Limits...
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.

Show MeSH

Related in: MedlinePlus

Carbon source modulation of cell size is totally lost when both Far1- and Sic1-dependent thresholds are disrupted. (A) Size of wild-type cells and isogenic single or double mutants exponentially growing in medium supplemented with ethanol (SCE, open distributions) or glucose (SCD, gray distributions) by FACS analysis of total cell protein after FITC staining. (B) Mean values of P distributions of wild-type cells and deletion mutant cells in A exponentially growing in glucose and in ethanol were determined by FACS analysis (the complete set of mean values and SDs from three independent experiments is reported in Fig. S2); the ratio between mean value in glucose, P(D), and mean value in ethanol, P(E), is indicated as D/E ratio and is shown in the histogram for wild-type and each deletion mutant (black bars). In the same way and for the same cells, Ps were determined by FACS analysis (see Fig. S2 for values and Materials and methods for details). The corresponding D/E ratios for Ps are shown in the histogram as white bars. Shaded areas cover a 20% variation of D/E ratio around ordinate values of 1 (absence of carbon source–dependent modulation of size) and 2 (full wild-type modulation). (C) At time 0 2% glucose was added to cells exponentially growing in SCE medium and samples were collected to evaluate the percentage of budded cells. Wild type (closed circles), sic1Δ (open circles), and cln3Δsic1Δ (open triangles).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2172493&req=5

fig5: Carbon source modulation of cell size is totally lost when both Far1- and Sic1-dependent thresholds are disrupted. (A) Size of wild-type cells and isogenic single or double mutants exponentially growing in medium supplemented with ethanol (SCE, open distributions) or glucose (SCD, gray distributions) by FACS analysis of total cell protein after FITC staining. (B) Mean values of P distributions of wild-type cells and deletion mutant cells in A exponentially growing in glucose and in ethanol were determined by FACS analysis (the complete set of mean values and SDs from three independent experiments is reported in Fig. S2); the ratio between mean value in glucose, P(D), and mean value in ethanol, P(E), is indicated as D/E ratio and is shown in the histogram for wild-type and each deletion mutant (black bars). In the same way and for the same cells, Ps were determined by FACS analysis (see Fig. S2 for values and Materials and methods for details). The corresponding D/E ratios for Ps are shown in the histogram as white bars. Shaded areas cover a 20% variation of D/E ratio around ordinate values of 1 (absence of carbon source–dependent modulation of size) and 2 (full wild-type modulation). (C) At time 0 2% glucose was added to cells exponentially growing in SCE medium and samples were collected to evaluate the percentage of budded cells. Wild type (closed circles), sic1Δ (open circles), and cln3Δsic1Δ (open triangles).

Mentions: It has long been known that cln3Δ cells are larger than their wild-type isogenic counterpart (Nash et al., 1988; Futcher, 1996). Protein distributions from wild-type, cln3Δ, far1Δ, and cln3Δ far1Δ double mutants are reported in the top row of Fig. 5 A for cells exponentially growing in ethanol and glucose supplemented media. Average P and average Ps were computed from these distributions and are reported in Fig. S2 (A and B; available at http://www.jcb.org/cgi/content/full/jcb.200405102/DC1). Fig. 5 B shows the ratios of P and Ps measured in glucose over that measured in ethanol (black and white bars, respectively). Both P and Ps in wild-type cells growing in glucose are about twice as much as those found in cells growing in ethanol. We consider that carbon source modulation is present for values of the ratio glucose/ethanol above 1.6 (Fig. 5 B, top shaded area) and lost for values of the ratio around 1.0 (Fig. 5 B, bottom shaded area). cln3Δ, far1Δ and cln3Δ far1Δ cells only marginally loose their ability to modulate P and Ps in response to carbon source (Fig. 5, A and B), thereby indicating that the Cln3/Far1-dependent cell sizer mechanism is not the only determinant in modulation of DNA synthesis initiation by carbon source.


