Membrane Domain Distributions: Analysis of Fluorescence Sterol Ex-change Kinetics

Abstract

It is well known that cholesterol is localized in a non-random distribution within and across biological membranes. The importance of the cholesterol-enriched domains, also termed rafts, is evident from the fact that non-receptormediated cholesterol uptake and reverse cholesterol transport also occur through select plasma membrane domains. However, despite much effort to resolve the mechanisms that explain the origin, function, and regulation of membrane cholesterol lateral and transbilayer asymmetric distribution into domains, these phenomena remain largely unresolved. As well, progress in understanding the pathways of intracellular cholesterol trafficking, and the ultimate cellular fate of that cholesterol, has been sparse. Understanding the above-named phenomena and processes is itself crucial to resolving the molecular mechanisms of cholesterol uptake, reverse cholesterol transport, modulation of membrane function, and steroidogenesis. These ongoing efforts to elucidate the nature of cholesterol distribution and dynamics within the cell, have necessitated devising new ways to investigate the trafficking of cholesterol. To that end, fluorescent kinetic exchange assays have been developed to probe the nature of sterol transfer between biological membranes, i.e. endoplasmic reticulum, lysosomes, mitochondria, plasma membrane, and caveolae/lipid rafts (i.e., distinct sub-domains of the plasma membrane). These exchange assays make use of spectroscopic properties, such as polarization, to investigate the nature and distribution of sterol within biological membranes. These assays demonstrate that: cholesterol is distributed within the plane of biomembrane layers into dynamic and static domains, with the latter predominating, and that regulation of the size and kinetics of biomembrane cholesterol domains might be determining factors in intracellular cholesterol trafficking, targeting, and efflux.