Cellular Control of Viscosity Counters Changes in Temperature and Energy Availability

Abstract

Cellular functioning requires the orchestration of thousands of molecular interactions in time and space. Yet most molecules in a cell move by diffusion, which is sensitive to external factors like temperature. How cells sustain complex, diffusion-based systems across wide temperature ranges is unknown. Here, we uncover a mechanism by which budding yeast modulate viscosity in response to temperature and energy availability. This "viscoadaptation" uses regulated synthesis of glycogen and trehalose to vary the viscosity of the cytosol. Viscoadaptation functions as a stress response and a homeostatic mechanism, allowing cells to maintain invariant diffusion across a 20°C temperature range. Perturbations to viscoadaptation affect solubility and phase separation, suggesting that viscoadaptation may have implications for multiple biophysical processes in the cell. Conditions that lower ATP trigger viscoadaptation, linking energy availability to rate regulation of diffusion-controlled processes. Viscoadaptation reveals viscosity to be a tunable property for regulating diffusion-controlled processes in a changing environment.

Keywords: ATP; diffusion; glycogen; heat shock; homeostasis; phase separation; starvation; stress response; trehalose; viscosity.

Figure 1. A viscosity-controlled model reaction is temperature-sensitive in cell lysates but not live cells.

(A) Biotinylation rate vs viscosity (1/t-half) for reactions carried out in 0%, 20%, 40%, or 60% sucrose solutions (●blue) or cell lysates (○orange). Reaction rates and t-half values were normalized to their respective values in H₂O. Horizontal error bars are s.e.m. (n> 3 FRAP experiments). Vertical error bars are the standard error of the slope derived from the best fit line for multiple timecourses (n> 5 timecourses/condition) (B) Biotinylation rates in lysates. Cell growth temperature (left of arrow) and reaction temperature (right of arrow) are indicated in the figure key. Normalized to max labeling at 30°C (nonlinear regression, difference between slopes, p<.0001) (C) Diffusion coefficients for GFP in pure H₂0 (n=4), lysate from cells grown at 30°C (n=3), and live cells growing at 30°C (n=19) (unpaired t-test, H₂0 vs cell lysate p=.0037; cell lysate vs live cells p<.0001; H₂O vs live cells p<.0001) (D) Biotinylation rates in live cells growing at 30°C (●blue) or 37°C (◼orange). Measurements are at the growth temperatures of the cells. Normalized to max labeling at 30°C (n=4 timecourses/temperature) (nonlinear regression, difference between slopes p=0.1) (E) Biotinylation rates in lysates from cells grown and measured at the indicated temperatures. Normalized to max labeling for 30°C→30°C (nonlinear regression difference between slopes, 30°C→30°C vs 30°C→37°C p>.0001; 30°C→30°C vs 37°C→30°C p>.0001; 30°C→37°C vs 37°C→30°C p>.0001) (F) Biotinylation rates measured at the same temperature of cell growth prior to lysis. Normalized to max labeling at 30°C→30°C (nonlinear regression, difference between slopes, 30°C→30°C vs 30°C→37°C p=.01) Error bars are s.e.m. Points without visible error bars have error within the size of the point. See also Figure S1.

Figure 2. Cells regulate intracellular viscosity in response to heat (“viscoadaptation”).

(A) Diffusion coefficients of GFP in lysates from cells grown at 30°C and measured at the indicated temperatures (n=4 lysates/temperature) (unpaired t-test, 22°C vs 42°C p=.0024; 22°C vs 37°C p=.0094; 22°C vs 30°C p=.083; 30°C vs 37°C p=.059; 30°C vs 42°C p=.0087; 37°C vs 42°C p=.432) (B) FRAP on live cells expressing eGFP, shifted from 22°C to the indicated temperature for >20 minutes before measurement. Striped bar represents cells measured within 5 minutes of temperature shift (n> 3 experiments/condition, >8 cells/experiment) (unpaired t-test, 22°C vs 42°C p<.0001; 22°C vs 40°C p<.0001; 22°C vs 37°C p<.0001; 22°C vs 30°C p=.0528; immediate 42°C vs delayed 42°C p=.0011; 22°C vs 42°C immediate p<.01) (C) Timecourse of FRAP measurements on live cells shifted from 22°C (T₀) to 42°C (n>3 cells/timepoint). Points without visible error bars have error within the size of the point (nonlinear regression, slope of best fit line=.005) (D) Cells expressing eGFP were grown to log phase (minimum 3 hours) at the indicated temperatures. FRAP was performed at the same temperature as cell growth. (n> 3 replicates/temperature, >10 cells/replicate)(unpaired t-test, p>.05 for all pairwise comparisons) (E) Diffusion coefficients of purified GFP in lysates from cells grown to steady state at 22°C, 30°C, 37°C, or 40°C, determined by FRAP. All measurements were at 22°C (n> 3 lysates/condition) (unpaired t-test, 22°C vs 40°C p=.168; 22°C vs 37°C p=.046; 22°C vs 30°C p=.857; 30°C vs 37°C p=.0048; 30°C vs 40°C p=.075; 37°C vs 40°C p=.372) Error bars are s.e.m. See also Figure S2.

