Cholesterol Depletion from a Ceramide/Cholesterol Mixed Monolayer: A Brewster Angle Microscope Study

Abstract

Cholesterol is crucial to the mechanical properties of cell membranes that are important to cells' behavior. Its depletion from the cell membranes could be dramatic. Among cyclodextrins (CDs), methyl beta cyclodextrin (MβCD) is the most efficient to deplete cholesterol (Chol) from biomembranes. Here, we focus on the depletion of cholesterol from a C16 ceramide/cholesterol (C16-Cer/Chol) mixed monolayer using MβCD. While the removal of cholesterol by MβCD depends on the cholesterol concentration in most mixed lipid monolayers, it does not depend very much on the concentration of cholesterol in C16-Cer/Chol monolayers. The surface pressure decay during depletion were described by a stretched exponential that suggested that the cholesterol molecules are unable to diffuse laterally and behave like static traps for the MβCD molecules. Cholesterol depletion causes morphology changes of domains but these disrupted monolayers domains seem to reform even when cholesterol level was low.

Figure 1

(A) Molecular structure of C16 Ceramide; Cholesterol and MβCD. (B) Schematic of BAM Experimental Set-up.

Figure 2. BAM images of C16 Cer/Chol mixed Langmuir film, capturing the monolayer morphology for different mole% of Chol, before and after the MβCD-injection.

For each mole% of Chol, we present the monolayer at three different surface pressures: (i) very low~1 mN/m (ii) Just before MβCD-injection, ~30 mN/m, (iii) After surface pressure decay due to depletion, ~12 mN/m. In the last case, note the disrupted monolayer domains due to Chol-depletion.

Figure 3

Effect of MβCD-injection in subphase: Surface pressure vs.Time for different mole% of choesterol: (a) The monolayer is compressed to ~32 mN/m (b) The monolayer is compressed to a pressure around 10 mN/m. (c) Fit of the surface pressure to a stretched exponential. (d) Decay constants (τ) vs. mole% of Chol, obtained form (c). (e) Fit parameter “β” for different mole%, obtained form (c). (f) Jump in surface pressures during domain restoration.

Figure 4

(a) Surface pressure decay rate (dπ/dt) as a function of time, for varying mole% of Chol. (b) Distribution of (dπ/dt); showing the normal Gaussian distribution centered on ~0.25 mN/m.