The Developmental Genetics of Tetrahymena thermophila.

描述(申请人提供)：这项工作旨在探索与单细胞模式生物中两个动态细胞器相关的发育组装和分子机制：嗜热四膜虫。核交换连接代表了一个研究膜运输和细胞骨架动力学的分子剧场，因为它们与细胞皮质的发育程序性重塑有关。膜转运通路与许多神经退行性疾病有关，并且在炎症期间对免疫系统的正常运作至关重要。Conjuome是一种非膜结合的电子致密细胞器，类似于后生动物胚胎的种质或核。因此，它代表了一个潜在的易于处理的系统，用于探索小RNA介导的生物体基因组重塑的机制，以服务于基因组防御。使用小RNA分子(RNAi现象)已成为治疗研究的一个领先领域。我们的长期目标包括：1)了解四膜虫在核交换和交配过程中细胞骨架和膜运输的动态，从而重塑细胞皮质。2)了解在纤毛虫模型中基因组重组期间组装的种质状细胞器四膜虫“Conjuome”的作用，以及它与高等真核细胞胚胎中这种结构的潜在进化关系，以及3)为开展嗜热四膜虫发育遗传学研究的本科生提供跨学科的培训。我们的具体目标包括：1)基因发现。我们计划进行蛋白质纯化和质谱分析，以确定四膜虫在交配过程中伴随核交换的皮质重塑以及交配后期类似种质的细胞器的组装所涉及的基因。2)功能分析。通过产生功能缺失突变并分析它们的表型，通过功能基因组学来研究这些候选基因。3)蛋白质定位。通过GFP标签、扫描激光共聚焦显微镜和免疫金电子断层扫描，确定通过蛋白质组学和生物信息学鉴定的候选基因产物的细胞内定位。4)本科生科研培训。利用扫描激光共聚焦荧光显微镜和电子断层扫描技术，开发一套用于大学生蛋白质定位分析的超微结构工作站。参与创建纤毛虫分子生物学本科生研究人员训练营。 公共卫生相关性：四膜虫核交换连接代表了一种新的系统，用于探索“循环内小体途径”的参与，该途径与许多医学疾病和免疫系统的正常运作有关。Conjuome是一种非膜结合的电子致密细胞器，类似于后生动物胚胎的种质或核，因此，它代表了一个潜在的易于处理的系统，用于探索小RNA介导的生物体基因组重塑的机制，为基因组防御服务。使用小RNA分子(RNAi现象)已成为治疗研究的一个领先领域。

DESCRIPTION (provided by applicant): This work aims to explore the developmental assembly and molecular mechanisms associated with two dynamic organelles within a unicellular model organism: Tetrahymena thermophila. The nuclear exchange junction represents a molecular theater for studying membrane trafficking and cytoskeletal dynamics as they pertain to developmentally programmed remodeling of the cell cortex. Membrane trafficking pathways have been implicated in numerous neuro-degenerative diseases (among others), and are critical to the proper functioning of the immune system during inflammation. The "conjusome" is a non-membrane bound electron-dense organelle resembling germ-plasm or nuage from metazoan embryos. As such, it represents a potentially tractable system for exploring mechanisms of small RNA-mediated remodeling of an organism's genome in service of genome defense. The use of small RNA molecules (the RNAi phenomenon) has become a leading area for therapeutic research. Our long term objectives include: 1) Understanding the dynamics of cytoskeletal and membrane trafficking that remodel the cell cortex during nuclear exchange and mating in Tetrahymena. 2) Understanding the role of the Tetrahymena "conjusome", a germ-plasm-like organelle assembled during a period of genome-reorganization in this ciliate model, and its potential evolutionary relationship to such structures in higher eukaryote embryos, and 3) providing interdisciplinary training for undergraduates conducting research into the develop- mental genetics of Tetrahymena thermophila. Our specific goals include: 1) Gene discovery. We plan to conduct protein purification and mass spectrometry to identify genes involved in the cortical remodeling that accompanies nuclear exchange during mating, and the assembly of a germ-plasm-like organelle during late stages of mating in Tetrahymena. 2) Functional analysis. Investigate these candidate genes through functional genomics by generating loss of function mutations and analyzing their phenotypes. 3) Protein localization. Determine the intracellular localization of candidate gene products identified via proteomics and bioinformatics by GFP-tagging, scanning-laser confocal micros-copy, and immuno-gold electron tomography. 4) Undergraduate research training. Develop an ultrastructure workstation for undergraduate analysis of protein localization through both scanning laser confocal fluorescence microscopy and electron tomography. Participate in creating a Ciliate Molecular Biology "Bootcamp" for undergraduate researchers. PUBLIC HEALTH RELEVANCE: The Tetrahymena nuclear exchange junction represents a novel system for exploring the involvement of the "recycling endosome pathway", a pathway implicated in numerous medical disorders and in the proper functioning of the immune system. The "conjusome" is a non-membrane-bound electron-dense organelle resembling germ-plasm or nuage from metazoan embryos, and as such, it represents a potentially tractable system for exploring mechanisms of small-RNA-mediated remodeling of an organism's genome in service of genome defense. The use of small RNA molecules (the RNAi phenomenon) has become a leading area for therapeutic research.