Membrane fusion is a vital process for many cellular activities such as exocytosis, endocytosis, intracellular membrane traffic, cell fertilization and cell division. These fusion phenomena are regulated by a variety of stimuli and their mechanism of action is largely unknown. In an attempt to understand the basic mechanism at the molecular level, we have focussed on the role of Ca2+, which is an essential requirement for many membrane fusion events and certainly for exocytosis and secretion. In earlier work, we have studied the interactions of Ca2+ with specific phospholipids and synexin and have established in vitro systems that demonstrate some of the properties of exocytotic fusion events. In this grant proposal, we focus on the function of a group of cytoplasmic Ca2+-binding proteins recently termed """"""""annexins"""""""", whose function has not been established as yet. We propose to study their respective ability to control fusion using in vitro models that involve liposomes, isolated intracellular granules, endocytotic vesicles and inverted plasma membrane vesicles. Using a sensitive assay systems for membrane fusion which mimic exocytotic conditions we will examine the role of lipid and protein specificity and determine the influence of other cytoplasmic factors. The availability of sensitive fusion assays and the existence of biochemical information on the protein structure makes this project a timely and exciting prospect. Our approach involves three different levels of experimental strategy: One: an initial screening of the different purified annexins with respect to their ability to cause fusion of specific liposome compositions at various Ca2+ concentrations. Two: for the proteins that show promising effects on the fusion assays (promotion or inhibition) we will undertake a detailed analysis of the mechanism of interaction with the lipid bilayer at the molecular level. Three: in order to validate their function at the cellular level, we will study the ability of these proteins to promote fusion of intracellular granules with inverted plasma membranes under physiological conditions reconstituting exocytotic events. Understanding the role of exocytotic fusion has wide health-related implications, since secretion of specific granule contents is important in host defense mechanisms, as well as in neutrophil emigration through the vascular endothelium. Uncontrolled exocytosis by neutrophils may be involved in pulmonary complications of adult respiratory distress syndrome, mucosal damage after myocardial infarction and may also affect mutagenesis. Therefore understanding exocytosis at the molecular level may clarify the mechanisms which control both the vital and pathological function of neutrophils and other secretory cells.
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