Degradation of cyclin Cln3 by exceeding CENs: Mad3 physical and functional interactions with SCF.

(A) Analysis of Cln3 stability by promoter shut-off experiments in the presence (orange circles) or absence (gray circles) of two YCp–CENGALp vectors in wild-type cells grown under permissive conditions. After tetracycline addition, cells were collected at the indicated times, and obtained Cln3–6FLAG levels are plotted relative to an unspecific cross-reacting band (asterisk) used as loading control. (B) Analysis of Cln3 stability in Mad3-deficient cells as in (A). (C) Analysis of mCitrine–Cln3–11A stability by time-lapse microscopy in the presence (orange circles) or absence (gray circles) of three YCp vectors. Nuclear levels of mCitrine–Cln3–11A in cells were determined at the indicated times after cycloheximide addition, and mean values (N = 100) are plotted. (D) Analysis of mCitrine–Cln3–11A accumulation in the nucleus in the presence (orange circles) or absence (gray circles) of three YCp vectors. Nuclear levels of mCitrine–Cln3–11A were determined in G1 daughter cells with 50–60 μm3 of volume. Individual data (N = 90) and median values are plotted. (E) Cell extracts (input) and GST PDs of cdc4ts grr1 cells expressing Cln3–13myc and GST fusions to Cdc4ΔFbox, Mad3, or Mad3ΔGLEBS were analyzed by immunoblotting with either αmyc (top panels) or αGST (bottom panel) antibodies. (F) Cell extracts (input) and GST PDs of cells expressing Mad3–3HA or Mad3 ΔGLEBS–3HA and either GST or GST–Cdc4 were analyzed by immunoblotting with either αHA (top panels) or αGST (bottom panel) antibodies. (G) Cells with the indicated genotypes carrying three YCp vectors were analyzed as in Fig 1B to determine cell size at budding as a function of copy number. Individual budding volumes (small dots) were binned, and mean values (large circles, N = 50) and a regression line are plotted. Correlation analysis and pairwise comparisons were performed with nonparametric tests as described in Materials and methods. Underlying data can be found in S1 Data. GST, glutathione S-transferase; PD, pulldown; YCp, yeast centromeric plasmid.</p

Exceeding CENs require centromeric Mad3/Bub3 signaling proteins to modulate cell size.

(A) Cells with the indicated genotypes carrying three YCp vectors were analyzed as in Fig 1B to determine cell size at budding as a function of copy number. Individual budding volumes (small dots) were binned, and mean values (large circles, N = 50) and a regression line are plotted. (B) Newborn daughter cells with the indicated genotypes were analyzed to determine cell size at budding. Mad3 overexpression (oMAD3) was attained by inducing a GAL1p–MAD3 construct with 1 mM estradiol in newborn cells expressing the Gal4–hER–VP16 transactivator. Individual data (N > 300) and median values are plotted. Correlation analysis and pairwise comparisons were performed with nonparametric tests as described in Materials and methods. Underlying data can be found in S1 Data. CEN, centromere; YCp, yeast centromeric plasmid.</p

Centromeric signaling proteins boost G1 cyclin degradation and modulate cell size in budding yeast

<div><p>Cell size scales with ploidy in a great range of eukaryotes, but the underlying mechanisms remain unknown. Using various orthogonal single-cell approaches, we show that cell size increases linearly with centromere (CEN) copy number in budding yeast. This effect is due to a G1 delay mediated by increased degradation of Cln3, the most upstream G1 cyclin acting at Start, and specific centromeric signaling proteins, namely Mad3 and Bub3. Mad3 binds both Cln3 and Cdc4, the adaptor component of the Skp1/Cul1/F-box (SCF) complex that targets Cln3 for degradation, these interactions being essential for the CEN-dosage dependent effects on cell size. Our results reveal a pathway that modulates cell size as a function of CEN number, and we speculate that, in cooperation with other CEN-independent mechanisms, it could assist the cell to attain efficient mass/ploidy ratios.</p></div

CEN signaling proteins cooperate with SCF–Cdc4 to enhance degradation of the yeast G1 cyclin and modulate cell size in budding yeast.

When present in excess, centromeres accelerate Cln3 degradation in the nucleus with the essential participation of Mad3, a centromeric signaling protein that interacts with Cdc4 and requires SCF function to modulate cell size as a function of centromere number. CEN, centromere.</p

Exceeding CENs modulate cell size in a Cln3-dependent manner.

<p>(<b>A</b>) Cells with the indicated genotypes carrying three YCp vectors (3YCp) or none (ctrl) were analyzed to determine cell size at budding. Individual data (<i>N</i> > 400), and median values are plotted. (<b>B</b>) Cells with the indicated genotypes carrying three YCp vectors were analyzed as in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2005388#pbio.2005388.g001" target="_blank">Fig 1B</a> to determine cell size at budding as a function of copy number. Individual budding volumes (small dots) were binned, and mean values (large circles, <i>N</i> = 50) and a regression line are plotted. Correlation analysis and pairwise comparisons were performed with nonparametric tests as described in Materials and methods. Underlying data can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2005388#pbio.2005388.s008" target="_blank">S1 Data</a>. CEN, centromere; YCp, yeast centromeric plasmid.</p

CEN number effects on cell size.

