Cholesterol Depletion by MβCD Enhances Cell Membrane Tension and Its Variations-Reducing Integrity

Abstract

Cholesterol depletion by methyl-β-cyclodextrin (MβCD) remodels the plasma membrane's mechanics in cells and its interactions with the underlying cytoskeleton, whereas in red blood cells, it is also known to cause lysis. Currently it's unclear if MβCD alters membrane tension or only enhances membrane-cytoskeleton interactions-and how this relates to cell lysis. We map membrane height fluctuations in single cells and observe that MβCD reduces temporal fluctuations robustly but flattens spatial membrane undulations only slightly. Utilizing models explicitly incorporating membrane confinement besides other viscoelastic factors, we estimate membrane mechanical parameters from the fluctuations' frequency spectrum. This helps us conclude that MβCD enhances membrane tension and does so even on ATP-depleted cell membranes where this occurs despite reduction in confinement. Additionally, on cholesterol depletion, cell membranes display higher intracellular heterogeneity in the amplitude of spatial undulations and membrane tension. MβCD also has a strong impact on the cell membrane's tenacity to mechanical stress, making cells strongly prone to rupture on hypo-osmotic shock with larger rupture diameters-an effect not hindered by actomyosin perturbations. Our study thus demonstrates that cholesterol depletion increases membrane tension and its variability, making cells prone to rupture independent of the cytoskeletal state of the cell.

Figure 1

Effect of MβCD-mediated cholesterol depletion on membrane topology. (a) Left: representative fluorescence images of Filipin III-stained control and MβCD-treated HeLa cells are shown. Right: shown is a box plot of mean intensity of Filipin III in cells in mentioned conditions (N = 30 cells each). Center lines of boxes show the medians; stars show the means; box limits indicate the 25th and 75th percentiles; and whiskers extend 1.5 times the interquartile range from the 25th to 75th percentiles. ^∗∗p < 0.001, Mann-Whitney U test. (b) Shown are representative DIC (first), IRM (second) images, kymographs of regions overlaid on the images in red (third, scale bars in y, 16 s, scale bars in x, 5 μm) and corresponding SD_time maps of control versus MβCD-treated cells (non-FBRs blacked out). Scale bars, 10 μm. See Table S3 for statistics. To see this figure in color, go online.

Figure 2

Cholesterol depletion by MβCD reduces temporal fluctuations and enhances membrane tension. (a) Shown are averaged PSDs of FBRs in MβCD-treated cells and their controls (solid lines) with their backgrounds (dashed lines); inset shows f (ratio of background subtracted PSDs). (b) Left: Shown are box plots of $\overline{\sigma \left(f_1,f_2\right)}$ in two different frequency regimes. Right: exponent in the two conditions is shown. N = 70 cells each. (c) Left: single FBR statistics of SD_space in control and cholesterol-depleted cells. n_control = 1683 FBRs, n_MβCD = 1471 FBRs, and N = 70 cell each. Right: correlation lengths are shown. (d) Single cell statistics of SD_space (left) and correlation lengths (right) in the two conditions are shown. N = 70 cell each. (e) Shown are box plots of mechanical parameters A, η_eff, γ, and σ in control and MβCD-treated cells. N = 70 cell each. ^∗p < 0.05, ^∗∗p < 0.001, Mann-Whitney U test. See Fig. S1 and Document S1. Supporting Discussion, Figs. S1–S10, and Tables S1–S2, Table S3. Statistical Parameters for Data Presented in Main Figures for statistics. To see this figure in color, go online.

