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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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A functional extracellular glucose sensing system is required to properly set cell size (P) and protein content required for S phase initiation (Ps). (A) Size of wild-type cells (thick line), isogenic gpa2Δ cells (dashed line), and gpr1Δ cells (thin line) exponentially growing 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) Immunoblot showing Cln3 protein level in wild-type and isogenic gpa2Δ and gpr1Δ cells grown in SCD and SCE (lanes labeled D and E, respectively). Extracts were loaded and bands quantified as detailed in Materials and methods. The values of average P, critical P at the beginning of DNA replication (Ps) determined by cytofluorimetric analysis (see Materials and methods for details), and duplication time (T) and length of budded phase (Tb) were determined for wild-type cells (black bars), gpa2Δ cells (white), and gpr1Δ cells (dashed) exponentially growing in SCE and SCD. (D) Cln3 protein levels determined as detailed above, were plotted as a function of Ps for wild-type cells (diamonds), gpa2Δ cells (circles), gpr1Δ cells (triangles), and wild-type cells overexpressing Cln3 (squares). Closed symbols indicate cells grown in SCD, open symbols indicate cells grown in SCE. Either average ± SD of data derived from experiments repeated at least three times (C and D, bar chart) or representative results from one of such experiments (A, B, and Western blot in C) are shown.
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fig1: A functional extracellular glucose sensing system is required to properly set cell size (P) and protein content required for S phase initiation (Ps). (A) Size of wild-type cells (thick line), isogenic gpa2Δ cells (dashed line), and gpr1Δ cells (thin line) exponentially growing 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) Immunoblot showing Cln3 protein level in wild-type and isogenic gpa2Δ and gpr1Δ cells grown in SCD and SCE (lanes labeled D and E, respectively). Extracts were loaded and bands quantified as detailed in Materials and methods. The values of average P, critical P at the beginning of DNA replication (Ps) determined by cytofluorimetric analysis (see Materials and methods for details), and duplication time (T) and length of budded phase (Tb) were determined for wild-type cells (black bars), gpa2Δ cells (white), and gpr1Δ cells (dashed) exponentially growing in SCE and SCD. (D) Cln3 protein levels determined as detailed above, were plotted as a function of Ps for wild-type cells (diamonds), gpa2Δ cells (circles), gpr1Δ cells (triangles), and wild-type cells overexpressing Cln3 (squares). Closed symbols indicate cells grown in SCD, open symbols indicate cells grown in SCE. Either average ± SD of data derived from experiments repeated at least three times (C and D, bar chart) or representative results from one of such experiments (A, B, and Western blot in C) are shown.

Mentions: Both carbon source and cAMP are known to modulate Ps (Broach, 1991; Baroni et al., 1992). Thus, it was of interest to investigate whether mutations in the glucose-sensing pathway that activates cAMP synthesis affect glucose modulation of cell size and Ps. An easy way to assess whether a cell has the ability to modulate cell size and Ps in response to carbon source is to measure the average P in glucose and ethanol media, for instance by analyzing protein distributions of exponentially growing populations (Alberghina and Porro, 1993). Mutants carrying a deletion in either the Gpr1 receptor or the cognate Gα encoding gene (gpr1Δ or gpa2Δ strain, respectively) were analyzed during exponential growth in synthetic complete (SC) medium supplemented with either 2% ethanol (SCE) or 2% glucose (SCD). During exponential growth on SCE, gpr1Δ and gpa2Δ strains do not differ significantly from isogenic wild-type cells in any of the measured cell cycle parameters. These include duplication time (T), the length of the budded phase (Tb), the average P, and the average Ps (Fig. 1, A and C). The latter is operationally defined as the average P of cells with a DNA content just above 1c, as determined by two-dimensional flow cytometry (Coccetti et al., 2004). Instead, gpr1Δ and gpa2Δ strains during exponential growth on SCD showed significantly altered protein distributions (Fig. 1 B), resulting in a reduction of both P and Ps (Fig. 1 C). The duplication time (T) and the length of the budded phase (Tb) were unaffected, consistent with the notion that signaling through the Gpr1–Gpa2 pathway specifically modulates Ps setting.


