COLL 139 |
| Steven P. Wrenn1, Gregory M. Troup1, Sum P. Lee2, and Thomas N. Tulenko3. (1) Chemical Engineering Dept, Drexel University, 481 CAT Bldg, 3141 Chestnut Street, Philadelphia, PA 19104, (2) School of Medicine, University of Washington, Seattle, WA 98195, (3) School of Medicien, Thomas Jefferson University, Philadelphia, PA 19106 |
| We present evidence that laterally phase separated cholesterol nano-domains constitute a new, equilibrium phase in biological membranes. The domains are characterized in multi-lamellar vesicles made of cholesterol and dimyristoylphosphatidylcholine (DMPC) but are also shown to exist in biologically relevant, egg lecithin systems containing a mixture of phospholipids. This work utilizes the fluorescent membrane probes 1-acyl-2-[12-[(5-dimethylamino-1-naphthalenesufonyl)amino]dodecanoyl]-sn-Glycero-3-phosphocholine (DANSYL), and ergosta-5,7,9(11),22-tetraen-3b-ol (ERGO), which have been shown to be minimally-invasive mimics of native membrane lipids. The highlight of the work is a heating-induced alleviation of a DANSYL blue shift at relatively high (but undersaturated) cholesterol loadings, which is reversible through at least three heating and cooling cycles. Comparison of the DANSYL spectral shifts with published DMPC-cholesterol phase diagrams shows that the spectral results cannot be explained in terms of previously understood phase behavior. Rather, a lateral phase separation occurs within the vesicle bilayer, giving rise to cholesterol nano-domains. The cholesterol domains appear to coexist with, and should not be confused with, the well-known liquid-order phase that arises because of the cholesterol condensation effect. Additional studies involving ERGO-to-DANSYL energy transfer show a sequestration of probes within the bilayer, confirming the DANSYL spectral data, and a model that includes domains provides the best description of measured energy transfer efficiencies. Best fits of the energy transfer data, using a mathematical model developed to account for the presence of domains, indicates the domain size to be in the range 10-20 nm. |
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Nanoscale Imaging of Biological Systems
Division of Colloid and Surface Chemistry |