Figure 2. The fission yeast nucleus behaves as an ideal osmometer.

(A) Images of cells expressing a plasma membrane marker mCherry-Psy1 (green) and a nuclear envelope marker Ish1-GFP (purple). Individual cells in isotonic medium (C_iso) were shifted to hypertonic or hypotonic medium and imaged for 3D volume measurements (Materials and methods). (B) Images of individual protoplasts in response to hypertonic and hypotonic shifts. See also Figure 2—figure supplement 1. Scale bar = 5 µm. (C–E) BVH plots of the effects of osmotic shifts on the volume of the cell and nucleus. (C) Theoretical predictions of effects of osmotic concentration in the medium (C_iso/C) on the volume of a cell or nucleus with zero (black) or large (green) membrane tension (σ). Dashed line (black) depicts the behavior of an ideal osmometer in which there is no effect of membrane tension. (D) Effect of osmotic shifts on the relative volumes (V/V_iso, mean ± STD) of whole fission yeast cells (N=707, three biological replicates) and protoplasts (N=441, from at least five biological replicates). (E) Effect of osmotic shifts on relative nuclear volume (V/V_iso, mean ± STD) in protoplasts (N=441, from at least five biological replicates). Note that the response of nuclei fits to the predicted behavior of an ideal osmometer.

Figure 2—figure supplement 1. 3D image analysis methods and use of an osmotic adaptation mutant allow for robust volume measurements.

Previous fission yeast cell studies estimated nuclear and cell volumes from length and single width measurements using assumptions of symmetric ellipsoid or cylindrical geometry (Facchetti et al., 2019; Kume et al., 2017; Lemière et al., 2021; Neumann and Nurse, 2007). As the shape of fission yeast cells are not perfectly symmetric ellipsoids, we determined volumes using a 3D segmentation approach (Machado et al., 2019). To minimize the adaptation responses to osmotic stress, most of these studies were done with cells with a gpd1∆ background, which is defective in glycerol synthesis responsible for the rapid volume adaptation to osmotic stresses (Hohmann, 2002). (A) N/C ratio is maintained in distinct cellular backgrounds. Scatter plot of cell size and nuclear size for asynchronous cells in growth medium. Right, Box and whisker plots of the N/C ratio for the three strains. Our 3D measurements of mean cell volume (97.5±27.1 µm³), nuclear volume (7.3±2.1 µm³), and the N/C ratio (7.5±0.8) of a population of asynchronous cells were consistent with previously reported values that used different image analysis methods. (Kume et al., 2017; Neumann and Nurse, 2007). For all box and whiskers plots, the horizontal line indicates the median, the box indicates the interquartile range (IQR) of the data set while the whiskers show the rest of the distribution within 1.5*IQR except for points that are defined as outliers. Statistical difference compared with an unpaired t-test. These data show that the N/C ratio measurements was not affected by the gpd1∆ background, or by use of different nuclear envelope markers ish1-GFP and cut11-GFP. (B&C) Time course of volume adaption in response to hyperosmotic shocks of 1 M sorbitol (B) and 0.5 M sorbitol (C). Normalized cell volume dynamics WT (chartreuse, N=12 cells) and gpd1Δ (green, N=12 cells) after 1 M sorbitol shock, mean ±STD. (C) Normalized cell and nucleus volume dynamics after 0.5 M sorbitol shock in WT (N=12) and gpd1Δ (N=10) background cells, mean values ±STD. These measurements of volume adaption enabled us to define time windows in which acute volume changes can be measured.

Figure 2—figure supplement 2. Additional evidence that protoplasts behave as ideal osmometers.

(A) Defining an isotonic medium for protoplasts. The osmotic pressure of the medium changes the protoplasts’ volume and concentration of proteins due to addition or removal of water from the cell. To assess cytoplasmic concentration, we monitored fluorescence intensity of a protein E2-mCrimson expressed from the ACT1 promoter, which has been shown to normally maintain a largely constant concentration in whole cells throughout the cell cycle (Al-Sady et al., 2016; Knapp et al., 2019). mCrimson fluorescence intensity and cellular volume in a population of whole cells in isotonic medium (black) were similar to those of protoplasts in same medium supplemented with 0.4 M sorbitol (red), demonstrating that this is the isotonic condition for these protoplasts. Right panels, mid focal plane image of a cell (top) and protoplast (bottom) expressing mCrimson. (B) In contrast, comparison of mCrimson fluorescence intensities in protoplasts in 1 M sorbitol (dark red) and 0.4 M sorbitol (red), showed that 1 M sorbitol led to higher protein concentration than in isotonic conditions. (C–G) Number of intracellular osmolytes (N) was measured as described in Methods. Consistent with an ideal osmometer, N is directly proportional to the change in cell volume under osmotic shocks. N is proportional to the cell initial volume and does not depend on the range of osmotic shock used to probe the cells (Methods). R² values are R-squared values for linear regression. Scale bar = 5 µm.