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A cell sizer network involving Cln3 and Far1 controls entrance into S phase in the mitotic cycle of budding yeast.

Alberghina L, Rossi RL, Querin L, Wanke V, Vanoni M - J. Cell Biol. (2004)

Bottom Line: Cells grown in glucose are larger than cells grown in ethanol.Here, we show that an increased level of the cyclin-dependent inhibitor Far1 increases cell size, whereas far1 Delta cells start bud emergence and DNA replication at a smaller size than wild type.A new molecular network accounting for the setting of Ps is proposed.

View Article: PubMed Central - PubMed

Affiliation: Department of Biotechnology and Biosciences, University of Milano-Bicocca, 20126 Milan, Italy. lilia.alberghina@unimib.it

ABSTRACT
Saccharomyces cerevisiae must reach a carbon source-modulated critical cell size, protein content per cell at the onset of DNA replication (Ps), in order to enter S phase. Cells grown in glucose are larger than cells grown in ethanol. Here, we show that an increased level of the cyclin-dependent inhibitor Far1 increases cell size, whereas far1 Delta cells start bud emergence and DNA replication at a smaller size than wild type. Cln3 Delta, far1 Delta, and strains overexpressing Far1 do not delay budding during an ethanol glucose shift-up as wild type does. Together, these findings indicate that Cln3 has to overcome Far1 to trigger Cln-Cdc28 activation, which then turns on SBF- and MBF-dependent transcription. We show that a second threshold is required together with the Cln3/Far1 threshold for carbon source modulation of Ps. A new molecular network accounting for the setting of Ps is proposed.

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FAR1 deletion partially uncouples budding and DNA synthesis and alters timing and cell size of S phase entry. Wild-type and far1Δ cells were grown in SCD, elutriated as reported in Materials and methods and small cells refed with fresh prewarmed SCD at time 0. (A) Percentage of budded cells determined by microscopic observation (a minimum of 300 cells were counted for each time point) for wild-type cells (closed circles) and far1Δ cells (open circles). (B) DNA FACS distributions of wild-type cells synchronized by centrifugal elutriation and released in fresh SCD medium were determined every 10 min after refeeding. The percentage of budded cells and of cells with DNA content greater then 1c (S+G2+M) is indicated on the right side. (C) Same as in B for far1Δ cells. The single black arrow in the left side of B and C indicates the time point in which wild-type and far1Δ cells had the same volume (26 fl) as determined by coulter counter analysis. (D) Percentage of S+G2+M cells determined FACS analysis for wild-type cells (closed circles) and far1Δ cells (open circles). (E) Cln3 protein level were evaluated by Western blot analysis in wild-type (W303-p strain) and in far1Δ cells (far1Δ-p strain) exponentially growing in glucose (SCD medium); quantities of crude extracts loaded are indicated. (F) The percentage of budded cells and of S+G2+M cells from A and D are plotted as function of relative cell volume, where the initial volume of both cell types at time 0 was made equal to 1. Experiments were repeated twice with superimposable results. Representative results from one of these experiments are shown.
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fig3: FAR1 deletion partially uncouples budding and DNA synthesis and alters timing and cell size of S phase entry. Wild-type and far1Δ cells were grown in SCD, elutriated as reported in Materials and methods and small cells refed with fresh prewarmed SCD at time 0. (A) Percentage of budded cells determined by microscopic observation (a minimum of 300 cells were counted for each time point) for wild-type cells (closed circles) and far1Δ cells (open circles). (B) DNA FACS distributions of wild-type cells synchronized by centrifugal elutriation and released in fresh SCD medium were determined every 10 min after refeeding. The percentage of budded cells and of cells with DNA content greater then 1c (S+G2+M) is indicated on the right side. (C) Same as in B for far1Δ cells. The single black arrow in the left side of B and C indicates the time point in which wild-type and far1Δ cells had the same volume (26 fl) as determined by coulter counter analysis. (D) Percentage of S+G2+M cells determined FACS analysis for wild-type cells (closed circles) and far1Δ cells (open circles). (E) Cln3 protein level were evaluated by Western blot analysis in wild-type (W303-p strain) and in far1Δ cells (far1Δ-p strain) exponentially growing in glucose (SCD medium); quantities of crude extracts loaded are indicated. (F) The percentage of budded cells and of S+G2+M cells from A and D are plotted as function of relative cell volume, where the initial volume of both cell types at time 0 was made equal to 1. Experiments were repeated twice with superimposable results. Representative results from one of these experiments are shown.

