Figure 1. Eisosomes disassemble to buffer increases in membrane tension.

(A) Representative walled yeast cells (left column) and protoplasts (right column) at steady state in 1.2 M sorbitol expressing eisosome core protein Pil1-mEGFP (inverted contrast). Note that the cellular concentration of Pil1p-mEGFP is the same in walled cells and protoplasts (panel E). (B) Eisosomes labeled with Pil1p-mEGFP (inverted contrast) in wild-type protoplasts at steady state in different sorbitol concentrations. From left to right: 0.25, 0.4, 0.8, and 1.2 M sorbitol. (C and D) Density of eisosomes at the plasma membrane (C), measured as the ratio between the intensity of Pil1p-mEGFP on the plasma membrane and the surface area of the protoplast and volume (D), at steady state in 0.25 M (N = 26), 0.4 M (N = 34), and 1.2 M (N = 39) sorbitol. Error bars: standard deviations. (E) The total amount of Pil1-mEGFP in walled cells (N = 32) and protoplasts in 0.4 M sorbitol (N = 19) are not significantly different (Mann-Whitney test, p=0.65). Each point represents one measurement; bars are the mean and SEM. (F) Micropipette aspiration was used to measure membrane tension. R_c: cell radius; R_p: micropipette radius; l: length of the tongue inside the micropipette. (G) Membrane tension of protoplasts at steady state in 0.8 M sorbitol and ~5 min after a hypotonic shock (ΔC = −0.2 M) for wild-type (blue bars, N = 28 for steady state and N = 5 for the shock) and pil1Δ protoplasts (red bars, N = 42 for steady state and N = 7 for the shock). Error bars: standard deviation. p-values: non-significant (ns), p>0.05; **p≤0.01; ***p≤0.001. (H and I) Eisosomes of wild-type protoplasts disassemble rapidly after a hypotonic shock. (H) Time course of a representative protoplast expressing Pil1p-mEGFP over 10 min after a hypotonic shock (ΔC = −0.2 M) and initially at steady state in 0.4 M sorbitol (just before time 0 min). (I) Evolution of the surface area covered by eisosomes over time, as a fraction of the surface area covered at time 0 min (normalized to 1). Data are from three independent experiments (N = 15) and presented as mean ± 95% confidence interval. Scale bars in (A), (B), (F), and (H): 5 µm.