Membrane fusion is an essential and characteristic function of all plant and animal cells. It involves a complex set of membrane interactions, few of which are understood in detail. The overall goals of this proposal are to understand membrane fusion in a simple lipid system so that the knowledge acquired can be applied to understanding biological membrane fusion, answering some fundamental problems of membrane electrostatics, and developing liposomes that efficiently deliver materials to cells. The proposal is prompted by the discovery that some o-alkyl-phosphtidylcholinium compounds, a new class of phospholipids with a net positive charge that very recently became available through another project, form giant vesicles that fuse with vesicles of anionic lipids. Because these vesicles can be manipulated electrophoretically under the fluorescence microscope, a unique opportunity arises to examine bilayer fusion in considerably greater detail than possible heretofore. Since this phenomenon was discovered, it has been found that a) bilayer vesicles can fuse without leakage, b) bilayers can undergo hemifusion (fusion of the two monolayers that come into contact when vesicles adhere to one another) in several morphologically different ways, c) the vesicle composition (kind and proportion of uncharged lipid included with charged lipid) has a controlling influence on the proportion of possible outcomes, i.e. whether fusion, one of the modes of hemifusion, or stable adhesion predominates, and d) the physical properties of cationic phospholipids change in fundamental ways when they are mixed with anionic lipids, in particular, the area occupied by a molecule in a monolayer is reduced and these mixtures can transiently form a three- dimensional pattern of interconnected bilayers (cubic phase). It is hypothesized that, in fusion of oppositely-charged vesicles, destabilization of the contact region between adherent membranes is due to charge neutralization, leading to reorganization of the contacting surfaces and fusion. Cationic lipids have been prepared that form phases which should promote fusion as well as in fluorescent versions that will facilitate monitoring details of membrane fusion.
The specific aims are: 1. To identify cationic phospholipids that are prone to formation of membrane-destabilizing phases under conditions of vesicle adhesion. 2. To characterize the process of fusion between negative vesicles and positive vesicles containing lipids identified as fusion-prone. 3. To measure membrane electrostatic properties relevant to these processes. 4. To apply information from aims 1-3 to fusion of liposomes with cells and to functional tests of viral membrane components.
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