Abstract
The formation and properties of liquid-ordered (Lo) lipid domains (rafts) in the plasma membrane are still poorly understood. This limits our ability to manipulate ordered lipid domain-dependent biological functions. Giant plasma membrane vesicles (GPMVs) undergo large-scale phase separations into coexisting Lo and liquid-disordered lipid domains. However, large-scale phase separation in GPMVs detected by light microscopy is observed only at low temperatures. Comparing Förster resonance energy transfer-detected versus light microscopy-detected domain formation, we found that nanodomains, domains of nanometer size, persist at temperatures up to 20°C higher than large-scale phases, up to physiologic temperature. The persistence of nanodomains at higher temperatures is consistent with previously reported theoretical calculations. To investigate the sensitivity of nanodomains to lipid composition, GPMVs were prepared from mammalian cells in which sterol, phospholipid, or sphingolipid composition in the plasma membrane outer leaflet had been altered by cyclodextrin-catalyzed lipid exchange. Lipid substitutions that stabilize or destabilize ordered domain formation in artificial lipid vesicles had a similar effect on the thermal stability of nanodomains and large-scale phase separation in GPMVs, with nanodomains persisting at higher temperatures than large-scale phases for a wide range of lipid compositions. This indicates that it is likely that plasma membrane nanodomains can form under physiologic conditions more readily than large-scale phase separation. We also conclude that membrane lipid substitutions carried out in intact cells are able to modulate the propensity of plasma membranes to form ordered domains. This implies lipid substitutions can be used to alter biological processes dependent upon ordered domains.
Highlights
The formation and properties of liquid-ordered (Lo) lipid domains in the plasma membrane are still poorly understood
We investigated the relationship between nanodomain and large-scale phase separation in giant plasma membrane vesicle (GPMV) and how the formation of both large-scale phases and nanodomains is affected by alteration of plasma membrane lipid composition
Using methods to modify plasma membrane composition, we found that both large-scale phase separation and nanodomain thermal stability respond to lipid composition in a fashion analogous to what has been previously reported for domain formation in artificial vesicles
Summary
The formation and properties of liquid-ordered (Lo) lipid domains (rafts) in the plasma membrane are still poorly understood. Lipid substitutions that stabilize or destabilize ordered domain formation in artificial lipid vesicles had a similar effect on the thermal stability of nanodomains and large-scale phase separation in GPMVs, with nanodomains persisting at higher temperatures than large-scale phases for a wide range of lipid compositions This indicates that it is likely that plasma membrane nanodomains can form under physiologic conditions more readily than large-scale phase separation. The lipids in GPMVs undergo a phase separation in which classical large-scale coexisting liquid-disordered (Ld) and liquidordered (Lo) phases form and are visualized by light microscopy These phases have properties very similar to those formed in phase-separating artificial vesicles with simple lipid mixtures in terms of their properties and association with specific proteins. These studies show that GPMVs are a highly useful system for examining the principles behind lipid domain formation in natural membranes
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