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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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Evolution of actin polarization in the L2C24d (cdc15-lyt1) strain at the restrictive temperature. Actin was stained with  rhodamine-conjugated phalloidine. The plot represents the percentage of each cell type within the population (y axis) versus  time of incubation at 37°C (x axis). Only live cells can be stained  for the visualization of actin, so no lysed cells were included in  this assay. (▴) Single unbudded cells with a random distribution  of actin patches. (♦) Doublets with a depolarized actin cytoskeleton, as corresponds to anaphase-arrested cells. (▪) Doublets with  a polarization of the actin cytoskeleton to the neck area for the  promotion of septum morphogenesis. (•) Cells showing a pattern of actin polarization towards the incipient, emergent or developing bud. (✖) Doublets or asymmetric doublets that polarize  the actin cytoskeleton towards the distal pole of one of the cells.  Bars, 5 μm.
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Figure 3: Evolution of actin polarization in the L2C24d (cdc15-lyt1) strain at the restrictive temperature. Actin was stained with rhodamine-conjugated phalloidine. The plot represents the percentage of each cell type within the population (y axis) versus time of incubation at 37°C (x axis). Only live cells can be stained for the visualization of actin, so no lysed cells were included in this assay. (▴) Single unbudded cells with a random distribution of actin patches. (♦) Doublets with a depolarized actin cytoskeleton, as corresponds to anaphase-arrested cells. (▪) Doublets with a polarization of the actin cytoskeleton to the neck area for the promotion of septum morphogenesis. (•) Cells showing a pattern of actin polarization towards the incipient, emergent or developing bud. (✖) Doublets or asymmetric doublets that polarize the actin cytoskeleton towards the distal pole of one of the cells. Bars, 5 μm.

Mentions: We incubated the cdc15-lyt1 mutant strain L2C24d at 24°C overnight and then shifted it to the restrictive temperature. Samples were taken every 30 min, fixed, and then stained with rhodamine-conjugated phalloidine to visualize the actin cytoskeleton. An average of 120 nonlysed cells from each sample was analyzed for actin polarization under a fluorescence microscope. The dynamics of actin polarization are represented graphically in Fig. 3. As expected in a log phase culture, only 17% of the cells (single cells or doublets) displayed a randomly distributed pattern of actin deposition, whereas 76% of the population showed a polarization of actin towards the incipient, emerging or developing bud, and ∼7% of the cells concentrated most actin around the septum area. Total depolarization of the actin cytoskeleton was observed after 30 min at 37°C. This is probably not due to the expression of the cdc15 phenotype but rather would be the normal cellular response to the stress caused by a temperature shift (Pringle et al., 1989). After 1 h, the actin cytoskeleton had been repolarized, but the pattern of localization to the septum area was not detected. The same was observed subsequently, despite the progressive accumulation of cells in doublets, which invariably had a delocalized actin cytoskeleton, as corresponds to the expected anaphase arrest.


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)

Evolution of actin polarization in the L2C24d (cdc15-lyt1) strain at the restrictive temperature. Actin was stained with  rhodamine-conjugated phalloidine. The plot represents the percentage of each cell type within the population (y axis) versus  time of incubation at 37°C (x axis). Only live cells can be stained  for the visualization of actin, so no lysed cells were included in  this assay. (▴) Single unbudded cells with a random distribution  of actin patches. (♦) Doublets with a depolarized actin cytoskeleton, as corresponds to anaphase-arrested cells. (▪) Doublets with  a polarization of the actin cytoskeleton to the neck area for the  promotion of septum morphogenesis. (•) Cells showing a pattern of actin polarization towards the incipient, emergent or developing bud. (✖) Doublets or asymmetric doublets that polarize  the actin cytoskeleton towards the distal pole of one of the cells.  Bars, 5 μm.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 3: Evolution of actin polarization in the L2C24d (cdc15-lyt1) strain at the restrictive temperature. Actin was stained with rhodamine-conjugated phalloidine. The plot represents the percentage of each cell type within the population (y axis) versus time of incubation at 37°C (x axis). Only live cells can be stained for the visualization of actin, so no lysed cells were included in this assay. (▴) Single unbudded cells with a random distribution of actin patches. (♦) Doublets with a depolarized actin cytoskeleton, as corresponds to anaphase-arrested cells. (▪) Doublets with a polarization of the actin cytoskeleton to the neck area for the promotion of septum morphogenesis. (•) Cells showing a pattern of actin polarization towards the incipient, emergent or developing bud. (✖) Doublets or asymmetric doublets that polarize the actin cytoskeleton towards the distal pole of one of the cells. Bars, 5 μm.
Mentions: We incubated the cdc15-lyt1 mutant strain L2C24d at 24°C overnight and then shifted it to the restrictive temperature. Samples were taken every 30 min, fixed, and then stained with rhodamine-conjugated phalloidine to visualize the actin cytoskeleton. An average of 120 nonlysed cells from each sample was analyzed for actin polarization under a fluorescence microscope. The dynamics of actin polarization are represented graphically in Fig. 3. As expected in a log phase culture, only 17% of the cells (single cells or doublets) displayed a randomly distributed pattern of actin deposition, whereas 76% of the population showed a polarization of actin towards the incipient, emerging or developing bud, and ∼7% of the cells concentrated most actin around the septum area. Total depolarization of the actin cytoskeleton was observed after 30 min at 37°C. This is probably not due to the expression of the cdc15 phenotype but rather would be the normal cellular response to the stress caused by a temperature shift (Pringle et al., 1989). After 1 h, the actin cytoskeleton had been repolarized, but the pattern of localization to the septum area was not detected. The same was observed subsequently, despite the progressive accumulation of cells in doublets, which invariably had a delocalized actin cytoskeleton, as corresponds to the expected anaphase arrest.

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