Components And Kinetics In Exocytosis

Child Health And Human Development

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Abstract

This project is centered on the mechanisms of calcium-triggered exocytosis, the ubiquitous eukaryotic process by which vesicles fuse to the plasma membrane and release their contents. Research is focused on the cortical fusion of secretory vesicles of the sea urchin egg, which occurs upon fertilization. The isolated cortex of the sea urchin egg is used as a minimal preparation for the study of exocytosis, as it requires neither ATP nor cytosol. Isolated cortical granules can be reconstituted to fuse with egg plasma membrane, other cortical granules, and even purely lipid membranes. Thus cortical granules themselves have sufficient protein machinery for fusion. Upon echinoderm egg fertilization, cortical secretory vesicle exocytosis is massive and synchronous. By combining physiological and molecular analyses with a variety of purified membrane isolates containing secretory vesicles that fuse to the plasma membrane or each other, they have characterized the final steps of this calcium-triggered exocytosis. Although immunoblotting, or Western blotting is widely used for detection of specific proteins, it is generally thought to be poor as a quantative tool for measuring the concentration of specific proteins. However, for testing hypotheses at the level of quantitative science, it is essential to have such a tool. For analysis and understanding of the molecular mechanism of exocytosis requires one to unambiguously identify and quantitatively assess the surface density of specific molecules. Here, using newly refined immunoblotting and analysis paradigms, we provide a fully quantitative analysis of the SNARE protein complement of functional secretory vesicles. These findings demonstrate the routine quantitation of femtomole to attomole amounts of known proteins and indicate that native secretory vesicles of the sea urchin egg, like other regulated secretory vesicles, undergo Ca2+-triggered fusion despite an endogenous SNARE complement that is ~50-fold lower than required for relatively inefficient fusion in model vesicle systems. At the heart of exocytosis is membrane fusion. The energetics of a fusion pathway was considered theoretically, starting from the contact site where two apposed membranes each locally protrude (as "nipples") toward each other. The equilibrium distance between the tips of the two nipples is determined by a balance of physical forces: repulsion caused by hydration and attraction generated by fusion proteins. The energy to create the initial stalk, caused by bending of cis monolayer leaflets, is much less when the stalk forms between nipples rather than parallel flat membranes. The stalk cannot, however, expand by bending deformations alone, because this would necessitate the creation of a hydrophobic void of prohibitively high energy. But small movements of the lipids out of the plane of their monolayers allow transformation of the stalk into a modified stalk. This intermediate, not previously considered, is a low-energy structure that can reconfigure into a fusion pore via an additional intermediate, the prepore. The lipids of this latter structure are oriented as in a fusion pore, but the bilayer is locally compressed. All membrane rearrangements occur in a discrete local region without creation of an extended hemifusion diaphragm. Importantly, all steps of the proposed pathway are energetically feasible.