Figure 3. Heat-induced accumulation of the carbohydrates glycogen and trehalose increases cellular viscosity.

(A) Glycogen and trehalose are produced from glucose through conversion of glucose-1-p to glucose-6-p by phosphoglucomutase (PGM) which is inhibited by LiCl. (B) Quantification of glycogen (top) and trehalose (bottom) during 42°C heat shock. T₀ is at 22°C. (n=3 timecourses each) (C) t-half values in the indicated strains at 22°C (leftmost, blue) or after >20 minutes at 42°C (red). Each dot represents a single cell. Corresponding iodine staining shown above (all iodine staining spots are 5 mm diameter) (unpaired t-test, WT 22°C vs WT 42°C p<.0001; WT 42°C vs ΔGlc3 42°C p=.4; WT 42°C vs ΔGsy2 42°C p=.924; WT 42°C vs ΔTps2 42°C p<.0001; WT 42°C vs ΔGΔGΔT 42°C p<.0001; WT 42°C vs WT 42°C +LiCl p<.0001) (D) Cells were treated with Validamycin A and DAB (10 ug/ml each) immediately prior to 42°C heat shock for 40 minutes. Each dot is a single cell (n=3 experiments)(unpaired t-test, vehicle 10 vs vehicle 90 p<.0001; +drug 10 vs +drug 90 p=.058; vehicle 10 vs +drug 10 p=.125; vehicle 90 vs +drug 90 p<.0001) (E) Cells expressing BirA and GFP-Avi were grown to steady state at 30°C (blue) or 37°C (red) with (▪) or without (●) 50 mM LiCl. Normalized to max labeling at 30°C -LiCl (nonlinear regression, difference between slopes, −LiCl 30°C vs +LiCl 30°C p=.48; −LiCl 30°C vs −LiCl 37°C p=.1; +LiCl 30°C vs +LiCl 37°C p<.0001; −LiCl 30°C vs +LiCl 37°C p<.0001) Error bars are s.e.m. Points without visible error bars have error within the size of the point. See also Figure S3.

Figure 4. Viscoadaptation occurs in response to nutrient limitation.

(A) Quantification of glycogen (left) and trehalose (right) for cells in 2% glucose, 0.1% glucose (30 minutes), and stationary phase for >24 hours (n=3 samples/condition). Below (bottom left) are iodine staining spots for the indicated conditions. (glycogen: unpaired t-test, log phase vs low glucose p=.004; log phase vs stationary phase p=.006) (trehalose: unpaired t-test, log phase vs low glucose p=.337; log phase vs stationary phase p=.005) (trehalose: unpaired t-test, log phase vs low glucose p=.337; log phase vs stationary phase p=.005) B) In vivo BirA labeling of GFP-Avi in 2% glucose (green) and 0% glucose (blue) (nonlinear regression, difference between slopes, p<.0001) (C) In vitro BirA labeling timecourse in lysates from cells grown in 2% glucose (green) and 0% glucose (blue) (nonlinear regression difference between slopes, p<.0001) (D) Fraction of GFP-Avi labeled by BirA after 30 minutes in 0% glucose with (checkered) and without (solid) 50mM LiCl. Normalized to the −LiCl condition (unpaired t-test, p=.01) (E) FRAP measurements on WT cells in the indicated growth conditions (n> 3 replicates, >9 cells/replicate) (unpaired t-test, 2% glucose vs low glucose p=.0006, 2% glucose vs low glucose +LiCl p>.05; 2% glucose vs stationary phase p=.0094; 2% glucose vs 0% glucose p>.05) (F) FRAP on the indicated strains in 2% glucose (leftmost) or 0.1% glucose for 30 minutes. Each dot represents one cell. (unpaired t-test, WT 2% glucose vs WT 0.1% glucose p<.0001; WT 0.1% glucose vs ΔGlc3 0.1% glucose p=.673; WT 0.1% glucose v. ΔGΔGΔT 0.1% glucose p=.006; WT 2% glucose vs ΔGΔGΔT 0.1% glucose p=.05) Error bars are s.e.m. See also Figure S4.

