Research ArticleCELL BIOLOGY

Budding yeast relies on G1 cyclin specificity to couple cell cycle progression with morphogenetic development

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Science Advances  04 Jun 2021:
Vol. 7, no. 23, eabg0007
DOI: 10.1126/sciadv.abg0007
  • Fig. 1 S phase delay in the Clns-Clb2S-M strain.

    (A) Schematic of cyclin waves in wild-type budding yeast and in the Clns-Clb2S-M strain. (B) Cell cycle progression of control and Clns-Clb2S-M cells. α-Factor–synchronized cells were followed through one cell cycle before rearrest in the following G1. Flow cytometry analysis of DNA content as well as Western blot analysis of Clb5 and Clb2 levels is shown. Clb2 expressed from the CLB5 promoter was fused to a 6×HA epitope tag, causing its slower migration. Tubulin served as a loading control. The fraction of budded cells over time is shown, as well as the fraction of cells with 2C DNA content. (C) Cdk-associated kinase activity against histone H1 was measured following Cdc28 immunoprecipitation by virtue of a Pk epitope tag. A representative autoradiogram and Western blot are shown. The results from three independent experiments are shown; the medians are connected by a line.

  • Fig. 2 Compromised S phase target phosphorylation in Clns-Clb2S-M cells.

    (A) Median normalized intensity profiles of Cdk targets, grouped by their phosphorylation timing in control cells. Phosphosite number in each category is given in parenthesis. The same groups are shown in the Clns-Clb2S-M strain. Phosphorylation midpoints of the 40 to 70 min categories are indicated by dashed arrows. A complete list of phosphosites can be found in data file S1. (B) Median normalized intensity profiles of phosphosites from biochemically identified Clb5-Cdk targets (9), as well as sites with similar control phosphorylation timing in targets lacking Clb5 specificity (9). Dashed arrows point to phosphorylation midpoints. (C and D) Normalized intensity profiles of Cdk sites in Sld2, Orc2, and Orc6. Line colors are chosen to approximate those of the time categories in (A). Western blot analyses of Orc2 and Orc6 phosphorylation during synchronous cell cycle progression of wild-type and Clns-Clb2S-M cells are also shown.

  • Fig. 3 Cyclin specificity shapes the Cdk phosphorylation landscape.

    (A) Contribution of Clb3-specific substrate interactions. Kar9 phosphorylation was assessed during synchronous cell cycle progression in control and Clns-Clb2S-M cells by Western blotting. Normalized intensity profiles from the phosphoproteome data of the indicated sites are also shown. (B) as in (A) but the effect of early Clb2 expression on Ndd1 phosphorylation was evaluated. The dashed arrows point to the phosphorylation midpoints.

  • Fig. 4 Signs of a DNA damage response in Clns-Clb2S-M cells.

    (A) Delayed securin degradation following its phosphorylation on Chk1 kinase sites. Securin levels in synchronized cultures of control and Clns-Clb2S-M cells were analyzed by Western blotting. Normalized intensity profiles of three Chk1 kinase target sites contained in the phosphoproteome data are shown. (B) Delayed Cdc14 release in Clns-Clb2S-M cells. Cdc14 fused to a Pk epitope tag was visualized by indirect immunofluorescence during a time course experiment as in (A). One hundred cells were scored for loss of discernible nucleolar Cdc14 accumulation at each time point, as well as for the presence of short metaphase or long anaphase spindles. (C) Compromised Net1 phosphorylation. The Net1 phosphorylation status was analyzed during synchronous cell cycle progression of control and Clns-Clb2S-M cells by Western blotting. Normalized intensity profiles of four Cdk phosphorylation sites that regulate Cdc14 release are shown. (D) Reduced fitness of Clns-Clb2S-M cells. Tenfold serial dilutions of cells with the indicated genotypes were spotted on YPD plates, with or without added hydroxyurea (HU), and grown for 3 days at the indicated temperatures.

  • Fig. 5 Cell cycle progression with a single cyclin.

    (A) Schematic of cyclin waves in the MET3prCln2-Clb2G1-S-M strain with active or repressed MET3 promoter, as well as in the repressed MET3prCln2-Clb2S-M strain. (B) Cell cycle characteristics with a single cyclin. α-Factor synchronized cells of the indicated genotypes were released into medium lacking (Cln2 ON) or containing methionine (Cln2 OFF). Flow cytometry analysis of DNA content is shown together with Western blots of cyclin expression and cell cycle markers. Clb2 expressed from the CLN2 promoter was fused to a 3×HA epitope tag, leading to migration between CLB5 promoter expressed 6×HA epitope–tagged Clb2 and endogenous untagged Clb2. Tubulin served as a loading control. (C) Mitosis inside single-cell bodies in the single-cyclin strain. Fields of MET3prCln2-Clb2G1-S-M and MET3prCln2-Clb2S-M cells with Cln2 OFF from the 300 min time point, stained for the mitotic spindle and DNA. One hundred cells at each time point were scored for elongated (E) or segregated (S) nuclei.

  • Fig. 6 Dissecting the essential nature of G1 cyclins.

    (A) Transcriptional inhibitors delay G1 progression. Cells of the indicated genotypes were synchronized by α-factor treatment and released. Flow cytometry analysis of DNA content was used to monitor G1 progression and entry into S phase. (B) Analysis of G1 cyclin features. Fourfold serial dilutions of cells with indicated genotypes were spotted onto CSM without methionine (Cln2 ON) or YPD (Cln2 OFF) plates and grown for 3 days at 25°C. Schematics represent the cyclin variants expressed under control of the CLN2 promoter. Cln2 (blue) and Clb2 (red) are divided into their N-terminal, cyclin core, and C-terminal parts. Two Cln2-specific loop insertions are highlighted by arrowheads. Locations of engineered gene alterations are highlighted in dark gray.

  • Fig. 7 Cln2 is required for cell polarization and budding pathway target phosphorylation.

    (A) Cln2 promotes budding pathway target phosphorylation. Electrophoretic mobility shifts, indicative of phosphorylation, of the denoted proteins were assessed during synchronous cell cycle progression of MET3prCln2-Clb2G1-S-M cells in CSM medium lacking (Cln2 ON) or containing (Cln2 OFF) methionine. Tubulin served as a loading control. (B) Cln2 is required for cell polarization. Samples from (A) were processed for rhodamine-phalloidin staining at the indicated time points. Fluorescence is shown next to differential interference contrast (DIC) images.

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