Stress-dependent cell stiffening by tardigrade tolerance proteins that reversibly form a filamentous network and gel
Fig 4
CAHS confers the mechanical resistance against deformation forces on cell-like microdroplets and insect cells.
(A) Representative fluorescent images of a microdroplet containing CAHS3-GFP in the absence or presence of additional NaCl. Scale bar, 5 μm. (B) Representative response curves of the elongation length of microdroplets containing CAHS3-GFP or GFP alone under a very small pressure (<<0.5 kPa). Continuous elongation exceeding 50 μm indicates not elastic and in a liquid phase. (C) Comparison of the elasticity (Young’s modulus) among droplets containing CAHS3-GFP or GFP with or without NaCl addition. Data are presented as average ± SE. (D) The effects of CAHS3-expression on the cortical elasticity of Drosophila S2 cells under hyperosmosis. CAHS3-stably expressing cells exhibited higher elasticity compared to the control cells transfected with empty vector under a hyperosmotic condition supplemented with 0.4 M trehalose for 3 h. Gray and red dots indicate the values of each measurement. Black dots and bars indicate averages and standard errors, respectively. (E) Comparison of cell volume changes by hyperosmotic stress between CAHS3-expressing cells and control cells. The relative cell volume was calculated by dividing the volume under hyperosmotic stress by the averaged cell volume under isosmotic conditions. Center bar and edges indicate 50th, 25th, and 75th percentiles, respectively, and whiskers correspond to the 1.5 interquartile range. (F and G) Comparison of cell viability between CAHS3-expressing cells and control cells under an isosmotic condition (F) and a hyperosmotic condition for 48 h (G). PI was used to determine dead cells. Survival rates were examined in 6 wells for each condition by counting >500 cells/well. Statistical analyses were performed with the Wilcoxon rank sum test in (D) and (E), and Student t test in (F) and (G); n.s. means not significant in the statistical tests. The underlying numerical data are available in S4 Data (B–G). CAHS, cytoplasmic-abundant heat-soluble; PI, propidium iodide.