Sterol Structure Determines Miscibility versus Melting Transitions in Lipid Vesicles

Abstract

Lipid bilayer membranes composed of DOPC, DPPC, and a series of sterols demix into coexisting liquid phases below a miscibility transition temperature. We use fluorescence microscopy to directly observe phase transitions in vesicles of 1:1:1 DOPC/DPPC/sterol within giant unilamellar vesicles. We show that vesicles containing the "promoter" sterols cholesterol, ergosterol, 25-hydroxycholesterol, epicholesterol, or dihydrocholesterol demix into coexisting liquid phases as temperature is lowered through the miscibility transition. In contrast, vesicles containing the "inhibitor" sterols androstenolone, coprostanol, cholestenone, or cholestane form coexisting gel (solid) and liquid phases. Vesicles containing lanosterol, a sterol found in the cholesterol and ergosterol synthesis pathways, do not exhibit coexisting phases over a wide range of temperatures and compositions. Although more detailed phase diagrams and precise distinctions between gel and liquid phases are required to fully define the phase behavior of these sterols in vesicles, we find that our classifications of promoter and inhibitor sterols are consistent with previous designations based on fluorescence quenching and detergent resistance. We find no trend in the liquid-liquid or gel-liquid transition temperatures of membranes with promoter or inhibitor sterols and measure the surface fraction of coexisting phases. We find that the vesicle phase behavior is related to the structure of the sterols. Promoter sterols have flat, fused rings, a hydroxyl headgroup, an alkyl tail, and a small molecular area, which are all attributes of "membrane active" sterols.

Yeast Lipids Can Phase-separate into Micrometer-scale Membrane Domains

“…(i) In the presence of SM, replacement of cholesterol by ergosterol results in a membrane order that is as high as the order of membranes of the well characterized raft mixture SM/PC/cholesterol. This result confirms previous reports showing the property of ergosterol to have a condensing effect on glycerophospholipids (43)(44)(45)(46). (ii) Yeast PI increases the order of membranes containing IPC, irrespective of whether cholesterol or ergosterol is used as the sterol in these mixtures.…”

“…Ergosterol differs from cholesterol in additional double bonds in the B ring and the hydrocarbon side chain and an additional methylation at C24, the latter two making the side chain stiffer and bulkier, respectively (59). These structural attributes were proposed to restrict the motions of acyl chains more efficiently and thus lead to stronger ordering by ergosterol as compared with cholesterol (43)(44)(45)(46). As judged from the higher order of IPC/ergosterol membranes, this effect is more pronounced for IPC, whereas the sterol structure does not seem to be critical for the ordering of C18-SM-containing bilayers (Fig.…”

Cholesterol homeostasis and the escape tendency (activity) of plasma membrane cholesterol

“…While the studies just mentioned document the particular ability of cholesterol to promote the formation of condensed liquid-ordered phases, these publications also provide evidence that various sterols (biosynthetic intermediates and otherwise) are as good as and sometimes better than cholesterol at building rafts [26,49,50,88,89,91]. In this set are ergosterol, 25-hydroxycholesterol, lathosterol, epicholesterol, 7-dehydrocholesterol and dihydrocholesterol.…”

“…Sterols deficient in this capacity include lanosterol, androstenolone, coprostanol, cholestane and cholest-4-en-3-one (i.e., cholestenone) [26,49,50]. There is evidence that sterols that fail to promote liquid-liquid phase separation nevertheless form phospholipid complexes that are presumably miscible with the bilayer continuum (Ratajczak, M.K., Steck, T.L., Lee, K-Y.…”

“…The data are generally in line with Bloch's hypothesis. They show that, compared to cholesterol, androstenolone, coprostanol, cholestane and cholestenone have weak or even negative effects on domain formation [26,49,50,88]. The same is true for lanosterol and succeeding sterol intermediates: dihydrolanosterol, desmosterol, zymosterol, and zymostenol [89].…”