The regulated pathway of secretion of proteins and polypeptides in eukaryotic cells is evolutionarily ancient and underlies numerous physiological processes as diverse as digestion, blood coagulation, endocrine function and neurotransmission. The key functional component of the regulated pathway is the dense-core vesicle (DCV), a phospholipid bounded organelle that contains the secreted cargo and the molecular machinery necessary for its biogenesis, exocytosis and trafficking in the cell. The DCV has functional and biochemical properties that overlap with the synaptic vesicle (SV) involved in classic neurotransmission but differ significantly in its biogenesis, cellular localization and its fate post-exocytosis. Studies in genetically tractable model organisms such as yeast have contributed considerably to our general knowledge of membrane traffic and exocytosis but yeast lacks a regulated secretory pathway and microtubule based motor system typical of DCVs and SVs in higher organisms. We propose to develop the nematode, C. elegans, as a model system for molecular cell biological studies of regulated secretion based upon preliminary data obtained with IDA-1, a C. elegans ortholog of the phogrin and ICA512 subfamily of receptor-type protein tyrosine phosphatases that are specifically localized to DCVs of mammalian neuroendocrine tissues.
We aim : to characterize the movement and localization of the IDA-1::GFP tagged vesicles in the axons and dendritic processes of transgenic nematodes using time-lapse epifluorescence microscopy combined with physiological and pharmacological interventions. These studies will be complemented by immuno-electron microscopical analysis of native IDA-1 and IDA-1::GFP distribution under these conditions. To analyze IDA-1::GFP expression in nematodes bearing mutations in genes affecting vesicle biogenesis, vesicle movement and associated membrane traffic. Mutants will include candidate motor proteins, adaptor proteins and other genes documented to affect the movement and localization of synaptic vesicles in these organisms. To generate a panel of new mutants affecting the regulated secretory pathway by mutagenizing IDA-1: GFP expressing transgenic animals and screening for mislocalization of the fluorophore and alteration of vesicle dynamics. To characterize the phenotype of an ida-1 loss-of-function mutant and perform gene rescue analysis using wild type and GFP tagged IDA-1, the mammalian homologs, ICA512 and phogrin, and mutant forms of the IDA-1 molecule. Use of the C. elegans model will advance our understanding of the biochemical properties and function of the regulated secretory pathway and provide insight into a class of molecules that are major targets of cellular and humoral autoimmunity in type 1 diabetes in man.
| Wasmeier, Christina; Burgos, Patricia V; Trudeau, Tammy et al. (2005) An extended tyrosine-targeting motif for endocytosis and recycling of the dense-core vesicle membrane protein phogrin. Traffic 6:474-87 |
| Zahn, Tobias R; Angleson, Joseph K; MacMorris, Margaret A et al. (2004) Dense core vesicle dynamics in Caenorhabditis elegans neurons and the role of kinesin UNC-104. Traffic 5:544-59 |