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)

A functional extracellular glucose sensing system is required to properly set cell size (P) and protein content required for S phase initiation (Ps). (A) Size of wild-type cells (thick line), isogenic gpa2Δ cells (dashed line), and gpr1Δ cells (thin line) exponentially growing 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) Immunoblot showing Cln3 protein level in wild-type and isogenic gpa2Δ and gpr1Δ cells grown in SCD and SCE (lanes labeled D and E, respectively). Extracts were loaded and bands quantified as detailed in Materials and methods. The values of average P, critical P at the beginning of DNA replication (Ps) determined by cytofluorimetric analysis (see Materials and methods for details), and duplication time (T) and length of budded phase (Tb) were determined for wild-type cells (black bars), gpa2Δ cells (white), and gpr1Δ cells (dashed) exponentially growing in SCE and SCD. (D) Cln3 protein levels determined as detailed above, were plotted as a function of Ps for wild-type cells (diamonds), gpa2Δ cells (circles), gpr1Δ cells (triangles), and wild-type cells overexpressing Cln3 (squares). Closed symbols indicate cells grown in SCD, open symbols indicate cells grown in SCE. Either average ± SD of data derived from experiments repeated at least three times (C and D, bar chart) or representative results from one of such experiments (A, B, and Western blot in C) are shown.
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Related In: Results  -  Collection

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fig1: A functional extracellular glucose sensing system is required to properly set cell size (P) and protein content required for S phase initiation (Ps). (A) Size of wild-type cells (thick line), isogenic gpa2Δ cells (dashed line), and gpr1Δ cells (thin line) exponentially growing 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) Immunoblot showing Cln3 protein level in wild-type and isogenic gpa2Δ and gpr1Δ cells grown in SCD and SCE (lanes labeled D and E, respectively). Extracts were loaded and bands quantified as detailed in Materials and methods. The values of average P, critical P at the beginning of DNA replication (Ps) determined by cytofluorimetric analysis (see Materials and methods for details), and duplication time (T) and length of budded phase (Tb) were determined for wild-type cells (black bars), gpa2Δ cells (white), and gpr1Δ cells (dashed) exponentially growing in SCE and SCD. (D) Cln3 protein levels determined as detailed above, were plotted as a function of Ps for wild-type cells (diamonds), gpa2Δ cells (circles), gpr1Δ cells (triangles), and wild-type cells overexpressing Cln3 (squares). Closed symbols indicate cells grown in SCD, open symbols indicate cells grown in SCE. Either average ± SD of data derived from experiments repeated at least three times (C and D, bar chart) or representative results from one of such experiments (A, B, and Western blot in C) are shown.
Mentions: Both carbon source and cAMP are known to modulate Ps (Broach, 1991; Baroni et al., 1992). Thus, it was of interest to investigate whether mutations in the glucose-sensing pathway that activates cAMP synthesis affect glucose modulation of cell size and Ps. An easy way to assess whether a cell has the ability to modulate cell size and Ps in response to carbon source is to measure the average P in glucose and ethanol media, for instance by analyzing protein distributions of exponentially growing populations (Alberghina and Porro, 1993). Mutants carrying a deletion in either the Gpr1 receptor or the cognate Gα encoding gene (gpr1Δ or gpa2Δ strain, respectively) were analyzed during exponential growth in synthetic complete (SC) medium supplemented with either 2% ethanol (SCE) or 2% glucose (SCD). During exponential growth on SCE, gpr1Δ and gpa2Δ strains do not differ significantly from isogenic wild-type cells in any of the measured cell cycle parameters. These include duplication time (T), the length of the budded phase (Tb), the average P, and the average Ps (Fig. 1, A and C). The latter is operationally defined as the average P of cells with a DNA content just above 1c, as determined by two-dimensional flow cytometry (Coccetti et al., 2004). Instead, gpr1Δ and gpa2Δ strains during exponential growth on SCD showed significantly altered protein distributions (Fig. 1 B), resulting in a reduction of both P and Ps (Fig. 1 C). The duplication time (T) and the length of the budded phase (Tb) were unaffected, consistent with the notion that signaling through the Gpr1–Gpa2 pathway specifically modulates Ps setting.

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