The long-term goal of the proposed research is to understand the mechanisms of membrane fusion. I propose to develop a new system for measuring the forces involved in the early stages of fusion between two lipid bilayers using atomic force microscopy (AFM). The general strategy is to produce a lipid bilayer on a solid support and coat the tip of an AFM with a second lipid bilayer, bring the two bilayers together, and apply fore to the bilayer-bilayer contact until fusion is initiated. Preliminary experiments indicate that for certain lipids there is at least fusion of the outer leaflets, thus allowing the initial stages of fusion to be examined. This system will provide the basis for a long-term project to study the mechanisms of membrane fusion and the factors that control fusion.
The first aim of the research is to develop routine and robust methods for preparing lipid coated surfaces appropriate for the proposed experiments.
The second aim i s to demonstrate that membrane fusion between the outer leaflets occurs and measure the force associated with fusion.
The third aim i s to investigate the effect of geometry and strain on the force of fusion. In the long-term this system will allow mechanisms of fusion to be studied by varying a range of parameters such as lipid type, ionic conditions, temperature, and interaction geometry. It may also become possible to incorporate proteins that facilitate fusion into the system.