Mentions: As shown in Fig. 3 A, during the first 90 min after fresh medium refeeding, no bud emergence is observed in wild-type cells, whereas mutant far1Δ cells started to bud as soon as fresh medium was refed. Only small buds were produced in this period (not depicted). During the same time window, no net DNA synthesis was observed in either wild-type (Fig. 3 B) or far1Δ cells (Fig. 3 C), although in the latter strain a constant low level of cells with DNA content higher than 1c was observed. Because DNA synthesis requires overcoming of a second, Sic1-dependent, threshold (Nash et al., 2001), these results may be due to partial uncoupling of budding and DNA synthesis, although limited sensitivity of flow cytometry may hamper detection with sufficient resolution of cells entering just the earliest stage of S phase. After 90 min, budding and DNA synthesis were activated in both wild-type and far1Δ cells. The kinetics of budding and DNA synthesis completion are directly compared in Fig. 3 (A and D), respectively. The initial rate of budding in far1Δ cells (time 0–90 min) was significantly lower than that observed in the same cells after 90 min, that paralleled that observed in the wild type. Right after elutriation, far1Δ cells showed a low level of cells with DNA content higher than 1c, but only after 90 min DNA synthesis started, with a rate very similar to that observed in wild type (Fig. 3 D). Some delay in the completion of S (or in the entry of all cells into S) was also apparent. The above data clearly indicate that budding is anticipated in the absence of Far1, as expected. In far1Δ cells bud emergence proceeds steadily, although with an initial slow kinetics that may depend at least in part upon the lower steady-state level of Cln3 in far1Δ cells (Fig. 3 E).


A cell sizer network involving Cln3 and Far1 controls entrance into S phase in the mitotic cycle of budding yeast.

Alberghina L, Rossi RL, Querin L, Wanke V, Vanoni M - J. Cell Biol. (2004)

FAR1 deletion partially uncouples budding and DNA synthesis and alters timing and cell size of S phase entry. Wild-type and far1Δ cells were grown in SCD, elutriated as reported in Materials and methods and small cells refed with fresh prewarmed SCD at time 0. (A) Percentage of budded cells determined by microscopic observation (a minimum of 300 cells were counted for each time point) for wild-type cells (closed circles) and far1Δ cells (open circles). (B) DNA FACS distributions of wild-type cells synchronized by centrifugal elutriation and released in fresh SCD medium were determined every 10 min after refeeding. The percentage of budded cells and of cells with DNA content greater then 1c (S+G2+M) is indicated on the right side. (C) Same as in B for far1Δ cells. The single black arrow in the left side of B and C indicates the time point in which wild-type and far1Δ cells had the same volume (26 fl) as determined by coulter counter analysis. (D) Percentage of S+G2+M cells determined FACS analysis for wild-type cells (closed circles) and far1Δ cells (open circles). (E) Cln3 protein level were evaluated by Western blot analysis in wild-type (W303-p strain) and in far1Δ cells (far1Δ-p strain) exponentially growing in glucose (SCD medium); quantities of crude extracts loaded are indicated. (F) The percentage of budded cells and of S+G2+M cells from A and D are plotted as function of relative cell volume, where the initial volume of both cell types at time 0 was made equal to 1. Experiments were repeated twice with superimposable results. Representative results from one of these experiments are shown.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172493&req=5

