Figure 3. The density of endocytic events rapidly adapts after acute osmotic shocks.

(A) Temporal evolution of density of endocytic events (average number of endocytic events at a given time in a cell divided by the cell length) in wild-type protoplasts initially at steady state in 0.4 M sorbitol and after an acute hypotonic shock of ΔC = −0.05 M (dark blue, N_cell ≥ 102), ΔC = −0.1 M (blue, N_cell ≥ 54), and ΔC = −0.2 M (light blue, N_cell ≥ 83). For ΔC = −0.1 and −0.2 M, the difference in the density of clathrin-mediated endocytosis (CME) events between steady state and 0 or 2 min after the shock is statistically significant (one-way ANOVA, p<10⁻⁴). In all conditions, the difference after 6 min is not significant (one-way ANOVA, p>0.12; details in Figure 3—source data 1). (B) Same as (A) but with pil1Δ protoplasts and hypotonic shocks of ΔC = −0.025 M (dark red, N_cell ≥ 70), ΔC = −0.05 M (red, N_cell ≥ 103), and ΔC = −0.1 M (light red, N_cell ≥ 78). In all conditions, the difference in the density of CME events between steady- state and any time after the shock is statistically significant (one-way ANOVA, p<10⁻³). For ΔC = −0.025 and −0.05 M, the differences between time points after 6 min are not significant (one-way ANOVA, p>0.09; details in Figure 3—source data 2). (A and B) Insets: relative volume increase after the hypotonic shocks (the volume at steady state is used as a reference). The numbers of cells used for each condition and each time point are given in Supplementary file 1a. The number of cells measured in the insets is the same as that in the main figures. *: the large majority of pil1Δ protoplasts were too damaged or dead 4 min after the hypotonic shocks at ΔC = −0.1 M (Figure 2B), which prevented us to measure the density of endocytic events and the volume after this time point. (C) Density of endocytic events in intact cells at steady state in different osmolarities, N_cell ≥ 80. In pil1Δ walled cells, the difference in the density of CME events between all pairs of conditions is statistically significant (one-way ANOVA, p<10⁻⁴). In wild-type walled cells, the difference is small but statistically significant (details in Figure 3—source data 3). The numbers of cells used for each condition and each time point are given in Supplementary file 1b. (D) Density of endocytic events in wild-type (blue circle) and pil1Δ (red triangle) walled cells initially at steady state in 1.2 M sorbitol and after an acute hypotonic shock of ΔC = −1.2 M, N_cell ≥ 44. The numbers of cells used for each condition and each time point are given in Supplementary file 1c. For wild-type and pil1Δ walled cells, the differences in the density of CME events after 2 min are not statistically significant (p>0.08; details in Figure 3—source data 4). (A), (B), (C), and (D): error bars are standard errors of the mean.

Figure 3—figure supplement 1. Separate plots for each condition shown in Figure 3A and B.

(A) Temporal evolution of density of endocytic events (average number of endocytic events at a given time in a cell divided by the cell length) in wild-type protoplasts initially at steady state in 0.4 M sorbitol and after an acute hypotonic shock of ΔC = −0.05 M (dark blue, N_cell ≥ 102), ΔC = −0.1 M (blue, N_cell ≥ 54), and ΔC = −0.2 M (light blue, N_cell ≥ 83). (B) Same as (A) but with pil1Δ protoplasts and hypotonic shocks of ΔC = −0.025 M (dark red, N_cell ≥ 70), ΔC = −0.05 M (red, N_cell ≥ 103), and ΔC = −0.1 M (light red, N_cell ≥ 78). (A and B) Insets: relative volume increase after the hypotonic shocks (the volume at steady state is used as a reference). The number of cells used for each condition and each time point is given in Supplementary file 1a. The number of cells measured in the insets is the same as in the main figures. (A and B) Error bars are standard errors of the mean. The number of cells used for each condition and each time point is given in Supplementary file 1b.