RUI: Membrane dynamics at the mating junction of Tetrahymena

RUI：四膜虫交配处的膜动力学

• 批准号: 1517624
• 负责人: Eric Cole
• 金额: $ 41.25万
• 依托单位: Saint Olaf College
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-07-15 至 2018-09-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1517624&HistoricalAwards=false
• 关键词: RUI; Membrane; dynamics; mating; junction

Tetrahymena thermophile, a single-celled, freshwater protozoan endemic to the eastern USA, is a genius at performing universal cellular activities in exaggerated ways. During sexual reproduction, Tetrahymena manufacture thousands of miniature chromosomes that must be "capped" at each end to preserve their integrity. This made Tetrahymena the ideal model organism for uncovering the mechanism of "telomere" capping leading to the 2009 Nobel Prize. Paradoxically, while Tetrahymena perform almost universal acts of cell biology, they are poised equidistant from plant and animal kingdoms, and often display novel approaches to solving fundamental biological problems. This evolutionary distance allows researchers to gain broad perspective into how life solves many of its functional dilemmas. The investigators in this research project will take advantage of this organism's unique place in the tree of life and its hugely accessible biology, to explore cellular mechanisms that resemble events triggered in "higher" organisms when sperm meets egg: how do cells communicate with one another to trigger changes that permit cell-cell fusion; how do cells attach to one another during mating and; how do cells protect one set of nuclei while simultaneously destroying another set of nuclei within a common cytoplasm. The results from this work will inform the understanding of cell biology associated with these processes in higher organisms. The accessibility of Tetrahymena makes this a model system for training undergraduates in the art of molecular and cellular biology and the practice of good science. Students in the investigator's laboratory will learn to culture live cells, isolate cellular organelles and their constituent proteins, identify these proteins by Mass Spectrometry, take the resultant data to clone genes of interest, and perform sophisticated microscopy and electron tomography experiments to examine localization of cellular components (proteins, cytoskeleton) during mating associated events. As students are trained, they will be encouraged to "cross-train", picking up multiple types of research expertise in both classroom and laboratory settings, while experiencing a truly collaborative and interdisciplinary research environment.The formal questions that constitute the Intellectual Merit of this research are: 1) Can cell-adhesion proteins that mediate pair-formation in Tetrahymena be identified and are they related to products of the Mating Type Locus? 2) Do Tetrahymena cells engaging in pre-mating encounters exhibit elevated levels of intracellular Ca++ reminiscent of those accompanying the fertilization reaction in metazoan gametes? 3) How could physical association of post-meiotic nuclei with membrane systems of the mating junction shield those nuclei from signals that trigger macro-autophagy, and 4) What membrane trafficking pathways lead to production and shedding of micro-vesicles into the extracellular space between mating cells, and do these shed micro-vesicles perform a signaling function? This study ultimately explores inter-cellular signaling via surface proteins and possibly shed micro-vesicles and the programmed cellular responses triggered by those signals. The work in this project will be performed by undergraduates engaged in formal scientific training.

嗜热四膜虫是美国东部特有的一种单细胞淡水原生动物，擅长以夸张的方式进行普遍的细胞活动。 在有性繁殖过程中，四膜虫制造了数千个微型染色体，这些染色体必须在两端“加盖”以保持其完整性。 这使得四膜虫成为揭示“端粒”加帽机制的理想模式生物，并获得了2009年诺贝尔奖。 奇怪的是，虽然四膜虫执行几乎普遍的细胞生物学行为，但它们与植物和动物王国的距离相等，并且经常展示解决基本生物学问题的新方法。 这种进化距离使研究人员能够获得广阔的视角，了解生命如何解决其许多功能困境。 该研究项目的研究人员将利用这种生物在生命树中的独特地位及其巨大的生物学特性，探索类似于精子与卵子相遇时在“高等”生物中触发的事件的细胞机制：细胞如何相互交流以触发允许细胞-细胞融合的变化;细胞如何在交配过程中相互附着;细胞如何在保护一组细胞核的同时破坏共同细胞质中的另一组细胞核。这项工作的结果将有助于了解与高等生物中这些过程相关的细胞生物学。 四膜虫的可访问性使其成为培养大学生分子和细胞生物学艺术以及良好科学实践的模型系统。 在研究者实验室的学生将学习培养活细胞，分离细胞器及其组成蛋白质，通过质谱鉴定这些蛋白质，将所得数据克隆感兴趣的基因，并进行复杂的显微镜和电子断层扫描实验，以检查交配相关事件期间细胞成分（蛋白质，细胞骨架）的定位。 作为学生的培训，他们将被鼓励“交叉培训”，拿起在课堂和实验室设置多种类型的研究专业知识，同时体验一个真正的协作和跨学科的研究环境.构成本研究的智力优点的正式问题是：1）在四膜虫中介导配对形成的细胞粘附蛋白能否被鉴定，它们是否与交配型基因座的产物相关？ 2)四膜虫细胞在交配前的遭遇表现出高水平的细胞内Ca++联想到那些伴随着后生动物配子的受精反应？ 3)减数分裂后的细胞核与交配连接处的膜系统的物理关联如何使这些细胞核免受触发大自噬的信号的影响，以及4）什么样的膜运输途径导致微泡的产生和脱落进入交配细胞之间的细胞外空间，这些脱落的微泡是否执行信号传导功能？ 这项研究最终探索了通过表面蛋白质和可能脱落的微泡以及由这些信号触发的程序化细胞反应的细胞间信号传导。 本项目的工作将由接受正规科学培训的本科生完成。