1 transition in cdc15 mutants is due to a septin-dependent checkpoint that couples initiation of the cell cycle to the completion of cytokinesis.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Microbiología II, Facultad de Farmacia.

ABSTRACT
The budding yeast lyt1 mutation causes cell lysis. We report here that lyt1 is an allele of cdc15, a gene which encodes a protein kinase that functions late in the cell cycle. Neither cdc15-1 nor cdc15-lyt1 strains are able to septate at 37 degreesC, even though they may manage to rebud. Cells lyse after a shmoo-like projection appears at the distal pole of the daughter cell. Actin polarizes towards the distal pole but the septins remain at the mother-daughter neck. This morphogenetic response reflects entry into a new round of the cell cycle: the preference for polarization from the distal pole was lost in bud1 cdc15 double mutants; double cdc15-lyt1 cdc28-4 mutants, defective for START, did not develop apical projections and apical polarization was accompanied by DNA replication. The same phenomena were caused by mutations in the genes CDC14, DBF2, and TEM1, which are functionally related to CDC15. Apical polarization was delayed in cdc15 mutants as compared with budding in control cells and this delay was abolished in a septin mutant. Our results suggest that the delayed M/G1 transition in cdc15 mutants is due to a septin-dependent checkpoint that couples initiation of the cell cycle to the completion of cytokinesis.

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Flow cytometry graphics of RNase-treated propidium  iodide-stained cells of the L2C24d strain. Cells were incubated at  37°C in osmotically stabilized media and samples were taken every 30 min. Peaks correspond to cell populations containing different amounts of DNA. The peak corresponding to a DNA content  of 1 nucleus per cell (G1 peak) appears at ∼15 fluorescence units  (see scale at the bottom), the G2/M peak (2 nuclei per unit) stands  at ∼30 units, and cells displaying a DNA content equivalent to 4  nuclei appear at 60 U. Upon expression of the cdc15 phenotype  (right), the G1 peak progressively disappears due to the accumulation of cells in anaphase. After 150 min, a new well-defined peak  reveals a new population of cells with a larger amount of DNA.  None of these phenomena occurred when the same strain was  transformed with a CDC15-containing plasmid (left).
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Figure 5: Flow cytometry graphics of RNase-treated propidium iodide-stained cells of the L2C24d strain. Cells were incubated at 37°C in osmotically stabilized media and samples were taken every 30 min. Peaks correspond to cell populations containing different amounts of DNA. The peak corresponding to a DNA content of 1 nucleus per cell (G1 peak) appears at ∼15 fluorescence units (see scale at the bottom), the G2/M peak (2 nuclei per unit) stands at ∼30 units, and cells displaying a DNA content equivalent to 4 nuclei appear at 60 U. Upon expression of the cdc15 phenotype (right), the G1 peak progressively disappears due to the accumulation of cells in anaphase. After 150 min, a new well-defined peak reveals a new population of cells with a larger amount of DNA. None of these phenomena occurred when the same strain was transformed with a CDC15-containing plasmid (left).

Mentions: To confirm that nuclei from cytokinetic-defective cells expressing the cdc15 phenotype were overcoming mitotic arrest, we decided to study the progression of the nuclear cell cycle in these cells. We performed quantitative DNA content assays by flow cytometry to determine whether the morphogenetic response was indeed accompanied by post-START events. As shown in Fig. 5, after incubation of the cells at the restrictive temperature in 1 M sorbitol, which retards lysis (as shown above), the peak corresponding to pre-S phase cells progressively decreased while the post-S phase peak increased due to the accumulation of anaphase-arrested cells, as expected. Interestingly, in longer exposures (times longer than 150 min) to the restrictive temperature a third peak was seen, indicating the presence of a new population bearing an amount of DNA equivalent to that of four nuclei. This last peak very likely represented cells that overcame the M phase arrest and started a new cell division, in agreement with the results reported above. When the same experiments were carried out on diploid cdc15/cdc15 strains, the graphics were difficult to interpret due to the appearance of subsequent diffuse peaks of higher fluorescence intensity, probably corresponding to chained cells (data not shown). In sum, these results suggest that a significant part of the population indeed overcomes the anaphase arrest at the restrictive temperature and initiates a new round of the cell cycle.


Morphogenesis beyond cytokinetic arrest in Saccharomyces cerevisiae.