fig3: FAR1 deletion partially uncouples budding and DNA synthesis and alters timing and cell size of S phase entry. Wild-type and far1Δ cells were grown in SCD, elutriated as reported in Materials and methods and small cells refed with fresh prewarmed SCD at time 0. (A) Percentage of budded cells determined by microscopic observation (a minimum of 300 cells were counted for each time point) for wild-type cells (closed circles) and far1Δ cells (open circles). (B) DNA FACS distributions of wild-type cells synchronized by centrifugal elutriation and released in fresh SCD medium were determined every 10 min after refeeding. The percentage of budded cells and of cells with DNA content greater then 1c (S+G2+M) is indicated on the right side. (C) Same as in B for far1Δ cells. The single black arrow in the left side of B and C indicates the time point in which wild-type and far1Δ cells had the same volume (26 fl) as determined by coulter counter analysis. (D) Percentage of S+G2+M cells determined FACS analysis for wild-type cells (closed circles) and far1Δ cells (open circles). (E) Cln3 protein level were evaluated by Western blot analysis in wild-type (W303-p strain) and in far1Δ cells (far1Δ-p strain) exponentially growing in glucose (SCD medium); quantities of crude extracts loaded are indicated. (F) The percentage of budded cells and of S+G2+M cells from A and D are plotted as function of relative cell volume, where the initial volume of both cell types at time 0 was made equal to 1. Experiments were repeated twice with superimposable results. Representative results from one of these experiments are shown.
Mentions: As shown in Fig. 3 A, during the first 90 min after fresh medium refeeding, no bud emergence is observed in wild-type cells, whereas mutant far1Δ cells started to bud as soon as fresh medium was refed. Only small buds were produced in this period (not depicted). During the same time window, no net DNA synthesis was observed in either wild-type (Fig. 3 B) or far1Δ cells (Fig. 3 C), although in the latter strain a constant low level of cells with DNA content higher than 1c was observed. Because DNA synthesis requires overcoming of a second, Sic1-dependent, threshold (Nash et al., 2001), these results may be due to partial uncoupling of budding and DNA synthesis, although limited sensitivity of flow cytometry may hamper detection with sufficient resolution of cells entering just the earliest stage of S phase. After 90 min, budding and DNA synthesis were activated in both wild-type and far1Δ cells. The kinetics of budding and DNA synthesis completion are directly compared in Fig. 3 (A and D), respectively. The initial rate of budding in far1Δ cells (time 0–90 min) was significantly lower than that observed in the same cells after 90 min, that paralleled that observed in the wild type. Right after elutriation, far1Δ cells showed a low level of cells with DNA content higher than 1c, but only after 90 min DNA synthesis started, with a rate very similar to that observed in wild type (Fig. 3 D). Some delay in the completion of S (or in the entry of all cells into S) was also apparent. The above data clearly indicate that budding is anticipated in the absence of Far1, as expected. In far1Δ cells bud emergence proceeds steadily, although with an initial slow kinetics that may depend at least in part upon the lower steady-state level of Cln3 in far1Δ cells (Fig. 3 E).

Bottom Line: Cells grown in glucose are larger than cells grown in ethanol.Here, we show that an increased level of the cyclin-dependent inhibitor Far1 increases cell size, whereas far1 Delta cells start bud emergence and DNA replication at a smaller size than wild type.A new molecular network accounting for the setting of Ps is proposed.

View Article: PubMed Central - PubMed

Affiliation: Department of Biotechnology and Biosciences, University of Milano-Bicocca, 20126 Milan, Italy. lilia.alberghina@unimib.it

ABSTRACT
Saccharomyces cerevisiae must reach a carbon source-modulated critical cell size, protein content per cell at the onset of DNA replication (Ps), in order to enter S phase. Cells grown in glucose are larger than cells grown in ethanol. Here, we show that an increased level of the cyclin-dependent inhibitor Far1 increases cell size, whereas far1 Delta cells start bud emergence and DNA replication at a smaller size than wild type. Cln3 Delta, far1 Delta, and strains overexpressing Far1 do not delay budding during an ethanol glucose shift-up as wild type does. Together, these findings indicate that Cln3 has to overcome Far1 to trigger Cln-Cdc28 activation, which then turns on SBF- and MBF-dependent transcription. We show that a second threshold is required together with the Cln3/Far1 threshold for carbon source modulation of Ps. A new molecular network accounting for the setting of Ps is proposed.

Show MeSH
Related in: MedlinePlus