The Development and Characterization of Scaffolded Vesicles as a Model Membrane

Grossutti, Michael
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University of Guelph

A novel model biological membrane system was developed and characterized. The general structure of the model was that of a giant vesicle supported by a spherical hydrogel scaffold. This model has been termed a scaffolded vesicle. Four different scaffolded vesicle systems were examined. Scaffolded vesicles consisting of a 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and cholesterol membrane in a 70:30 molar ratio supported on Sephadex G10 hydrogel beads were studied using ATR-IR spectroscopy and fluorescence techniques. The DMPC acyl chains were found to have a tilt angle of ~21°-25°, consistent with a bilayer structure. Fluorescence microscopy studies revealed that these scaffolded vesicles lacked durability. Fluorescence spectroscopic membrane permeability studies found that the DMPC:cholesterol coating acted as a permeability barrier to some degree, but exhibited high permeability near the main phase transition temperature of the membrane. In order to overcome these difficulties, three scaffolded vesicle systems were also developed using Sephadex A50 as the hydrogel support. The membrane compositions were as follows: 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC), DPhPC with 10 mol% 1,2-diphytanoyl-sn-glycero-3-phospho-L-serine (DPhPS), and a DMPC, cholesterol, ganglioside GM1 membrane in a 40:30:30 DMPC:cholesterol:GM1 molar ratio. Each of these scaffolded vesicles were characterized using ATR-IR spectroscopy, fluorescence microscopy, fluorescence quenching, fluorescence recovery after photobleaching (FRAP), and electrophysiological techniques. These techniques provided complementary information regarding the structure and properties of the membranes studied. The DPhPC scaffolded vesicle coating was found to be a single bilayer. However, this bilayer lacked lateral lipid mobility. The 90:10 DPhPC:DPhPS scaffolded vesicles were found to have a multibilayer membrane structure with a mobile fraction (0.61) and diffusion coefficient of 1.4 x 10-9 cm2/s. The 40:30:30 DMPC:cholesterol:GM1 scaffolded vesicle coating was found to be a single bilayer, lacking lateral mobility. The molecular order and orientation of the DMPC and GM1 acyl chains were in close agreement with one another. The tilt angle of the DMPC acyl chains increased ~10° relative to the 70:30 DMPC:cholesterol scaffolded vesicles.

biological membranes, model membranes, attenuated total reflection infrared spectroscopy, fluorescence, patch-clamp