Fig 1.
Control of yeast cell size using optogenetics.
(A) Exogenous PhyB (phytochrome B; dark green) is fused to the C-terminus of S. cerevisiae Tom20, anchoring it to the mitochondria outer membrane (orange). PIF (phytochrome-interacting factor; light green) is fused to endogenous Bem1 (blue). Illumination with red light drives a conformational change in PhyB allowing it to bind PIF-Bem1. Conversely, illumination with infrared (IR) light drives the reverse reaction, releasing PIF-Bem1. (B) Production of ‘giant’ yeast using reversible optogenetic-based Bem1 disruption. (C) Cells were illuminated with red light for 8 h (indicated by red borders) and imaged every 10 min using bright-field microscopy. (D) Following red light illumination for 6–10 hours as in Fig 1C, large cells were illuminated with IR light (indicated by grey borders) and imaged every 5–10 min using bright-field microscopy.
Fig 2.
Effects of continuous isotropic growth on yeast physiology.
(A) Yeast were prepared and imaged as in Fig 1C and 1D with media containing indicated concentrations of sorbitol. Each bar indicates the percentage of cells surviving the entire 12-h time-course. (B) Average normalized diameter of yeast grown in media with or without 1 M sorbitol. N > 40 cells for each condition. Error bars, SD. (C) Cell wall staining. Cells were prepared as in Fig 2A, treated with 100 μg/mL Alexa488-labeled concanavalin-A, and imaged every 15 min for 8 h. Each panel is a maximum intensity projection of multiple focal planes acquired via confocal microscopy. Yeast grow isotropically following Bem1 deactivation. (D) Average growth trajectory for Bem1-arrested cells calculated as in Fig 2B for yeast in 1 M sorbitol from experiments described in Fig 2A. Each blue dot represents an average of 48 cells. Expected trajectories for exponential, constant, and surface-area-proportional growth are indicated by grey, green, and red lines, respectively. (E) Analysis of Bem1-disrupted cells by flow cytometry. Representative plots, left; averaged data from 2 independent experiments, right. Error bars, SD. (F) Average growth trajectory for cdk1-ts cells at 37°C. The relationship between diameter and time remained linear for the first 5 h of growth (r2 = 0.998), but growth rapidly stalled at later timepoints. N = 126 cells.
Fig 3.
Convergence of yeast to set-point volume is inconsistent with an ‘adder’.
(A) Cells were incubated under red light illumination for 8 h followed by IR light illumination for 18 h. At indicated timepoints (every 2 h during IR light illumination), cell volumes were measured by microscopy. Each point represents a single cell. (B) Budding yeast cell cycle with labels depicting volume and growth intervals measured in Fig 3C, 3D and 3E. Blue-shaded areas, volume added as a newly-born cell grows. (C) OptoBem1 cells were illuminated for 8–10 h with red light (to generate giant yeast), then switched to IR light (allowing giant yeast to bud and divide) and imaged every 5 min for ~8 h. cdk1-ts cells were incubated at 37°C for 8 h, then switched to 25°C prior to imaging. Exogenously-expressed Cdc10-GFP was used to mark septin rings (green). Panels depict representative optoBem1 cells. Time, HH:MM. (D) Each point represents a single cell. Colored lines, best-fit line by linear regression analysis. (E) Cells were binned by volume at birth, as indicated. N = 28 optoBem1 cells.
Fig 4.
Comparison of sizer and timer mechanisms for regulation of bud growth.
(A) Schematic depicting the use of our optogenetics system for discriminating between bud ‘sizer’ and bud ‘timer’ mechanisms for specifying daughter cell volume. (B and C) Cells were binned by mother volume in 200-μm3 increments. The average volume within each bin is plotted. Measurements of ‘budding duration’, ‘mother volume’, and daughter volume’ were obtained as described in Fig A in S3 Fig. N = 73 optoBem1 cells and 80 cdk1-ts cells, with each bin containing at least 5 cells. Error bars, SD. rs, Spearman’s rho.