Abstract
Plasma membrane-derived vesicles are a good model system for studies of membrane protein interactions in mammalian membranes. In this project, our goal is to characterize the lipid asymmetry in the vesicles over time. The phospholipid distribution in the plasma membrane is asymmetric. Phosphatidylserine (PS) is predominantly found in the inner leaflets of the intact plasma membrane. Exposure of PS on the outer leaflet of the plasma membrane can be detected by annexin V, which preferentially binds to negatively charged phospholipids like PS. An assay is developed to study the lipid asymmetry in plasma membrane-derived vesicles using fluorescently labeled annexin V. Cells are vesiculated and FITC labeled annexin V is added to vesicles at different time points, and imaged using a confocal microscopy. A matlab program is developed to quantitatively analyze the intensity of FITC on the vesicles. As a control, the vesicles are scrambled completely by subjecting to freeze-thaw cycles and imaged. The results suggest that lipid asymmetry is lost during the vesiculation process.
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