Figure 3

MβCD treatment does not abrogate signatures of activity. (a) Top: shown are the typical temporal ACFs of single 2 × 2 pixels FBRs in control and MβCD-treated cells, along with their fits to a three-term exponential function. Bottom: shown is the weighted distribution of correlation timescales (τ) obtained from the temporal ACFs. The green arrow marks the timescales for active fluctuations. Inset shows a plot of the fraction of curves with features versus threshold used to detect the features (n_control = 2890 fits, n_MβCD = 3071 fits, and N = 21 cells each). (b) Shown are FBRs overlaid in yellow on IRM images and their corresponding whole cell (Scale bars, 10 μm) and FBR (Scale bars, 1 μm) p value maps (Kolmogorov-Smirnov hypothesis testing). Right: Shown is the p value for FBRs in control versus cholesterol-depleted cells. n_control = 53,568 pixels, and n_MβCD = 42,480 pixels. (c) Shown are the representative IRM images of control, ATP dep., and ATP dep. + MβCD cells. Scale bars, 10 μm. (d) Box plot for SD_time (left) and σ (middle) for these conditions. N = 10 cells. Right: Shown are single FBR statistics of σ in the three conditions. nc_ontrol = 305 FBRs, n_ATPdep. = 207 FBRs, n_{ATPdep.+MβCD} = 229 FBRs, and N = 10 cells each. ^∗p < 0.05, ^∗∗p < 0.001, ns p > 0.05, Mann-Whitney U test. See Fig. S3 and Document S1. Supporting Discussion, Figs. S1–S10, and Tables S1–S2, Table S3. Statistical Parameters for Data Presented in Main Figures for statistics. To see this figure in color, go online.

Figure 4

Enhanced spatial and mechanical intracellular heterogeneity on MβCD treatment. (a) Intra-FBR fluctuations heterogeneity measured by SD (SD_time) for the two conditions are shown. (b) Shown are intracellular long-range fluctuations heterogeneity measured by box plots of the number of dissimilar FBR pairs. Dissimilarity is evaluated by comparing SD_time. N = 70 cells each. (c) Lines in magenta and black connect FBRs that are dissimilar and similar in SD values, respectively. The lines are overlaid on the SD_time maps in representative control and MβCD-treated cells. Each node in the maps represents the center of an FBR. Scale bars, 10 μm. (d) Shown are histograms and box plots of intracellular heterogeneity of cell averaged SD_time, SD_space, and σ in the two conditions. N = 70 cells each. (e) Representative single pixel maps of tension (σ) and goodness of fit (R²) for a control and MβCD-treated cell are shown. The white dashed line marks the boundary of the cell. Fitting was performed for pixels inside this boundary. Scale bars, 10 μm. ^∗p < 0.05, ^∗∗p < 0.001, ns p > 0.05, Mann-Whitney U test. See Figs. S1 and S4 and Document S1. Supporting Discussion, Figs. S1–S10, and Tables S1–S2, Table S3. Statistical Parameters for Data Presented in Main Figures for statistics. To see this figure in color, go online.

Figure 5

Membrane rupture induced by hypo-osmotic/iso-osmotic medium in the presence of MβCD. (a) Top: shown are DIC and epifluorescent images of Calcein AM-labeled HeLa cells before and after the administration of a 95% hypo-osmotic shock (Scale bars, 100 μm). Among others, the cells in the box are representatives of membrane rupture. Bottom: shown is a box plot of rupture propensity of cells due to change in hypo-osmotic stress, change in temperature, and ATP depletion (N = 3 experiments each). (b) Top: shown are time lapse images of Calcein AM-loaded HeLa cells undergoing rupture (arrowheads in red) (Scale bars, 50 μm). (c) (i) Shown is the intensity and ratio $\left(Intensityofframe/Intensityofprevio\text{us}\text{frame}\right)$ map of a rupturing cell followed in time to show single point rupture. (ii) Shown are representative ratio maps of six different cells showing an asymmetric spread of fluorescence after rupture (Scale bars, 30 μm). (d) A time profile of normalized mean intensity of a ruptured cell is shown; inset shows the double exponential fit to the profile. (e) Shown are box plots of rupture propensity (left) and rupture diameter (right) of 95% hypo-osmotic shock administered HeLa cells under control, Cyto D (2 h post-treatment), MβCD (50 min post-treatment), and Cyto D + MβCD conditions (N = 3 experiments each). (f) Shown are box plots of rupture propensity of cells under Cyto D, MβCD, and dual drug treatments (N = at least three experiments each) in the absence of hypo-osmotic shock. “+” denotes 60 min treatment (for Cyto D) and 50 min (for MβCD), and “++” denotes 120 min treatment. ^∗p < 0.05, ^∗∗p < 0.001, ns p > 0.05, Mann-Whitney U test. See Fig. S7 and Table S3 for statistics. To see this figure in color, go online.