Figure 5. Glycogen and trehalose alter molecular movement in vitro.

(A) FRAP on purified GFP in aqueous solutions of glycogen and trehalose at 22°C and 40°C reported as t-half (n>3)(unpaired t-test, H₂O 22°C vs H₂O 40°C p=.0023; 45%Tre 22°C vs 45%Tre 40°C p=.636; 30%Gly 22°C vs 30%Gly 40°C p=.0013; Gly/Tre 22°C vs Gly/Tre 40°C p=.0286; H₂O 22°C vs 45%Tre 22°C p=.005; H₂O 22°C vs 30%Gly 22°C p<.0001; H₂O 22°C vs Gly/Tre 22°C p<.0001) (B) FRAP on GFP in serial dilutions of glycogen and trehalose, measured at 22°C and reported as the diffusion coefficient of GFP (n=3)(unpaired t-test, 11.25/7.5 vs 45/30 p=.0001; 11.25/7.5 vs 22.5/15 p=.0011; 22.5/15 vs 45/30 p<.0001) C) Aqueous solutions of trehalose at the indicated concentrations in liquid droplets (top row) or after solidification(bottom row). Initial droplet diameter is 4 mm. (D) Summary of phase observations for glycogen and trehalose droplets of varying concentrations at 22°C, see Figure S5B for phase designation criteria. (E) Fluorescent images of purified GFP in glycogen and trehalose solutions. See also Figure S5.

Figure 6. Viscoadaptation affects phase separation and the solubility of biomolecules in vivo.

(A) Pab1-GFP in cells at 22°C (top) or after 40 minutes at 42°C (bottom) in the presence or absence of 50 mM LiCl (B) 10x brightfield images of the interior of dried lysate droplets produced from the strains and conditions indicated (C) (left) 10x phase contrast microscopy on lysate from cells heat shocked at 45°C for 30 minutes. Top and bottom images are different z-planes of the same region. (right) Diffusion was measured by microrheology in regions interior and exterior to the inclusions (n=3 exterior/3 interior measurements)(paired t-test, p<.0001) (D) Biotinylation of GFP-Avi in lysates from unstressed cells (blue) or cells heated at 45°C for 30 minutes (red). Normalized to max labeling in unstressed lysate (nonlinear regression, difference between slopes, p<.0001) (E) Lysates from the previous panel were diluted 1:50 in H₂0. Normalized to max labeling in the diluted unstressed lysate (n=3 timecourses/condition)(nonlinear regression, difference between slopes, p=.14) (F) The mobile fraction (MF) of GFP in WT and ΔReg1 cells classified as low (<0.8) or high (>0.8) in log phase and stationary phase cultures (unpaired t-test, WT log vs WT stationary phase p>.05; WT log vs ΔReg1 log p=.001; ΔReg1 log vs ΔReg1 stationary phase p=.0214)(n>3 replicates, >8 cells/replicate). Error bars are s.e.m. See also Figure S6.

Figure 7. Viscoadaptation occurs in cells with low ATP.

(A) FRAP measurements on WT cells untreated (UT) or treated with sodium azide, sodium arsenite, or a combination of oligomycin (O), antimycin A (A), and FCCP (F) +/− 50 mM LiCl (n>3 experiments, >10 cells/experiment)(unpaired t-test, UT vs Azide p=.0002, UT vs Arsenite p=.001, UT vs AOF p<.0001; Azide vs Azide +Li p=.054, Arsenite vs Arsenite +Li p=.025, AOF vs AOF +Li p=.016) Error bars are s.e.m. (B) WT cells expressing a FRET-based ATP nanosensor were switched from 2% to 0.1% glucose for 30–60 minutes. Each point is one cell. ATP levels for single cells were determined by the ratio of 528:488 fluorescence (see Figure S7A). Mobility was assessed by performing FRAP of the fluorescent sensor on the same cells. Orange dots represent cells treated with 50 mM LiCl during starvation (without LiCl R= −0.597 p<.0001; with LiCl R= −0.361 p=0.0063) (C) WT cells in 0% glucose SD media. Measurements occur between 30–60 minutes after starvation onset (R= −0.047, p=.38) (D) t-half values and ATP levels for unstressed WT cells in 2% glucose SD media. Cells were sampled to capture a range of ATP levels. (R= −0.555 p<.0001) (E) Cells expressing the ATP FRET sensor were heated over the course of an hour from 22°C to 42°C. ATP level and t-half was recorded for each cell. X-axis is time in minutes. Data is plotted as a rolling average of every 3 values and normalized on a scale of 0 to 1 to facilitate comparison. Dotted lines represent s.e.m. See also Figure S7.