Vesicular traffic and regulated secretion in C. elegans

描述（由申请人提供）： 真核生物蛋白质和多肽分泌的调控途径 细胞在进化上是古老的，是许多生理过程的基础。 如消化、凝血、内分泌功能， 神经传递调节途径的关键功能组分是 致密核心囊泡（DCV），一种磷脂结合的细胞器，含有 分泌的货物和其生物发生所必需的分子机制， 胞吐作用和细胞内的运输。DCV具有功能性和生化性 与突触囊泡（SV）重叠的特性涉及经典的 神经传递，但在其生物起源，细胞 定位及其胞吐作用后的命运。遗传学研究 模式生物，如酵母，对我们的一般 膜运输和胞吐的知识，但酵母缺乏一个受调节的 分泌途径和微管为基础的运动系统典型的DCV和SV在 更高的生物。我们建议开发线虫，C。作为一个模特， 用于调节分泌的分子细胞生物学研究的系统， IDA-1、C.线虫的同源物， ICA 512受体型蛋白酪氨酸磷酸酶亚家族， 特异性定位于哺乳动物神经内分泌组织的DCV。我们的目标： 表征IDA-1：：GFP标记的囊泡在细胞中的移动和定位。 用延时摄影观察转基因线虫的轴突和树突 荧光显微镜结合生理药理学 干预措施。这些研究将通过免疫电子 在这些条件下，对天然IDA-1和IDA-1：：GFP分布进行显微镜分析。 条件为了分析IDA-1：：GFP在携带突变的线虫中的表达， 影响囊泡生物发生、囊泡运动和相关膜的基因 交通突变体将包括候选马达蛋白、衔接蛋白和 其他基因记录影响突触的运动和定位， 在这些生物体中。为了产生一组新的突变体， 通过诱变IDA-1：GFP表达转基因调节分泌途径 动物和筛选荧光团的错误定位和 囊泡动力学描述ida-1功能丧失的表型 突变体并使用野生型和GFP标记的IDA-1进行基因拯救分析， 哺乳动物同源物ICA 512和phogrin，以及IDA-1的突变形式 分子。 使用C。elegans模型将推进我们对生物化学的理解， 调节分泌途径的性质和功能，并提供见解 转化为一类分子，它们是细胞和体液免疫的主要目标， 1型糖尿病的治疗方法

DESCRIPTION (provided by applicant): 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.