Endocytosis, excretory processes, cell division, and membrane biogenesis are some of the many cell functions thought to require local disruption of the basic lipid bilayer structure of cell membranes. The proposed research will focus on two related processes in well-defined phospholipid vesicles: poly(ethylene glycol) (PEG)-induced membrane fusion and Mn2+ -induced phosphatidylglycerol (PG) transbilayer migration. Results from the previous funding period have suggested that both of these events proceed through an intermediate state of intimately juxtaposed bilayers. This proposal aims to provide direct evidence for close approach of two bilayers and, in addition, to define the structural mechanism responsible for bilayer destabilization in the juxtaposed state. The experimental approach will be to relate the kinetics of the fusion and transbilayer migration processes to the structural features of different model membranes in which these processes occur. Since the structural intermediates being sought are transitory in nature, coordinated kinetic and structural analysis will be a central and unique element of the research. Kinetics of vesicle fusion will be followed using fluorescence-intensity and - lifetime-based assays for the mixing of vesicle components and trapped contents. PG transbilayer migration will be followed using a chemical assay for surface PG. Data obtained at different vesicle concentrations will be analyzed in terms of a mass action model to obtain independent rate constants for reversible vesicle aggregation as well as for the irreversible events associated with fusion or transbilayer lipid migration. Membrane structure will be examined by differential scanning calorimetry to monitor membrane phase behavior, and frequency-resolved fluroescence to characterize the dynamic behavior of membrane probes.
An aim of the fluorescence measurements will be to resolve a probe subpopulation reflective of the destabilized state. Interbilayer contact will be detected directly by X-ray diffraction and freeze- fracture electron microscopy. Results of these experiments will be used to distinguish between several possible structural mechanisms that might account for local bilayer destabilization in membranes, such as formation of local """"""""hexagonal"""""""" phase, lateral domain formation, locally enhanced membrane curvature, or bilayer hydration layer overlap.
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