Cholesterol Depletion Suppresses the Translational Diffusion of Class II Major Histocompatibility Complex Proteins in the Plasma Membrane

Abstract

Glycosylphosphatidylinositol (GPI)-linked and native major histocompatibility complex class II I-E(k) were used as probes to determine the effect of varying cholesterol concentration on the mobility of proteins in the plasma membrane. These proteins were imaged in Chinese hamster ovary cells using single-molecule fluorescence microscopy. Observed diffusion coefficients of both native and GPI-linked I-E(k) proteins were found to depend on cholesterol concentration. As the cholesterol concentration decreases the diffusion coefficients decrease by up to a factor of 7 for native and 5 for GPI-linked I-E(k). At low cholesterol concentrations, after sphingomyelinase treatment, the diffusion coefficients are reduced by up to a factor of 60 for native and 190 for GPI-linked I-E(k). The effect is reversible on cholesterol reintroduction. Diffusion at all studied cholesterol concentrations, for both proteins, appears to be predominantly Brownian for time lags up to 2.5 s when imaged at 10 Hz. A decrease in diffusion coefficients is observed for other membrane proteins and lipid probes, DiIC12 and DiIC18. Fluorescence recovery after photobleaching measurements shows that the fraction of immobile lipid probe increases from 8 to approximately 40% after cholesterol extraction. These results are consistent with the previous work on cholesterol-phospholipid interactions. That is, cholesterol extraction destroys liquid cholesterol-phospholipid complexes, leaving solid-like high melting phospholipid domains that inhibit the lateral diffusion of membrane components.

The effect of cellular cholesterol on membrane-cytoskeleton adhesion

“…5B) cholesterol depletion increases the immobile fraction of HLA I proteins (Kwik et al, 2003) and decreases the diffusion coefficient of both raft and non-raft proteins (Kenworthy et al, 2004;Vrljic et al, 2005). Cholesterol enrichment was shown to have no effect on the lateral diffusion coefficients of either membrane proteins or membrane lipids (Kenworthy et al, 2004;Vrljic et al, 2005). Also, consistently with the earlier studies, disassembly of the actin network abrogated the differences in the diffusion coefficients between cholesteroldepleted, cholesterol-enriched and control cells (Fig.…”

“…Our study shows that after cholesterol depletion, the lateral diffusion of DiIC 12 decreased by about 30%, whereas cholesterol enrichment had no significant effect (Fig. 5B) cholesterol depletion increases the immobile fraction of HLA I proteins (Kwik et al, 2003) and decreases the diffusion coefficient of both raft and non-raft proteins (Kenworthy et al, 2004;Vrljic et al, 2005). Cholesterol enrichment was shown to have no effect on the lateral diffusion coefficients of either membrane proteins or membrane lipids (Kenworthy et al, 2004;Vrljic et al, 2005).…”

The endocytic pathway and therapeutic efficiency of doxorubicin conjugated cholesterol-derived polymers

“…Consistent with previous MβCD treatments, the cell morphologies slightly changed, which was attributed to cholesterol depletion and rearrangement of the cytoskeleton. 41 Based on the cell viability data and confocal images 2.5 mM MβCD for 2 hours was the preferential settings for inhibiting CIE. In accordance with MβCD treatments, genistein also blocked LacCer uptake to the cells ( Fig.…”

“…Polymer uptake was governed solely by their hydrophobic moieties which is very common with lipidbased systems. 10,[40][41][42] It should be mentioned that a preliminary quantitative analysis on inhibitory effects was undertaken by flow cytometry. However, the assay was not feasible as the desired amount of Alexa Fluor labelled polymer, for sufficient detection via flow cytometry, was too large to chemically synthesize in a cost-effective and efficient manner.…”

Single-particle tracking of murine polyoma virus-like particles on live cells and artificial membranes

“…That cholesterol depletion would lead to immobilization of plasma membrane components is not unprecedented in the literature. A reduction in lateral mobility of several glycosylphosphatrdylinositolanchored proteins, transmembrane proteins, and lipids has been reported (35)(36)(37)(38). One mechanism put forward to explain the effect is actin-independent, reversible agglutination of lipids into large, stable, ordered lipid domains (35,36,38).…”