Panel A shows a pathway diagram illustrating Ccr4, Bck2, Cln3, Whi5, SBF, and cell-cycle regulation. Panel B shows a box plot comparing Whi5 messenger RNA concentration across wild type, bck2 delta, and ccr4 delta. Panel C shows a box plot comparing Whi5 messenger RNA counts across wild type, whi5 delta, bck2 delta, and ccr4 delta. Panel D shows microscopy images with bright field, DAPI, Quasar-570, and analysis views of cell-cycle stages. Panel E shows microscopy images of wild type, whi5 delta, bck2 delta, ccr4 delta and double mutant cells with outlined boundaries. Panel F shows box plots comparing cell volume under SCD and SCGE conditions across strains. Panel G shows bar graphs comparing coefficient of variation of cell volume under SCD and SCGE conditions across strains.
Bck2 acts independently of Whi5. whi5Δbck2Δ has WT size and more efficient size homeostasis than whi5Δ and bck2Δ in glucose medium. (A) Schematic illustration of key regulators of cell cycle progression and cell size at the budding yeast G1/S transition. (B) mRNA concentration of WHI5 relative to ACT1 in cells grown in SCD, as determined from five independent RT-qPCR experiments. (C)WHI5 mRNA count per cell from S-phase cells pooled from two independent smFISH experiments performed in SCD (nWT = 172, nwhi5Δ = 182, nbck2Δ = 211, nccr4Δ = 83). (D) Representative WT smFISH images: phase contrast; nuclear DNA stained with DAPI; WHI5 mRNA stained with Quasar-570–labeled smFISH probes; the spots were detected using a custom routine in Python in yellow and cell contours in red. Dashed yellow lines connect mother–bud pairs. Cell cycle stage is annotated in black. Scale bars represent 5 µm. (E) Phase-contrast images from steady-state live-cell microscopy in SCD. Representative daughter cells (first-generation cells) from different strains are shown just before division and highlighted with red contours. Scale bars represent 5 µm. (F) Cell volume distributions obtained from steady-state live-cell microscopy in SCD and SCGE. For each strain, all living cells present in the last frame of each imaging position of two independent experiments were pooled. Cell numbers (n) for SCD: nWT = 382, nwhi5Δ = 370, nbck2Δ = 376, nwhi5Δbck2Δ = 463, nccr4Δ = 241, nwhi5Δccr4Δ = 280. Cell numbers for SCGE: nWT = 533, nwhi5Δ = 370, nbck2Δ = 429, nwhi5Δbck2Δ = 409. ccr4Δ and whi5Δccr4Δ cells growing in SCGE were excluded from analysis due to poor growth and high death rate. (G) Same dataset was used for calculating the CV of cell volume, as a measure of size homeostasis efficiency. •, ■, and x symbols are used to show the CVs of cell volume calculated from independent experimental replicates. For G, statistical analyses were performed by comparing overlaps between 10,000 bootstrap samples, explained in detail in the Materials and methods. Statistical comparisons were performed using two-sided Mann–Whitney U tests. Each strain was compared with WT and, where applicable, with whi5Δbck2Δ. For F and G, comparisons were made across both medium conditions separately (P < 0.001: not indicated for clarity; **: P < 0.01; *: P < 0.05; ns: not significant, P ≥ 0.05). For boxplots in B, C, and F, box limits show the IQR, whiskers extend to the most extreme data points within 1.5× IQR, the median is shown as a line, and the mean is shown as a cross. WT, wild type; IQR, interquartile range.