MEMBRANE TRANSPORT AND FUSION

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We have continued our research on fusion, the fundamental step in viral and parasitic infection, fertilization, secretion, and neuro- transmission. By developing an extensive set of computer programs, we can now measure the fusion pore conductance in a number of different ways that take more experimental parameters into account than possible previously. In addition, we developed methods for measuring very fast latencies between the time of solution application and fusion pore opening. With direct current, double whole-cell recording, measurement of the fusion pore is model-independent. Using this technique in baculovirus-induced cell-cell fusion, a large distribution of individual fusion pore conductances was measured with cell capacitance, and found to be statistically indistinguishable from that measured by direct recording, thus further validating the capacitance technique. We found that the latency to fusion after triggering was distributed about a mean of 627 ms, much shorter than that of influenza virus. This suggests either a higher fusion protein density or the absence of an appreciable delay, usually attributed to fusion protein aggregation. Preliminary experiments on the electrophysiology of invasion of cells with the protozoan parasite Toxoplasma gondii indicate that the initial response of the host cell is a transient increase in cell membrane conductance. A new preparation for measuring sperm-egg fusion pores during fertilization is currently being developed. Using light-scattering, we are studying the sea urchin planar isolated cortex because it is the only model system that exists for studying exocytosis in which fusion kinetics are not initially substrate limited, since cortical granules are both predocked to the target membrane and in great abundance. In both Lytechinus pictus and Strongylocentrotus purpuratus, we found stable, sub-maximal responses with micromolar concentrations of calcium, with no further fusion at that calcium concentration regardless of mode, duration, or frequency of calcium application. Rather, more fusion was always seen upon raising calcium concentration. Thus the calcium sensitivity of secretory granule exocytosis in vitro is heterogeneous.