Fig 1.
CEN number effects on cell size.
(A) Scheme showing the different approaches used to assess and manipulate CEN number. (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, N = 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). (C) 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. (D) Cells carrying YCp–CENGALp (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 CENGALp CEN. (E) Cells carrying a YAC–CENGALp artificial chromosome were grown at restrictive conditions for the conditional CENGALp 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, N = 50) and a regression line are plotted. The mean budding size for wild-type diploid cells is also plotted (black diamond). (F) Newborn daughter cells carrying additional conditional CENGALp CENs in chromosomes 4 and 7 were grown under permissive conditions until they entered the cell cycle. Individual budding volumes (N = 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 S1 Data. CEN, centromere; GFP, green fluorescent protein; YCp, yeast centromeric plasmid; YEp, yeast episomal plasmid.
Fig 2.
Exceeding CENs modulate cell size in a Cln3-dependent manner.
(A) Cells with the indicated genotypes carrying three YCp vectors (3YCp) or none (ctrl) were analyzed to determine cell size at budding. Individual data (N > 400), and median values are plotted. (B) 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. CEN, centromere; YCp, yeast centromeric plasmid.
Fig 3.
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.
Fig 4.
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.
Fig 5.
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.