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Biophysical Journal 86:2910-2922 (2004)
© 2004 The Biophysical Society

Liquid Domains in Vesicles Investigated by NMR and Fluorescence Microscopy

S. L. Veatch *, I. V. Polozov {dagger}, K. Gawrisch {dagger} and S. L. Keller *

* Departments of Chemistry and Physics, University of Washington, Seattle, Washington; and {dagger} Laboratory of Membrane Biochemistry and Biophysics, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Rockville, Maryland

Correspondence: Address reprint requests to Sarah L. Keller, University of Washington, Dept. of Chemistry, Seattle, WA 98195-1700. Tel.: 206-543-9613; E-mail: slkeller{at}chem.washington.edu.

We use 2H-NMR, 1H-MAS NMR, and fluorescence microscopy to detect immiscibility in three particular phospholipid ratios mixed with 30% cholesterol: 2:1 DOPC/DPPC, 1:1 DOPC/DPPC, and 1:2 DOPC/DPPC. Large-scale (>>160 nm) phase separation into liquid-ordered (Lo) and liquid-crystalline (L{alpha}) phases is observed by both NMR and fluorescence microscopy. By fitting superimposed 2H-NMR spectra, we quantitatively determine that the Lo phase is strongly enriched in DPPC and moderately enriched in cholesterol. Tie-lines estimated at different temperatures and membrane compositions are based on both 2H-NMR observations and a previously published ternary phase diagram. 2H- and 1H-MAS NMR techniques probe significantly smaller length scales than microscopy experiments (submicron versus micron-scalp), and complex behavior is observed near the miscibility transition. Fluorescence microscopy of giant unilamellar vesicles shows micrometer-scale domains below the miscibility transition. In contrast, NMR of multilamellar vesicles gives evidence for smaller (~80 nm) domains just below the miscibility transition, whereas large-scale demixing occurs at a lower temperature, Tlow. A transition at Tlow is also evident in fluorescence microscopy measurements of the surface area fraction of ordered phase in giant unilamellar vesicles. Our results reemphasize the complex phase behavior of cholesterol-containing membranes and provide a framework for interpreting 2H-NMR experiments in similar membranes.




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