The transcription factor Swi4 is target for PKA regulation of cell size at the G₁ to S transition in Saccharomyces cerevisiae

Figures & data

Figure 1. Effect of cAMP on growth and cell size in cyr1Δ msn2Δ msn4Δ pde2Δ mutant. Asynchronous culture of the GG104 strain growing exponentially (1.7–2 × 10⁶ cells/ml) in YPD medium was splitted into 2 fractions and 2mM cAMP was added to one of them (▪), while the other was the untreated control (♦). The black arrows indicate the time of cAMP addition. Samples were harvested every 30 minutes to measure the cell number (panel A), the absorbance at 600 nm (panel B), the percentage of budded cells (panel C) and the mean cell size (panel D).

Figure 2. Protein and DNA distribution of GG104 cells. Histograms of protein and DNA distributions of control cultures and of cultures treated with 2 mM cAMP collected at the indicated time after cAMP addition. Yeast cells were collected fixed with 70% ethanol, stored at 4°C, and subsequently processed for flow cytometry, using a Becton Dickinson FACStarPlus. Fluorescein isothiocianate (FITC) for protein staining and Propidium Iodide (PI) for DNA staining were used. Approx 30.000 events were analyzed for each sample. Plot generation and analysis were performed with WinMDI2.9 software.

Table 1 Growth and Cell cycle parameters for GG104 strain growing without cAMP and 5 hours after addition of cAMP.

	−cAMP	+cAMP
K(h-1)	0.35	0.39^*
%G1	32	17
%B	60	76
TB(min)	80	95
P(channel)	251	472
Ps	216	363
Pm	346	639

* The exponential growth rate constant ( k, h⁻¹) was calculated from the increase of OD at 600 nm between 1 and 7 hrs after cAMP addition. The % of G₁ cells was calculated from histograms of DNA distributions acquired by flow-cytometry after staining with Propidium iodide, using a software based on the method of Slater.⁴⁹ T_B, the length of budded phase was calculated using the formula T_B = DT × log(F_B+1) / log2.⁹ P (channel number) is the average of the protein distributions acquired by flow cytometry after staining with Fluoresceine isothiocyanate.⁹ Ps, the relative protein content (channel number) at budding, was estimated knowing the relevant cell cycle parameters of the population using a model for yeast population and a software previously developed in our laboratory, ^9,32 while the value of Pm (protein content at cell division) was calculated with the formula: Pm = Ps ( 1+ F_B).⁵⁰

Figure 3. Evaluation of cells size at budding (V_s) and at cell division (V_m). GG104 cells exponentially growing were treated with 2 mM cAMP, stained with DAPI and photographed with a fluorescence microscope. Panel A: sample image of the cells growing without cAMP and after 3.5 hr of cAMP treatment. The image is taken in light transmission merged with DAPI fluorescence. Panel B: Cell size at budding (V_s) and at cell division (V_m) measured on the photographs taken at different times after cAMP addition. The average size of at least 30 cells with small buds (V_s) and of at least 30 binucleate cells (V_m) is reported. Error bars indicate Standard Deviation. *p < 0.05, **p < 0.01 is related to a comparison with the preceding mean value.

Figure 4. CLN1 but not CLN2 is required for G1 delay induced by cAMP addition. Asynchronous cultures of the GG104 cln1Δ (A) and GG104 cln2Δ (B) strains growing exponentially (1.7–2 × 10⁶ cells/ml) in YPD medium were splitted into 2 fractions and 2mM cAMP was added to one of them (▪), while the other was the untreated control (♦). Samples were harvested every 30 minutes to assay cell number, cell size and budding index.

Figure 5. Cell size at budding and at cell division for cln1Δ strain. GG104 cln1Δ cells exponentially growing were treated with 2 mM cAMP, stained with DAPI and photographed with a fluorescence microscope. Cell size at budding (V_s) and at cell division (V_m) were measured on the images taken at different times after cAMP addition. The average size of at least 30 cells with small buds (V_s) and of at least 30 binucleate cells (V_m) is reported. Error bars indicate Standard Deviation. * p < 0.05, ** p < 0.01 is related to a comparison with the preceding mean value.

Figure 6. cAMP addition caused a CLN1 repression. Quantitative RealTime-PCR assays on CLN1 (hatched bars) and CLN2 (white bars) mRNA prepared from samples obtained during kinetic growth analysis, collected at the indicated time after cAMP addition. Graphic represents the mean values of 3 independent experiments, error bars indicate standard deviations.

Figure 7. GG104 swi4Δ mutant does not show G1/S transient arrest after PKA activation. Asynchronous cultures of the GG104 swi4Δ growing in YPD medium, GG104 swi4Δ [YCplac111-SWI4^S159A], GG104 swi4Δ [YCplac111-SWI4] strains growing exponentially in selective medium were splitted into 2 fractions and 2mM cAMP was added to one of them (▪), while the other was the untreated control (Δ). Samples were harvested every 30 minutes to assay cell number, cell size and budding index.

Table 2 Yeast strains used in this study

	Genotype
StrainsGG104	MATa W303 with pde2::TRP1 cdc35::KanMX2 msn2::HIS3 msn4::TRP1	SourceRoosen et al.^31
GG104 cln1Δ	GG104 cln1::URA3	This study
GG104 cln2Δ	GG104 cln2::URA3	This study
GG104 cln3Δ	GG104 cln3::URA3	This study
GG104 whi5Δ	GG104 whi5::URA3	This study
GG104 swi4Δ	GG104 swi4::KlURA3	This study
GG104 Swi4–9myc	GG104 SWI4–9myc::Hph	This study
GG104 Swi4WT	GG104 swi4Δ [YCplac111-SWI4^WT]	This study
GG104 Swi4S159A	GG104 swi4Δ [YCplac111-SWI4^S159A]	This study

Mariani L, Martegani E, Alberghina L. Yeast population models for monitoring and control of biotechnical processes. IEE Proc. 1986; 133:PT-D, 210-216; http://dx.doi.org/10.1049/ip-d.1986.0035

 Web of Science ®Google Scholar

Roosen J, Engelen K, Marchal K, Mathys J, Griffioen G, Cameroni E, Thevelein JM, De Virgilio C, De Moor B, Winderickx J. PKA and Sch9 control a molecular switch important for the proper adaptation to nutrient availability. Mol Microbiol 2005; 55:862-80; PMID:15661010

 PubMed Web of Science ®Google Scholar