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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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Visualization of the  septin ring by fluorescence  (a–d) and confocal (e–g) microscopy of the L2C24d strain  (cdc15-lyt1) transformed with  the pLA10 plasmid bearing a  CDC10–GFP fusion (Cid et  al., 1998a) (a, b, and e–g) and  MY3 strain (cdc15-lyt1/cdc15-1  CDC10–GFP) (c and d). Cells  were kept at 37°C for 4.5 h to  allow expression of the characteristic morphogenetic phenotype. (a and b) Fluorescence microscopy shows that  the septins remain at the  mother–daughter neck in  asymmetric doublets. (c and  d) The formation of chains of  cells in diploid cdc15/cdc15  mutants is supported by the formation of novel septin rings, but the first ring in the neck between the oldest cells persists. (e–g) Simultaneous staining with rhodamine-conjugated phalloidin allows the visualization of both the actin and septin cytoskeletons. Red, actin-rich  areas; green, corresponds to the septin–GFP fusion. In e and g, the fluorescence image is overlapped to a phase contrast micrograph for  better definition of cell shape. Bars, 5 μm.
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Figure 4: Visualization of the septin ring by fluorescence (a–d) and confocal (e–g) microscopy of the L2C24d strain (cdc15-lyt1) transformed with the pLA10 plasmid bearing a CDC10–GFP fusion (Cid et al., 1998a) (a, b, and e–g) and MY3 strain (cdc15-lyt1/cdc15-1 CDC10–GFP) (c and d). Cells were kept at 37°C for 4.5 h to allow expression of the characteristic morphogenetic phenotype. (a and b) Fluorescence microscopy shows that the septins remain at the mother–daughter neck in asymmetric doublets. (c and d) The formation of chains of cells in diploid cdc15/cdc15 mutants is supported by the formation of novel septin rings, but the first ring in the neck between the oldest cells persists. (e–g) Simultaneous staining with rhodamine-conjugated phalloidin allows the visualization of both the actin and septin cytoskeletons. Red, actin-rich areas; green, corresponds to the septin–GFP fusion. In e and g, the fluorescence image is overlapped to a phase contrast micrograph for better definition of cell shape. Bars, 5 μm.

Mentions: Septins constitute another cytoskeletal structure assumed to play crucial roles in morphogenetic events along the cell cycle, such as the localization of septa (Longtine et al., 1996; Cid et al., 1998a). Septins assemble at the bud neck as a ring before bud emergence and remain there until cytokinesis is complete. We studied the localization of the septin-based ring in cdc15-lyt1 mutants using a Cdc10p–green fluorescent protein (GFP) fusion. As shown in Fig. 4, a and b, septins remain as a double ring at the mother–daughter neck well after the apical structure has grown. Using confocal microscopy, we were able to simultaneously visualize rhodamine-stained actin and Cdc10p– GFP (Fig. 4, e–g). From this experiment we concluded that septins are never observed at the base of the distal actin-rich projections. Therefore, the aberrant shmoo-like structure characteristic of cdc15 mutants is supported by the actin but not by the septin cytoskeleton, which remains at the site of the aborted septation. It is likely that the lytic phenotype of cdc15ts mutants would be due to a budding attempt that has been aborted because of the absence of essential morphogenetic components that rely on the presence of the septin ring. Consistent with this hypothesis, septins are present at the neck of the successful new buds that originate chains of cells in diploid cdc15/cdc15 mutants at 37°C, as shown in Fig. 4, b and c.


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)

Visualization of the  septin ring by fluorescence  (a–d) and confocal (e–g) microscopy of the L2C24d strain  (cdc15-lyt1) transformed with  the pLA10 plasmid bearing a  CDC10–GFP fusion (Cid et  al., 1998a) (a, b, and e–g) and  MY3 strain (cdc15-lyt1/cdc15-1  CDC10–GFP) (c and d). Cells  were kept at 37°C for 4.5 h to  allow expression of the characteristic morphogenetic phenotype. (a and b) Fluorescence microscopy shows that  the septins remain at the  mother–daughter neck in  asymmetric doublets. (c and  d) The formation of chains of  cells in diploid cdc15/cdc15  mutants is supported by the formation of novel septin rings, but the first ring in the neck between the oldest cells persists. (e–g) Simultaneous staining with rhodamine-conjugated phalloidin allows the visualization of both the actin and septin cytoskeletons. Red, actin-rich  areas; green, corresponds to the septin–GFP fusion. In e and g, the fluorescence image is overlapped to a phase contrast micrograph for  better definition of cell shape. Bars, 5 μm.
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Related In: Results  -  Collection

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Figure 4: Visualization of the septin ring by fluorescence (a–d) and confocal (e–g) microscopy of the L2C24d strain (cdc15-lyt1) transformed with the pLA10 plasmid bearing a CDC10–GFP fusion (Cid et al., 1998a) (a, b, and e–g) and MY3 strain (cdc15-lyt1/cdc15-1 CDC10–GFP) (c and d). Cells were kept at 37°C for 4.5 h to allow expression of the characteristic morphogenetic phenotype. (a and b) Fluorescence microscopy shows that the septins remain at the mother–daughter neck in asymmetric doublets. (c and d) The formation of chains of cells in diploid cdc15/cdc15 mutants is supported by the formation of novel septin rings, but the first ring in the neck between the oldest cells persists. (e–g) Simultaneous staining with rhodamine-conjugated phalloidin allows the visualization of both the actin and septin cytoskeletons. Red, actin-rich areas; green, corresponds to the septin–GFP fusion. In e and g, the fluorescence image is overlapped to a phase contrast micrograph for better definition of cell shape. Bars, 5 μm.
Mentions: Septins constitute another cytoskeletal structure assumed to play crucial roles in morphogenetic events along the cell cycle, such as the localization of septa (Longtine et al., 1996; Cid et al., 1998a). Septins assemble at the bud neck as a ring before bud emergence and remain there until cytokinesis is complete. We studied the localization of the septin-based ring in cdc15-lyt1 mutants using a Cdc10p–green fluorescent protein (GFP) fusion. As shown in Fig. 4, a and b, septins remain as a double ring at the mother–daughter neck well after the apical structure has grown. Using confocal microscopy, we were able to simultaneously visualize rhodamine-stained actin and Cdc10p– GFP (Fig. 4, e–g). From this experiment we concluded that septins are never observed at the base of the distal actin-rich projections. Therefore, the aberrant shmoo-like structure characteristic of cdc15 mutants is supported by the actin but not by the septin cytoskeleton, which remains at the site of the aborted septation. It is likely that the lytic phenotype of cdc15ts mutants would be due to a budding attempt that has been aborted because of the absence of essential morphogenetic components that rely on the presence of the septin ring. Consistent with this hypothesis, septins are present at the neck of the successful new buds that originate chains of cells in diploid cdc15/cdc15 mutants at 37°C, as shown in Fig. 4, b and c.

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