Cell Size and Growth Rate Are Modulated by TORC2-Dependent Signals Rafael Lucena, Maria Alcaide-Gavilán, Katherine Schubert, Maybo He, Matthew G. Domnauer, Catherine Marquer, Christian Klose, Michal A. Surma, Douglas R. Kellogg  Current Biology  Volume 28, Issue 2, Pages 196-210.e4 (January 2018) DOI: 10.1016/j.cub.2017.11.069 Copyright © 2017 Elsevier Ltd Terms and Conditions

Current Biology 2018 28, 196-210.e4DOI: (10.1016/j.cub.2017.11.069) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 1 PP2ARts1 Controls the TORC2 Signaling Network via the PI(4)P Kinase Mss4 (A) A summary of signaling events in the TORC2 network. (B) Wild-type and rts1Δ cells were grown to early log phase at 22°C in YPD medium. Mss4-3XHA was detected by western blot. (C) rts1Δ and wild-type control cells containing Mss4-GFP were grown at 22°C in the same culture in complete synthetic medium containing dextrose. Wild-type controls cells were marked with Spc42-mRuby2 so they could be distinguished from rts1Δ cells. Cells were imaged by fluorescence microscopy in a field of view that includes both strains. Signal intensity was quantified along a line that bisected the plasma membrane. (D) The Mss4-GFP signal at the plasma membrane was quantified in wild-type (21 cells) and rts1Δ (22 cells). ∗∗∗p < 0.0001 by Student’s t test. (E) Bar plots showing levels of PI(4,5)P2 in wild-type and rts1Δ cells grown to early log phase at 22°C in YPD medium. Measurements were expressed as a mole percentage of total anionic phospholipids normalized to wild-type levels. Error bars represent the standard deviation of the mean of two biological replicates. ∗p = 0.025 by Student’s t test. (F) Wild-type and rts1Δ cells were grown to early log phase at 22°C in YPD medium. Western blotting with phosphospecific antibodies was used to detect a TORC2-dependent phosphorylation site (Ypk-pT662) and a Pkh1/2-dependent site (Ypk-pT504) on Ypk1/2. Total Ypk1 was detected using an anti-Ypk1 antibody. (G) Cells of the indicated genotypes were grown overnight to early log phase at 22°C in YPD medium. The cultures were then shifted to 30°C for 2 hr. Western blotting with phosphospecific antibodies was used to detect a TORC2-dependent phosphorylation site on Ypk1/2 (Ypk-pT662) and a Pkh1/2-dependent site (Ypk-pT504). Total Ypk1 was detected using an anti-Ypk1 antibody. The asterisk indicates a non-specific band. See also Figure S1 and Table S1. Current Biology 2018 28, 196-210.e4DOI: (10.1016/j.cub.2017.11.069) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 2 The TORC2 Network Is Modulated by Nutrients (A) Wild-type and rts1Δ cells were grown to early log phase at 22°C in YPD medium. Cells were washed into YPG/E medium, and the behavior of Mss4-3XHA was assayed by western blot at the indicated times. (B) Wild-type and rts1Δ cells were shifted from rich to poor carbon as in (A). Ypk-pT662, Ypk-pT504, and Ypk1 were assayed by western blot. (C) Wild-type and rts1Δ cells were grown at 22°C to early log phase in YEP media containing glucose (Glu), galactose (Gal), or glycerol/ethanol (GE). Ypk-pT662, Ypk-pT504, and Ypk1 were assayed by western blot. An asterisk indicates a non-specific band. See also Figure S2 and Table S1. Current Biology 2018 28, 196-210.e4DOI: (10.1016/j.cub.2017.11.069) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 3 Ypk1/2 Signaling Strongly Influences Cell Size Cells of the indicated genotypes were grown to log phase at 22°C and cell-size distributions were determined using a Coulter counter. Each plot is the average of 3 biological replicates. For each biological replicate, 3 technical replicates were analyzed and averaged. (A) Cells were grown in YPD medium. (B) Cells were grown in YPD medium for 16 hr in the presence of 0.1 μM 3-MOB-PP1 or DMSO control. (C) Cells were grown in YPD medium. (D) Cells were grown in YPGal medium. See also Figure S3 and Table S1. Current Biology 2018 28, 196-210.e4DOI: (10.1016/j.cub.2017.11.069) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 4 Ceramide Is Required for Normal Control of Cell Size (A) Summary of sphingolipid synthesis pathways. Small-molecule inhibitors are indicated in red. DHS, dihydrosphingosine; PHS, phytosphingosine; DHS-P/PHS-P, dihydrosphingosine/phytosphingosine-1-phosphate. (B and C) Wild-type (B) or rts1Δ (C) cells were grown in YPD media at 22°C for 16 hr to early log phase in the presence of varying