Cholesterol organization in membranes at low concentrations: effects of curvature stress and membrane thickness

Cholesterol is often found distributed nonrandomly in domains in biological and model membranes and has been reported to be distributed heterogeneously among various intracellular membranes. Although a large body of literature exists on the organization of cholesterol in plasma membranes or membranes with high cholesterol content, very little is known about organization of cholesterol in membranes containing low amounts of cholesterol. Using a fluorescent cholesterol analog (25-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-methyl]amino]-27-norcholesterol, or NBD-cholesterol), we have previously shown that cholesterol may exhibit local organization even at very low concentrations in membranes, which could possibly be attributable to transbilayer tail-to-tail dimers. This is supported by similar observations reported by other groups using cholesterol or dehydroergosterol, a naturally occurring fluorescent cholesterol analog which closely mimics cholesterol. In this paper, we have tested the basic features of cholesterol organization in membranes at low concentrations using spectral features of dehydroergosterol. More importantly, we have investigated the role of membrane surface curvature and thickness on transbilayer dimer arrangement of cholesterol using NBD-cholesterol. We find that dimerization is not favored in membranes with high curvature. However, cholesterol dimers are observed again if the curvature stress is relieved. Further, we have monitored the effect of membrane thickness on the dimerization process. Our results show that the dimerization process is stringently controlled by a narrow window of membrane thickness. Interestingly, this type of local organization of NBD-cholesterol at low concentrations is also observed in sphingomyelin-containing membranes. These results could be significant in membranes that have very low cholesterol content, such as the endoplasmic reticulum and the inner mitochondrial membrane, and in trafficking and sorting of cellular cholesterol.