<p>(<b>A</b>) Scheme showing the different approaches used to assess and manipulate CEN number. (<b>B</b>) Yeast cells endogenously expressing mCherry were transformed with one (small purple dots) or three (small blue dots) GFP-expressing YCp vectors, and cell size at budding was determined as a function of vector copy number (GFP/mCherry ratio). Individual budding volumes were binned, and mean values (large orange circles, <i>N</i> = 50) and a regression line are plotted. The mean budding size for wild-type diploid cells (which have 16 additional CENs compared to haploid cells) is also plotted (black diamond). (<b>C</b>) Cells carrying YEp (green circles) or YCp (orange circles) vectors were analyzed as in (B) to determine cell size at budding as a function of copy number. (<b>D</b>) Cells carrying YCp–CEN^<i>GALp</i> (red circles) or YCp (orange circles) vectors were analyzed as in (B) to determine cell size at budding as a function of copy number under permissive conditions for the additional conditional CEN^<i>GALp</i> CEN. (<b>E</b>) Cells carrying a YAC–CEN^<i>GALp</i> artificial chromosome were grown at restrictive conditions for the conditional CEN^<i>GALp</i> CEN to obtain a wide range of copies per cell, returned to permissive conditions, and analyzed as in (B) to determine cell size at budding as a function of copy number. Individual budding volumes (small blue dots) were binned, and mean values (large orange circles, <i>N</i> = 50) and a regression line are plotted. The mean budding size for wild-type diploid cells is also plotted (black diamond). (<b>F</b>) Newborn daughter cells carrying additional conditional CEN^<i>GALp</i> CENs in chromosomes 4 and 7 were grown under permissive conditions until they entered the cell cycle. Individual budding volumes (<i>N</i> = 100) and median values are plotted. Correlation analysis and pairwise comparisons were performed with nonparametric tests as described in Materials and methods. Underlying data can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2005388#pbio.2005388.s008" target="_blank">S1 Data</a>. CEN, centromere; GFP, green fluorescent protein; YCp, yeast centromeric plasmid; YEp, yeast episomal plasmid.</p

Yeast strains.

Yeast strains.</p

Cyclin waves in <i>S</i>. <i>cerevisiae</i> growing in normal lab conditions (YPD at 30°C).

G1 cyclin waves as determined in this research. From top to bottom, the panels show the Cdc28 G1 cyclins, the Pho85 G1 cyclins and the different molecular markers (Clb5 and Sic1) and morphological markers (budding percentage and size for elutriation). START, determined as the moment in which Sic1 and Clb5 amounts were identical, is extrapolated as a dashed line to all the panels. A) Cells were synchronized by centrifugal elutriation (left panels) or α-factor (right panels). Time 0 corresponds to the moment of cell removal from the elutriation device or α-factor removal, after which cells were incubated under agitation at 30°C. Note that the time elapsed before the cells resumed the cell cycle was greater after elutriation than after α-factor treatment. B) Since the amount of Cln3-3HA is very low, it is plotted both with the other Clns and individually so as to clearly represent levels. In the case of α-factor synchronization, at least three independent western blot experiments as in Fig 2B were quantified, standardized using loading control and relativized to the maximum expression amounts (Cln2). Values are expresed as mean±SEM.</p

Cell cycle progression and thermosensitivity of <i>pcl2</i>Δ cells.

A) FACS analysis of wild-type and pcl2Δ cells from the BY4741 background. Cells were grown exponentially at 30°C in YPD, synchronized in G1 with α-factor and released in fresh YPD medium at 30°C or 37°C. B) Quantification of cells with 2n DNA content from A). C) Spot assay. W303 background cells were grown in YPD or a malt-based medium to the exponential phase and diluted to an optical density of 0.05 (wavelength 660 nm). Spotted on plates were 5 μl of tenfold sequential dilution for incubation at the indicated temperature. D) Nomarski images of the strains after 5 h at 37°C. Arrows indicate cells showing a ‘mickey mouse’ phenotype and the bar represents 10 μm. The ‘mickey mouse’ cells were quantified (mean±SEM) for three different experiments. E) Relative cell volume (mean±SEM), based on measurement of some 30 cells from each of the three independent experiments.</p

Experimental setup for determining G₁ cyclin amounts.

A) Workflow and pooling scheme. Strains were grown overnight in YPD at 30°C in a water shaker, diluted in fresh medium, mixed in three different sets according to the molecular weight of the tagged proteins, grown in the exponential phase, synchronized, released in fresh medium and subjected to the designed stress or treatment (see Materials and Methods). B) Two representative and independent western blot images (to show reproducibility) used to quantify G1 cyclin amounts. Cells were synchronized using α-factor and samples were obtained as described in A), separated by SDS-PAGE and blotted and developed (see Materials and Methods). The different cyclins are indicated by numbers in the upper image. C) Same procedure as in B), except that cells were synchronized by elutriation, with time 0 corresponding to the moment the cells were retrieved from the elutriation device, after which the cells were incubated under agitation at 30°C.</p