Cholesterol depletion induces large scale domain segregation in living cell membranes

Abstract

Local inhomogeneities in lipid composition play a crucial role in regulation of signal transduction and membrane traffic. Nevertheless, most evidence for microdomains in cells remains indirect, and the nature of membrane inhomogeneities has been difficult to characterize. We used lipid analogs and lipid-anchored proteins with varying fluidity preferences to examine the effect of modulating cellular cholesterol on domain formation. We show that lowering cholesterol levels induces formation of visible micrometer-scale domains in the plasma membrane of several mammalian cell types with complementary distributions of fluorescent lipid analogs with preferences for fluid or ordered domains. A uniform distribution is restored by cholesterol repletion. Unexpectedly, cholesterol depletion does not visibly alter the distribution of a crosslinked or uncrosslinked glycosylphosphatidylinositol-anchored protein (the folate receptor). We also examined the effect of varying cholesterol content on the cold Triton X-100 solubility of several membrane constituents. Although a cholesterol analog, dehydroergosterol, and a glycosylphosphatidylinositol-anchored protein are largely retained after extraction, a lipid analog with saturated 16-carbon acyl chains is largely removed when the cellular cholesterol level is lowered. This result indicates that after cholesterol depletion molecules in the more ordered domains can be extracted differentially by cold nonionic detergents.

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

“…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). In contrast, it has been proposed that cholesterol depletion causes a drop in the PI(4,5)P 2 -level, which in turn affects the cortical actin cytoskeleton and thus lowers lateral mobility of membrane components (37).…”

“…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).…”

Cytoskeletal Modulation of Lipid Interactions Regulates Lck Kinase Activity

“…1A. Specifically, the C 16 tail targets the probes to domains composed of ordered lipids, whereas C 12 -anchored probes favor less-ordered L d phase lipid environments (28,29). Accordingly, rafts composed of ordered lipids co-label with C 16 -anchored DiO (C 16 -DiO) and C 16 -anchored DiI (C 16 -DiI), minimizing their intermolecular distance to increase the FRET efficiency relative to that between C 16 -DiO and C 12 -anchored DiI (C 12 -DiI) (21).…”

Limited cholesterol depletion causes aggregation of plasma membrane lipid rafts inducing T cell activation

“…That cholesterol depletion can lead to large scale membrane domain segregation has been shown in several cell types containing caveolae [53]. However, unlike the present study, where the lipid raft marker GM1 stained with CT--B resided in large aggregates, after limited cholesterol depletion, the folate receptor, a GPI--anchored protein and thus also a lipid raft marker, only formed small aggregates [53]. It could be that the levels of cholesterol depletion differed or that there is a limit to the maximum domain size a receptor destined for internalisation could reside in or that only a fraction of GPI--anchored proteins form clusters [54].…”

“…This would also explain why cholesterol loading did not result in T cell signalling and why cholesterol loading does not trigger phase separation [52]. That cholesterol depletion can lead to large scale membrane domain segregation has been shown in several cell types containing caveolae [53]. However, unlike the present study, where the lipid raft marker GM1 stained with CT--B resided in large aggregates, after limited cholesterol depletion, the folate receptor, a GPI--anchored protein and thus also a lipid raft marker, only formed small aggregates [53].…”