We have continued to characterize the fusion pore which initiates exocytosis. Using beige mouse mast cells, electron microscopy, and electrophysiology, it is possible to determine the sequence of events during exocytosis. After stimulation, a dimple in the plasma membrane forms a small contact area with secretory granule membrane. Within this zone, which has no ordered proteinaceous specializations, a small fusion pore forms which widens rapidly to 1 nS in conductance. Thereafter, the fusion pore remains at semi-stable conductances between 1 and 20 nS for a wide range of times, between 10 and 15000 ms. These conductances correspond to pore diameters of up to 25 nm. Ultrastructural data show small pores of hourglass morphology composed of biological membrane coplanar with both the plasma and granular membranes. Later, the fusion pore rapidly increases in conductance, consistent with the observed morphology of omega-figures. Channel-like fluctuations were not seen. Since the morphology of small pores show contiguous fracture planes, the electrical data represent pores which contain lipid, consistent with our previously proposed model of lipid/protein complexes mediating fusion. To better understand the role of fusion protein structure in fusion pore structure, we have been developing expression systems for fusion proteins. The baculovirus insect cell system was tested, and discovered to have a very active, pH-dependent fusion activity. Only a 10 second pulse of acidic medium is needed to trigger syncytia formation. Dye transfer, capacitance changes, and morphological changes are readily detected. This new system is very convenient for studying pH activated cell-cell fusion. We have begun electrophysiological studies to characterize fusion pore development in this system. To fully investigate biological membrane fusion, we have initiated two projects to purify membrane proteins mediating GTP-triggered rat liver microsome-microsome fusion and the sea urchin cortex, based upon inhibition of membrane fusion by covalent modification. We are also studying the massive fusion of cytoplasmic membranes at the end of mitosis.
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