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. 2022 Jul 20;11:e76075. doi: 10.7554/eLife.76075

Figure 4. Macromolecular crowding is affected similarly in the nucleus and cytoplasm under osmotic shocks.

(A) Images of protoplasts (left) and whole cells (right) expressing cytoplasmic 40 nm GEMs and nucleoplasmic 40 nm GEMs. Top, single time point image; bottom, maximum projection of 100 frames. Dashed lines, the cell boundary. Scale bar = 5 µm. (B) GEMs effective diffusion coefficient (mean ± SEM) is slower in the cytoplasm (green) than in the nucleoplasm (purple) in whole cells in YE medium. Numbers indicate the number of tracks, p-value <0.0001 Mann-Whitney U test. (C) Effective diffusion coefficient of cytoplasmic GEMs (mean ± SEM) in protoplasts shifted to various sorbitol concentrations in the medium. Dashed lines, predictions of Phillies’ model for diffusion with a power law λ=1. NGEMs tracked = 4058, from at least two biological replicates per condition. (D) Effective diffusion coefficient of cytoplasmic GEMs (mean ± SEM) plotted against cell volume under hypotonic and hypertonic shock (light blue and blue background respectively). Volumes represent mean distribution of an asynchronous culture ± STD. Dashed line, fit of Phillies’ model for self-diffusing trackers in a polymer solution. Black arrow indicates Deff for a population of cells in YE and protoplasts in isotonic condition. Protoplasts: NGEMs = 3355, NVolume = 2216 cells, whole cells: NGEMs = 9849, NVolume = 981 cells. (E) Effects of hyperosmotic shifts on the relative effective diffusion coefficients (mean ± SEM) of cytoplasmic and nuclear GEMs; no statistically significant difference was detected (F-test, p-value = 0.90). Cytoplasm NGEMs = 9365,, nuclear NGEMs = 3732, from at least two biological replicates per condition. See also Figure 4—figure supplement 1.

Figure 4—source data 1. Effective diffusion of cytGEMs and nucGEMs.
Related to Figure 4B.
Figure 4—source data 2. Effective diffusion of cytGEMs in protoplasts in various sorbitol concentrations.
Related to Figure 4C, Figure 4—figure supplement 1.
Figure 4—source data 3. Effective diffusion of cytGEMs plotted against cell volume under hypotonic and hypertonic.
Related to Figure 4D.
Figure 4—source data 4. Normalized effective diffusion of cytGEMs and nucGEMs in various sorbitol concentrations.
Related to Figure 4E.

Figure 4.

Figure 4—figure supplement 1. Comparison of the cytoplasmic and nucleoplasmic GEMs diffusion and anomalous exponent under osmotic shocks.

Figure 4—figure supplement 1.

(A) Example of MSD plots for cytoplasmic GEMs in whole cells under isotonic condition (0 M) or hypertonic shock (0.1–1.0 M of sorbitol). (B) Cytoplasmic (green) and nucleoplasmic (purple) GEM anomalous diffusion exponents for whole cells under osmotic shock. (C) Effects of hyperosmotic shifts on the effective diffusion coefficients (mean ± SEM) of cytoplasmic and nuclear GEMs; no statistically significant difference was detected (F-test, p-value = 0.90). Cytoplasm NGEMs = 9365, nuclear NGEMs = 3732, from at least two biological replicates per condition. (D) Example of cytoplasmic GEMs MSD plots in protoplasts in isotonic condition (0.4 M) or osmotically shocked (0.1–0.2 M and 0.6–1.0 M). (E) Anomalous diffusion exponent ⍺ obtained from linear fits of the MSD plots presented in (D). (F) Effective diffusion coefficient of cytoplasmic GEMs in protoplasts for various medium concentrations. Dashed lines, Phillies’ model for diffusion from three power law values: λ=0.5 (black), λ=1 (green), and λ=1.5 (gray).
Figure 4—figure supplement 1—source data 1. cytGEMs MSD and anomalous diffusion exponent plots in whole cells.
Related to Figure 4—figure supplement 1.
Figure 4—figure supplement 1—source data 2. Effective diffusion of cytGEMs and nucGEMs in whole cells.
Related to Figure 4—figure supplement 1.
Figure 4—figure supplement 1—source data 3. cytGEMs MSD plots and anomalous diffusion exponent in protoplasts.
Related to Figure 4—figure supplement 1.