Temperature-reversible eruptions of vesicles in model membranes studied by NMR

Deuterium (2H) and phosphorus (31P) nuclear magnetic resonance (NMR) and freeze-fracture electron microscopy were used to study spontaneous vesiculation in model membranes composed of POPC:POPS with or without cholesterol. The NMR spectra indicated the presence of a central isotropic line, the intensity of which is reversibly and linearly dependent upon temperature in the L alpha phase, with no hysteresis when cycling between higher and lower temperatures. Freeze-fracture microscopy showed small, apparently connected vesicles that were only present when the samples were frozen (for freeze-fracture) from an initial temperature of 40-60 degrees C, and absent when the samples are frozen from an initial temperature of 20 degrees C. Analysis of motional narrowing was consistent with the isotropic lines being due to lateral diffusion in (and tumbling of) small vesicles (diameters approximately 50 nm). These results were interpreted in terms of current theories of shape fluctuations in large unilamellar vesicles which predict that small daughter vesicles may spontaneously "erupt" from larger parent vesicles in order to expel the excess area created by thermal expansion of the bilayer surface at constant volume. Assuming that all the increased area due to increasing temperature is associated with the isotropic lines, the NMR results allowed a novel estimate of the coefficient of area expansion alpha A in multilamellar vesicles (MLVs) which is in good agreement with micromechanical measurements upon giant unilamellar vesicles of similar composition. Experiments performed on unilamellar vesicles, which had been placed upon glass beads, confirmed that alpha A determined in this way is unchanged compared with the MLV case. Addition of the highly positively charged (extrinsic) myelin basic protein (MBP) to a POPC:POPS system showed that membrane eruptions of the type described here occur in response to the presence of this protein.