concentrations of myriocin. Cell-size distributions were determined using a Coulter counter. (D–G) Cells were grown to early log phase in YPG/E medium, and small unbudded cells were isolated by centrifugal elutriation. Cells were released into YPD medium in the presence of varying concentrations of myriocin at 25°C, and samples were taken at 10-min intervals. (D) Mean cell volume was measured with a Coulter counter and plotted as a function of time. (E) The percentage of budded cells was plotted as a function of time. (F) A plot of growth rate versus myriocin concentration. (G) A plot of cell size at cell-cycle entry versus myriocin concentration. See also Figure S4 and Table S1. Current Biology 2018 28, 196-210.e4DOI: (10.1016/j.cub.2017.11.069) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 5 Ceramides Are Required for Nutrient Modulation of Growth Rate and Cell Size (A) Wild-type and lac1Δ lag1Δ cells were grown to log phase in YPD medium at 22°C. Cell-size distributions were determined using a Coulter counter. (B) lac1Δ lag1Δ cells were grown to log phase in YPD (Rich) or YPG/E (Poor) medium for 16 hr at 22°C. Cell-size distributions were determined using a Coulter counter. (C–H) Wild-type or lac1Δ lag1Δ cells were grown to log phase in YPG/E medium. Small unbudded cells in G1 were isolated by centrifugal elutriation and released into either YPD medium (Rich) or YPG/E medium (Poor) at 25°C. (C and E) Mean cell volume was analyzed in wild-type (C) or lac1Δ lag1Δ cells (E) using a Coulter counter and plotted as a function of time. (D and F) The percentage of budded cells in wild-type (D) or lac1Δ lag1Δ cells (F) was plotted as a function of time. (G) A plot showing the effects of carbon source on growth rate. (H) A plot showing the effects of carbon source on cell volume at cell-cycle entry. See also Figure S5 and Table S1. Current Biology 2018 28, 196-210.e4DOI: (10.1016/j.cub.2017.11.069) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 6 Ceramides Are Required for Negative Feedback in the TORC2 Network (A) Cells were grown to early log phase in YPD medium. Varying concentrations of phytosphingosine (PHS) were added and cells were incubated at 25°C. Samples were taken 15 min after addition of phytosphingosine and Mss4-3XHA was detected by western blot. (B) Wild-type and rts1Δ cells were grown to early log phase in YPD. 20 μM phytosphingosine (PHS) was added to each culture followed by incubation at 25°C. Samples were taken at the indicated times and Mss4-3XHA was detected by western blot. (C) Wild-type and lac1Δ lag1Δ cells were grown to early log phase in YPD at 22°C. Mss4-3XHA was detected by western blot. (D) lac1Δ lag1Δ cells were grown to early log phase in YPD at 25°C. Samples were taken at the indicated times after addition of 20 μM phytosphingosine (PHS), and Mss4-3XHA was detected by western blot. (E) Wild-type and lac1Δ lag1Δ cells were grown to early log phase in YPD at 22°C. Ypk-pT662, Ypk-pT504, and Ypk1 were assayed by western blot. (F) Wild-type and lac1Δ lag1Δ cells were grown to early log phase in YPD at 22°C. Samples were taken at the indicated times after addition of 20 μM phytosphingosine (PHS), and Ypk-pT662, Ypk-pT504, and Ypk1 were assayed by western blot. For (E) and (F), an asterisk indicates a non-specific band. See also Figure S6 and Table S1. Current Biology 2018 28, 196-210.e4DOI: (10.1016/j.cub.2017.11.069) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 7 Ceramides Are Required for Nutrient Modulation of the TORC2 Signaling Network (A and B) Wild-type and lac1Δ lag1Δ cells were grown to early log phase in YPD at 22°C. After washing into YPG/E, cells were incubated at 25°C and samples were taken at the indicated times. (A) Mss4-3XHA was detected by western blot. (B) Ypk-pT662, Ypk-pT504, and Ypk1 were assayed by western blot. An asterisk indicates a non-specific band. (C) Quantification of ceramides and complex sphingolipids in wild-type and rts1Δ cells growing in rich or poor carbon. Data represent the average of three biological replicates. Welch two-sample t test was used to estimate the p values: ∗p < 0.05, ∗∗p < 0.01. (D) A model for how nutrient-dependent modulation of the TORC2 signaling network could influence cell growth and size. See also Figure S7, Table S1, and Data S1. Current Biology 2018 28, 196-210.e4DOI: (10.1016/j.cub.2017.11.069) Copyright © 2017 Elsevier Ltd Terms and Conditions