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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.

SUBMITTER: Biswas A

PROVIDER: S-EPMC6486507 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The effect of cholesterol depletion on the activity of phosphatidylinositol/phosphatidylinositol 4-phosphate and diacylglycerol kinases and polyphosphoinositide phosphodiesterase has been studied in isolated membranes of human normal and cholesterol-depleted erythrocytes. Polyphosphoinositide synthesis (phosphatidylinositol/phosphatidylinositol 4-phosphate kinase activities) were found to depend on the permeability and sidedness characteristics of the membrane vesicles, which could limit the accessibility of ATP for the enzymes. When measured under proper conditions, phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate synthesis were decreased in cholesterol-depleted membranes as compared with control membranes. The same level of synthesis could be obtained in both membranes by the addition of phosphatidylinositol (and Triton X-100) or of phosphatidylinositol 4-phosphate. Phosphatidic acid synthesis (diacylglycerol kinase activity) was also decreased in cholesterol-depleted membranes as compared with control membranes when measured in the presence of Ca2+. Addition of diolein (and Triton X-100) caused a large increase in phosphatidic acid synthesis which reached approximately the same level in both membranes. This showed that the apparent inhibition of polyphosphoinositide and phosphatidic acid synthesis was not due to a loss or to an inactivation of the kinases. Ca2+-activated polyphosphoinositide phosphodiesterase promoted the hydrolysis of 65-70% of the polyphosphoinositides in control and of only 45-55% in cholesterol-depleted membranes without changing the Ca2+ concentration for half-maximum hydrolysis (1 microM). Upon addition of sodium oleate, the extent of polyphosphoinositide hydrolysis became identical in both membranes, indicating again that there was no loss nor inactivation of the polyphosphoinositide phosphodiesterase in the cholesterol-depleted membranes. Since the concentration of the polyphosphoinositides was not changed by cholesterol depletion [Giraud, M'Zali, Chailley & Mazet (1984) Biochim. Biophys. Acta 778, 191-200], the reduction in both their synthesis and degradation observed here could be attributed to a reorganization of the phosphoinositides in membrane domains where they were not accessible to the kinases and phosphodiesterase. The reduction in phosphatidic acid synthesis was likely caused by a reduction in the total amount of the substrate diacylglycerol in cholesterol-depleted membranes as already shown [Giraud, M'Zali, Chailley & Mazet (1984) Biochim. Biophys. Acta 778, 191-200].

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

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