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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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The FAR1 gene is involved in cell size setting under both steady-state and transient growth conditions. (A) Size of wild-type cells (thick line) and far1Δ cells overexpressing FAR1 under the control of a Tet-repressible promoter (FAR1tet strain, thin line) during exponential growth in ethanol (SCE medium) determined by FACS analysis of total cell protein after FITC staining. (B). Same as A for cells exponentially growing in glucose (SCD medium). (C) FAR1 expression in a FAR1tet strain exponentially growing in SCE was switched off by addition of doxocycline at time 0. Far1 protein levels (top) and the average P (FACS analysis protein profile) were measured at the indicated time after doxocycline addition. (D) Average P from FACS analysis of untreated cells (closed circles), and after treatment with 0.02 μg/ml doxocycline (open circles) and 2 μg/ml doxocycline (open triangles). (E) Correlation between cellular P and intracellular Far1 level determined from quantification of immunoblot bands as detailed in Materials and methods. (F) Size of wild-type, FAR1tet, and the FAR1N393-tet strains, the latter overexpressing a truncated form of Far1, during exponential growth in SCE determined by FACS analysis of total cell protein after FITC staining. Experiments were repeated twice with superimposable results. Representative results from one of these experiments are shown.
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fig2: The FAR1 gene is involved in cell size setting under both steady-state and transient growth conditions. (A) Size of wild-type cells (thick line) and far1Δ cells overexpressing FAR1 under the control of a Tet-repressible promoter (FAR1tet strain, thin line) during exponential growth in ethanol (SCE medium) determined by FACS analysis of total cell protein after FITC staining. (B). Same as A for cells exponentially growing in glucose (SCD medium). (C) FAR1 expression in a FAR1tet strain exponentially growing in SCE was switched off by addition of doxocycline at time 0. Far1 protein levels (top) and the average P (FACS analysis protein profile) were measured at the indicated time after doxocycline addition. (D) Average P from FACS analysis of untreated cells (closed circles), and after treatment with 0.02 μg/ml doxocycline (open circles) and 2 μg/ml doxocycline (open triangles). (E) Correlation between cellular P and intracellular Far1 level determined from quantification of immunoblot bands as detailed in Materials and methods. (F) Size of wild-type, FAR1tet, and the FAR1N393-tet strains, the latter overexpressing a truncated form of Far1, during exponential growth in SCE determined by FACS analysis of total cell protein after FITC staining. Experiments were repeated twice with superimposable results. Representative results from one of these experiments are shown.

Mentions: The Cln3/Far1 threshold hypothesis, predicts a direct correlation between FAR1 expression and population cell size. Data reported in Fig. 2 in fact show that overexpression of Far1 driven by a tetracycline-repressible promoter resulted in a shift of the protein distribution leading to increased average P in ethanol-grown cells (Fig. 2 A) and, to a much smaller extent, in glucose-grown cell (Fig. 2 B). Moreover, when FAR1 expression was shut off by addition of tetracycline to SCE-growing cells, a time-dependent reduction of Far1 level and of the total P was observed (Fig. 2, C and D). Fig. 2 E shows that a linear relation between cellular P and the log of intracellular Far1 concentration can be observed. Finally, the ability to increase cell size upon overexpression is retained, at least partially, by the Far11-393 truncated protein (Fig. 2 F), which still contains the minimal Far1 domain able to elicit G1 arrest after α factor treatment (Valtz et al., 1995; Gartner et al., 1998).


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)

The FAR1 gene is involved in cell size setting under both steady-state and transient growth conditions. (A) Size of wild-type cells (thick line) and far1Δ cells overexpressing FAR1 under the control of a Tet-repressible promoter (FAR1tet strain, thin line) during exponential growth in ethanol (SCE medium) determined by FACS analysis of total cell protein after FITC staining. (B). Same as A for cells exponentially growing in glucose (SCD medium). (C) FAR1 expression in a FAR1tet strain exponentially growing in SCE was switched off by addition of doxocycline at time 0. Far1 protein levels (top) and the average P (FACS analysis protein profile) were measured at the indicated time after doxocycline addition. (D) Average P from FACS analysis of untreated cells (closed circles), and after treatment with 0.02 μg/ml doxocycline (open circles) and 2 μg/ml doxocycline (open triangles). (E) Correlation between cellular P and intracellular Far1 level determined from quantification of immunoblot bands as detailed in Materials and methods. (F) Size of wild-type, FAR1tet, and the FAR1N393-tet strains, the latter overexpressing a truncated form of Far1, during exponential growth in SCE determined by FACS analysis of total cell protein after FITC staining. Experiments were repeated twice with superimposable results. Representative results from one of these experiments are shown.
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fig2: The FAR1 gene is involved in cell size setting under both steady-state and transient growth conditions. (A) Size of wild-type cells (thick line) and far1Δ cells overexpressing FAR1 under the control of a Tet-repressible promoter (FAR1tet strain, thin line) during exponential growth in ethanol (SCE medium) determined by FACS analysis of total cell protein after FITC staining. (B). Same as A for cells exponentially growing in glucose (SCD medium). (C) FAR1 expression in a FAR1tet strain exponentially growing in SCE was switched off by addition of doxocycline at time 0. Far1 protein levels (top) and the average P (FACS analysis protein profile) were measured at the indicated time after doxocycline addition. (D) Average P from FACS analysis of untreated cells (closed circles), and after treatment with 0.02 μg/ml doxocycline (open circles) and 2 μg/ml doxocycline (open triangles). (E) Correlation between cellular P and intracellular Far1 level determined from quantification of immunoblot bands as detailed in Materials and methods. (F) Size of wild-type, FAR1tet, and the FAR1N393-tet strains, the latter overexpressing a truncated form of Far1, during exponential growth in SCE determined by FACS analysis of total cell protein after FITC staining. Experiments were repeated twice with superimposable results. Representative results from one of these experiments are shown.
Mentions: The Cln3/Far1 threshold hypothesis, predicts a direct correlation between FAR1 expression and population cell size. Data reported in Fig. 2 in fact show that overexpression of Far1 driven by a tetracycline-repressible promoter resulted in a shift of the protein distribution leading to increased average P in ethanol-grown cells (Fig. 2 A) and, to a much smaller extent, in glucose-grown cell (Fig. 2 B). Moreover, when FAR1 expression was shut off by addition of tetracycline to SCE-growing cells, a time-dependent reduction of Far1 level and of the total P was observed (Fig. 2, C and D). Fig. 2 E shows that a linear relation between cellular P and the log of intracellular Far1 concentration can be observed. Finally, the ability to increase cell size upon overexpression is retained, at least partially, by the Far11-393 truncated protein (Fig. 2 F), which still contains the minimal Far1 domain able to elicit G1 arrest after α factor treatment (Valtz et al., 1995; Gartner et al., 1998).

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