The work undertaken in this thesis was directed towards understanding the organization of lipids in cell membranes and the lipid specificity of perforin. Three aspects of the organization of membrane lipids were investigated, (1) the asymmetrical distribution of lipids between the outer and inner leaflets of the membrane, (2) the bobbing motion of lipids perpendicular to the plane of the membrane, and (3) the spacing of lipids in the external leaflet of the membrane. The binding of perforin to membranes was studied by (1) comparing the ability of liposomes constructed from defined lipids to bind perforin, and (2) the ability of perforin to bind to and lyse lipid symmetric and lipid asymmetric erythrocyte membranes. The asymmetrical distribution of lipids in the plasma membrane of erythrocytes was investigated by examining two mechanisms for maintenance of lipid asymmetry: binding of internal lipids to cytoskeletal proteins, and pumping of internal lipids from the outside to the inside of the cell. Analysis of the kinetics of lipid internalization suggested the presence of an ATP-dependent lipid flip/flop catalyst. The thermal energy of lipids results in oscillatory motion perpendicular to the plane of the membrane, and in movement in the plane of the membrane. The vertical distribution of lipids arising from this oscillatory motion was calculated. The motion of lipids in the plane of the membrane results in an average spacing between the lipid molecules. Previous reports, had demonstrated that the fluorescent dye merocyanine 540 bound preferentially to widely spaced phosphatidylcholine lipids. MC540 was shown to have little headgroup, sidechain or cholesterol specificity per se, but bound preferentially to membranes whose lipids are widely spaced, suggesting its use as a probe of lipid spacing. Comparison of perforin binding to liposomes constructed from defined lipids revealed that perforin bound preferentially to liposomes whose lipids were widely spaced. Using erythrocytes as a model, it was observed that increasing the spacing of lipids in the outer leaflet of the plasma membrane resulted in increased perforin binding and susceptibility to perforin-mediated lysis, confirming the conclusions derived from the liposome studies and further suggesting that increased lipid spacing increases overall susceptibility to the more complex processes involved in cellular lysis by perforin. The external phospholipids of the plasma membrane of the CTL were shown to be more tightly packed than the lipids of the more-susceptible target cell, suggesting that the resistance of CTL to perforin is at least in part due to the presence of closely spaced lipids in its membrane.
| Identifer | oai:union.ndltd.org:UMASS/oai:scholarworks.umass.edu:dissertations-8052 |
| Date | 01 January 1991 |
| Creators | Antia, Rustom |
| Publisher | ScholarWorks@UMass Amherst |
| Source Sets | University of Massachusetts, Amherst |
| Language | English |
| Detected Language | English |
| Type | text |
| Source | Doctoral Dissertations Available from Proquest |
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