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、内分泌功能和 神经传递。受调控途径的关键功能组件是 致密核小泡(DCV)，一种磷脂结合的细胞器，包含 秘密货物及其生物发生所需的分子机制， 胞吐作用和细胞内的贩运。DCV具有功能性和生化性 与经典研究中涉及的突触小泡(SV)重叠的特性 神经传递，但在生物发生、细胞 胞吐后的定位及其命运。关于遗传易感性的研究 酵母菌等模式生物对我们的总体发展做出了巨大贡献 膜运输和胞吐作用的知识，但酵母缺乏调控 DCVs和SVS典型的分泌途径和基于微管的运动系统 高等有机体。我们建议开发线虫，线虫，作为模式 调节分泌的分子细胞生物学研究系统 用IDA-1获得的初步数据，线虫Pogrin的直系同源基因和 ICA512受体类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。我们的目标是： IDA-1：：GFP标记囊泡在体内的运动和定位特征 用时间推移法研究转基因线虫的轴突和树突 结合生理学和药理学的荧光显微镜 干预措施。这些研究将得到免疫电子技术的补充。 中国本土IDA-1和IDA-1：：GFP分布的显微分析 条件。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 堵车。突变体将包括候选马达蛋白、接头蛋白和 其他有文献记载的影响突触运动和定位的基因 这些生物体中的小泡。以产生一组新的突变体，影响 诱变IDA-1：GFP表达基因调控的分泌途径 动物及荧光团错位和改变的筛选 囊泡动力学。鉴定IDA-1功能丧失的表型 并利用野生型和GFP标记的IDA-1进行基因拯救分析， IDA-1的哺乳动物同源物、ICA512和phogrin及其突变形式 分子。 线虫模型的使用将促进我们对生物化学的理解 调节分泌途径的性质和功能，并提供洞察力 转化为一类分子，这些分子是细胞和体液的主要目标 男性1型糖尿病患者的自身免疫。

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.