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)

Carbon source modulation of cell size is totally lost when both Far1- and Sic1-dependent thresholds are disrupted. (A) Size of wild-type cells and isogenic single or double mutants exponentially growing in medium supplemented with ethanol (SCE, open distributions) or glucose (SCD, gray distributions) by FACS analysis of total cell protein after FITC staining. (B) Mean values of P distributions of wild-type cells and deletion mutant cells in A exponentially growing in glucose and in ethanol were determined by FACS analysis (the complete set of mean values and SDs from three independent experiments is reported in Fig. S2); the ratio between mean value in glucose, P(D), and mean value in ethanol, P(E), is indicated as D/E ratio and is shown in the histogram for wild-type and each deletion mutant (black bars). In the same way and for the same cells, Ps were determined by FACS analysis (see Fig. S2 for values and Materials and methods for details). The corresponding D/E ratios for Ps are shown in the histogram as white bars. Shaded areas cover a 20% variation of D/E ratio around ordinate values of 1 (absence of carbon source–dependent modulation of size) and 2 (full wild-type modulation). (C) At time 0 2% glucose was added to cells exponentially growing in SCE medium and samples were collected to evaluate the percentage of budded cells. Wild type (closed circles), sic1Δ (open circles), and cln3Δsic1Δ (open triangles).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172493&req=5

fig5: Carbon source modulation of cell size is totally lost when both Far1- and Sic1-dependent thresholds are disrupted. (A) Size of wild-type cells and isogenic single or double mutants exponentially growing in medium supplemented with ethanol (SCE, open distributions) or glucose (SCD, gray distributions) by FACS analysis of total cell protein after FITC staining. (B) Mean values of P distributions of wild-type cells and deletion mutant cells in A exponentially growing in glucose and in ethanol were determined by FACS analysis (the complete set of mean values and SDs from three independent experiments is reported in Fig. S2); the ratio between mean value in glucose, P(D), and mean value in ethanol, P(E), is indicated as D/E ratio and is shown in the histogram for wild-type and each deletion mutant (black bars). In the same way and for the same cells, Ps were determined by FACS analysis (see Fig. S2 for values and Materials and methods for details). The corresponding D/E ratios for Ps are shown in the histogram as white bars. Shaded areas cover a 20% variation of D/E ratio around ordinate values of 1 (absence of carbon source–dependent modulation of size) and 2 (full wild-type modulation). (C) At time 0 2% glucose was added to cells exponentially growing in SCE medium and samples were collected to evaluate the percentage of budded cells. Wild type (closed circles), sic1Δ (open circles), and cln3Δsic1Δ (open triangles).
Mentions: It has long been known that cln3Δ cells are larger than their wild-type isogenic counterpart (Nash et al., 1988; Futcher, 1996). Protein distributions from wild-type, cln3Δ, far1Δ, and cln3Δ far1Δ double mutants are reported in the top row of Fig. 5 A for cells exponentially growing in ethanol and glucose supplemented media. Average P and average Ps were computed from these distributions and are reported in Fig. S2 (A and B; available at http://www.jcb.org/cgi/content/full/jcb.200405102/DC1). Fig. 5 B shows the ratios of P and Ps measured in glucose over that measured in ethanol (black and white bars, respectively). Both P and Ps in wild-type cells growing in glucose are about twice as much as those found in cells growing in ethanol. We consider that carbon source modulation is present for values of the ratio glucose/ethanol above 1.6 (Fig. 5 B, top shaded area) and lost for values of the ratio around 1.0 (Fig. 5 B, bottom shaded area). cln3Δ, far1Δ and cln3Δ far1Δ cells only marginally loose their ability to modulate P and Ps in response to carbon source (Fig. 5, A and B), thereby indicating that the Cln3/Far1-dependent cell sizer mechanism is not the only determinant in modulation of DNA synthesis initiation by carbon source.

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