The Developmental Genetics of Tetrahymena thermophila.

描述（由申请人提供）：本工作旨在探索单细胞模式生物：嗜热四膜虫（Tetrahymena thermomila）中与两个动态细胞器相关的发育组装和分子机制。核交换连接代表了研究膜运输和细胞骨架动力学的分子剧场，因为它们与细胞皮层的发育程序性重塑有关。膜运输途径与许多神经退行性疾病（以及其他疾病）有关，并且对炎症期间免疫系统的正常功能至关重要。“同体”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绒毛。因此，它代表了一个潜在的可处理系统，用于探索小rna介导的生物体基因组重塑机制，以服务于基因组防御。小RNA分子的使用（RNAi现象）已经成为治疗研究的一个领先领域。我们的长期目标包括：1)了解在核交换和交配过程中重塑细胞皮质的细胞骨架和膜运输的动力学。2)了解在这种纤毛虫模型中基因组重组期间组装的一种种质样细胞器“同质体”的作用，及其在高等真核生物胚胎中与这种结构的潜在进化关系。3)为从事嗜热四膜虫发育遗传学研究的本科生提供跨学科培训。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，以确定参与四膜虫交配过程中核交换过程中皮质重塑的基因，以及交配后期种质样细胞器的组装。通过功能基因组学研究这些候选基因，产生功能突变损失并分析其表型。3)蛋白质定位。确定候选基因产物的细胞内定位，通过蛋白质组学和生物信息学鉴定，通过gfp标记，扫描激光共聚焦显微复制和免疫金电子断层扫描。4)本科生科研训练。通过扫描激光共聚焦荧光显微镜和电子断层扫描技术，建立一个用于大学生蛋白质定位分析的超微结构工作站。参与为本科生研究人员创建一个纤毛虫分子生物学“训练营”。

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.