RUI: Membrane dynamics at the mating junction of Tetrahymena

嗜热四膜虫是一种美国东部特有的单细胞淡水原生动物，是一种以夸张的方式进行普遍细胞活动的天才。在有性繁殖过程中，四膜虫制造出成千上万的微型染色体，这些染色体必须在每一端“封顶”以保持其完整性。这使得四膜虫成为揭示“端粒”封顶机制的理想模式生物，并最终获得2009年诺贝尔奖。矛盾的是，虽然四膜虫几乎完成了细胞生物学的普遍行为，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从更广阔的角度来看待生命是如何解决许多功能困境的。该研究项目的研究人员将利用这种生物在生命之树中的独特地位及其非常容易获得的生物学特性，探索类似于“高等”生物中精子与卵子相遇时触发的事件的细胞机制：细胞如何相互沟通以触发允许细胞-细胞融合的变化；细胞是如何在交配和；细胞如何在保护一组细胞核的同时破坏同一细胞质内的另一组细胞核。这项工作的结果将告知与这些过程相关的细胞生物学在高等生物的理解。四膜虫的可及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典范系统。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，通过质谱法鉴定这些蛋白质，利用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中细胞成分（蛋白质，细胞骨架）的定位。随着学生的训练，他们将被鼓励进行“交叉训练”，在课堂和实验室环境中学习多种类型的研究专业知识，同时体验真正的合作和跨学科的研究环境。构成本研究智力价值的正式问题是：1)在四膜虫中介导配对形成的细胞粘附蛋白能否被识别，它们是否与交配型位点的产物有关？2)参与预交配的四膜虫细胞是否表现出细胞内钙离子水平的升高，这与后生动物配子中伴随受精反应的钙离子水平相似？3)减数分裂后细胞核与交配接点的膜系统的物理关联如何保护这些细胞核免受触发大自噬的信号的影响？ 4)什么膜运输途径导致微囊泡的产生和脱落到交配细胞之间的细胞外空间，这些脱落的微囊泡是否执行信号功能？本研究最终探索了通过表面蛋白和可能脱落的微泡以及这些信号引发的程序化细胞反应的细胞间信号。本项目的工作将由从事正式科学训练的本科生完成。

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.