Over the course of this long-standing project the continuing objective has been to determine the mechanisms and consequences of the processes that regulate neuropeptide expression in the bag cells of Aplysia californica. These cells afford unusual experimental access and may yield insights into fundamental cell biological processes at work in other neuropeptidergic systems. Since our interest is centered on understanding the determinants of functional expression of the bag cell system our work has attempted to relate cellular biochemistry with the regulation of secretory output. This approach has increasingly brought the neurosecretory granule (NSG) to the center of our attention. The NSG is the structure that links the biochemistry of peptide biosynthesis with the functional output of the cell. In this way the contents, distribution, and exocytosis of NSG define the physiological boundaries of secretory plasticity available to the cells as well as providing a window through which the antecedent processes of peptide production can be glimpsed.
The specific aims of the present proposal will continue our efforts to understand the principles determining the filling, transport, and secretion of bag cell NSLG. Six experimental approaches are proposed to evaluate these and related issues: 1. Axonal transport of precursor-derived peptides will be studied to determine if all peptides are transported and if all transported peptides have the same destinations. 2. Secretion of NSG contents will be evaluated under conditions designed to test for both time-dependent and stimulus-dependent variation in peptide output. 3. Precursor converting activity of density-gradient fractions from the bag cells will be tested to determine if enzymatic activities are concentrated in the NSG fraction more widely distributed among other organelles. 4. Neurosecretory granules prepared by density gradient will be subjected to membrane permeant, organic crosslinking in an effort to assess content specificity. 5. The sequence of precursor converting steps will be explored by identifying molecular intermediates through sequential, N-terminal Edman degradations. 6. Transfection of heterologous cells with the ELH precursor will be undertaken to test the generality of precursor conversion determinants and to explore the effects of selective mutations of precursor structure on the conversion process.