Date of Award

2007

Level of Access Assigned by Author

Campus-Only Thesis

Degree Name

Master of Science (MS)

Department

Physics

Advisor

Sam T. Hess

Second Committee Member

Donald Mountcastle

Third Committee Member

James McClymer

Abstract

Viral infection, bacterial infection, and certain diseases such as Alzheimer's and Parkinson's are known to involve specific cell membrane domains referred to as "rafts." Rafts, which also play a role in cellular signaling, endocytosis, and membrane trafficking, are sub-micrometer sized domains of the cell membrane enriched in saturated lipids and cholesterol. In this experiment, unsaturated and saturated lipids, cholesterol, and minute amounts (total < 0.5 mole%) of two different fluorescent dyes are used to create model cell membranes called Giant Unilamellar Vesicles (GUVs). These GUVs separate into two distinct phases, a liquid disordered (La) domain enriched in unsaturated lipids and a liquid ordered (L0) domain, analogous to the raft phase, enriched in saturated lipids and cholesterol. Equatorial cross sections of GUVs are imaged with confocal fluorescence microscopy and analyzed with custom programs. Three different GUV compositions were examined at various cholesterol concentrations, with equi-molar unsaturated lipid, dioleoylphosphatidylcholine (DOPC), and saturated lipid, egg sphingomyelin (ESM). Computer programs analyze the distribution of fluorescent dye intensity, the area fraction, and distribution of meridional curvature in each phase of the GUVs and line tension values at the phase boundary between the Ld and L0 domains. The results indicate that GUVs with higher concentrations of cholesterol feature significant negative curvature due to similar bending rigidities between the two phases, a large area fraction of L0 phase, and a wide distribution of line tension values in the range of 10"13N. The GUVs with the lowest cholesterol concentration contain very little negative curvature especially in the L0 phase, significantly less overall L0 area, and much lower fluorescent probe partitioning in the L0 phase. These results give some insight into the structure and shape of interior cell organelles such as the Golgi and the possible effects on rafts in live cells at various cholesterol concentrations.

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