Degranulation is one of the most important response of neutrophils in inflammation and bacterial killing. Membrane fusion, which is required for degranulation, is a process which is virtually ubiquitous, as it must be employed by any cell engaged in endocytosis or exocytosis. Yet, fusion has been difficult to model in the laboratory and is consequently poorly understood. Several recent advances, such as isolation of endogenous fusogens and development of contents-mixing fusion assays have permitted an investigation of membrane fusion and secretion by human neutrophils. Working Hypothesis--Both membrane and cytosolic (annexin and non-annexin) components regulate fusion in neutrophils. We propose to study membrane fusion in neutrophils using a variety of established and novel techniques, emphasizing thoroughness and rigor, and minimizing assumptions. 1. Continued Isolation of Endogenous Neutrophil Fusogens Using a """"""""first principles"""""""" approach, we have isolated one endogenous protein fusogen from neutrophil cytosol. At least three more fusogenic activities have been found that require purification and identification. This will permit us to determine the potential of the cytosol to support degranulation. 2. DEVELOPMENT OF EXPERIMENTAL SYSTEMS FOR MEASURING GRANULE-LIPOSOME AND GRANULE-MEMBRANE FUSION We propose to develop contents-mixing assays suitable for studying the fusion of neutrophil specific granules (SG). This is necessary since lipid-mixing assays currently used to study fusion ar fraught with experimental limitations. These studies will allow us to explore the potential of neutrophil membranes to fuse. 3. USE OF """"""""MODEL"""""""", RECONSTITUTED GRANULES TO DISSECT FUSION We have found that isolated neutrophil granules, in contrast to plasma membrane (PM) vesicles, are extremely resistant to fusion. We therefore propose to isolate membrane-associated components from SG and reconstitute them into liposomes, to study the involvement of granule membrane components in fusion or regulating fusion. In particular, we will look at both the lipid and protein components of SG. These studies will allow us to separately dissect and reconstruct the elements of the fusion apparatus. 4. RECONSTITUTION OF SECRETION IN PERMEABILIZED NEUTROPHIL SYSTEMS As an intermediate step towards applying our findings from Specific Aims 1-3 to intact cells, we will attempt to reconstitute secretion in permeabilized neutrophils. The permeabilized cell system will be used as a tool for 1) identifying potential fusion modulators for use in vitro studies; and 2) testing fusion modulators identified in vitro in a more physiologically relevant system. The development of these in vitro model systems and the isolation of endogenous members of the fusion regulator system will also help clarify the mechanisms of secretion in intact and permeabilized cells.
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