Jiménez J, Cid VJ, Cenamor R, Yuste M, Molero G, Nombela C, Sánchez M - J. Cell Biol. (1998)

Flow cytometry graphics of RNase-treated propidium  iodide-stained cells of the L2C24d strain. Cells were incubated at  37°C in osmotically stabilized media and samples were taken every 30 min. Peaks correspond to cell populations containing different amounts of DNA. The peak corresponding to a DNA content  of 1 nucleus per cell (G1 peak) appears at ∼15 fluorescence units  (see scale at the bottom), the G2/M peak (2 nuclei per unit) stands  at ∼30 units, and cells displaying a DNA content equivalent to 4  nuclei appear at 60 U. Upon expression of the cdc15 phenotype  (right), the G1 peak progressively disappears due to the accumulation of cells in anaphase. After 150 min, a new well-defined peak  reveals a new population of cells with a larger amount of DNA.  None of these phenomena occurred when the same strain was  transformed with a CDC15-containing plasmid (left).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2132980&req=5

Figure 5: Flow cytometry graphics of RNase-treated propidium iodide-stained cells of the L2C24d strain. Cells were incubated at 37°C in osmotically stabilized media and samples were taken every 30 min. Peaks correspond to cell populations containing different amounts of DNA. The peak corresponding to a DNA content of 1 nucleus per cell (G1 peak) appears at ∼15 fluorescence units (see scale at the bottom), the G2/M peak (2 nuclei per unit) stands at ∼30 units, and cells displaying a DNA content equivalent to 4 nuclei appear at 60 U. Upon expression of the cdc15 phenotype (right), the G1 peak progressively disappears due to the accumulation of cells in anaphase. After 150 min, a new well-defined peak reveals a new population of cells with a larger amount of DNA. None of these phenomena occurred when the same strain was transformed with a CDC15-containing plasmid (left).
Mentions: To confirm that nuclei from cytokinetic-defective cells expressing the cdc15 phenotype were overcoming mitotic arrest, we decided to study the progression of the nuclear cell cycle in these cells. We performed quantitative DNA content assays by flow cytometry to determine whether the morphogenetic response was indeed accompanied by post-START events. As shown in Fig. 5, after incubation of the cells at the restrictive temperature in 1 M sorbitol, which retards lysis (as shown above), the peak corresponding to pre-S phase cells progressively decreased while the post-S phase peak increased due to the accumulation of anaphase-arrested cells, as expected. Interestingly, in longer exposures (times longer than 150 min) to the restrictive temperature a third peak was seen, indicating the presence of a new population bearing an amount of DNA equivalent to that of four nuclei. This last peak very likely represented cells that overcame the M phase arrest and started a new cell division, in agreement with the results reported above. When the same experiments were carried out on diploid cdc15/cdc15 strains, the graphics were difficult to interpret due to the appearance of subsequent diffuse peaks of higher fluorescence intensity, probably corresponding to chained cells (data not shown). In sum, these results suggest that a significant part of the population indeed overcomes the anaphase arrest at the restrictive temperature and initiates a new round of the cell cycle.

Bottom Line: This morphogenetic response reflects entry into a new round of the cell cycle: the preference for polarization from the distal pole was lost in bud1 cdc15 double mutants; double cdc15-lyt1 cdc28-4 mutants, defective for START, did not develop apical projections and apical polarization was accompanied by DNA replication.Apical polarization was delayed in cdc15 mutants as compared with budding in control cells and this delay was abolished in a septin mutant.Our results suggest that the delayed M/G1 transition in cdc15 mutants is due to a septin-dependent checkpoint that couples initiation of the cell cycle to the completion of cytokinesis.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Microbiología II, Facultad de Farmacia.

ABSTRACT
The budding yeast lyt1 mutation causes cell lysis. We report here that lyt1 is an allele of cdc15, a gene which encodes a protein kinase that functions late in the cell cycle. Neither cdc15-1 nor cdc15-lyt1 strains are able to septate at 37 degreesC, even though they may manage to rebud. Cells lyse after a shmoo-like projection appears at the distal pole of the daughter cell. Actin polarizes towards the distal pole but the septins remain at the mother-daughter neck. This morphogenetic response reflects entry into a new round of the cell cycle: the preference for polarization from the distal pole was lost in bud1 cdc15 double mutants; double cdc15-lyt1 cdc28-4 mutants, defective for START, did not develop apical projections and apical polarization was accompanied by DNA replication. The same phenomena were caused by mutations in the genes CDC14, DBF2, and TEM1, which are functionally related to CDC15. Apical polarization was delayed in cdc15 mutants as compared with budding in control cells and this delay was abolished in a septin mutant. Our results suggest that the delayed M/G1 transition in cdc15 mutants is due to a septin-dependent checkpoint that couples initiation of the cell cycle to the completion of cytokinesis.

Show MeSH
Related in: MedlinePlus