Figure 3. The N/C ratio is maintained in osmotic shifts and upon cell-wall removal.

(A) The N/C ratio of the same cells before and after cell wall digestion (mean ± STD) reveals no statistical differences (paired t test, p=0.36). Right panel, overlay of the plasma membrane (green) and nuclear membrane (purple) of the same cell middle plane before and after cell-wall digestion. For all box and whiskers plots, the horizontal line indicates the median, the box indicates the interquartile range of the data set (IQR) while the whiskers show the rest of the distribution within 1.5*IQR except for points that are defined as outliers. Scale bar = 5 µm. From six biological replicates. (B) Scatter plot of cell size and nuclear size for protoplasts under isotonic conditions (YE +0.4 M sorbitol) and immediately following osmotic shocks. Black and dashed red lines, measured N/C ratio of cells under isotonic conditions and osmotically shocked, respectively. From at least two biological replicates. (C) Protoplasts with the individual N/C ratio per osmotic condition described in (B). (D) Same as (B) for whole cells in YE under isotonic condition. Black line, measured N/C ratio of cells in isotonic conditions. From two biological replicates. (E) Same as (C) for whole cells with the individual N/C ratio per osmotic condition described in (D). See also Figure 3—figure supplement 1.

Figure 3—figure supplement 1. Measurements of cellular and nuclear volumes under osmotic shocks in protoplasts and whole cells.

(A) During protoplast preparation, a small portion of the cytoplasm was sometimes lost when the protoplast extruded out of the remaining cell wall. Image depicts a sum projection image of a protoplast with a portion of the plasma membrane (green) left behind (arrow). This behavior led to initial measurements that N/C ratio was slightly increased in some protoplasts compared to walled cells Figure 3B and D. To address this issue, for Figure 3 we therefore focused our analysis on cells that did not show any loss of cytoplasm during protoplasting. (B) Box and whisker plots for protoplasts showing individual cells volumes per osmotic condition described in main Figure 3. (C) (E) Same as (B) for protoplasts nuclei. (D) Separated plot from Figure 3B. Scatter plot of cell size and nuclear size for protoplasts under isotonic conditions (YE +0.4 M sorbitol, left panel) and immediately following osmotic shocks (right panel). Black and dashed red lines, measured N/C ratio of cells under isotonic conditions and osmotically shocked, respectively. (E) Box and whisker plots for whole cells showing individual cells volumes per osmotic condition described in main Figure 3. Mean ±STD are indicated under each condition. (F) Same as (E) for whole cells nuclei. (G) Separated plot from Figure 3D. Scatter plot of cell size and nuclear size for whole cells under isotonic conditions (YE, top panel) and immediately following osmotic shocks (bottom panel). Black and red lines, measured N/C ratio of cells under isotonic conditions and hypo-osmotically shocked, respectively. From two biological replicates. (H) Scatter plot of cell size and nuclear size for whole cells under isotonic conditions (YE) and immediately following hypo-osmotic shock. Black and blue lines, measured N/C ratio of cells under isotonic conditions and hypo-osmotically shocked, respectively. paired t test, p=0.0215 indicates a small difference in the N/C ratio. From two biological replicates. (I) Box and whisker plots for whole cells showing individual cells volumes per osmotic condition. Mean ±STD are indicated under each condition. (J) Same as (G) for whole cells nuclei. (I–J) Statistical differences indicate significant increase in nuclear and cell size under hypotonic cells (Wilcoxon paired t test, p<0.0001). 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.