RUI: Membrane dynamics at the mating junction of Tetrahymena

嗜热性四膜虫是美国东部特有的一种单细胞淡水原生动物，它是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，四膜虫制造数以千计的微型染色体，必须在每一端“封顶”，以保持它们的完整性。这使得四膜虫成为揭示导致2009年诺贝尔奖的“端粒”封顶机制的理想模式生物。矛盾的是，虽然四膜虫执行几乎普遍的细胞生物学行为，但它们与动植物王国的距离相等，并且经常展示解决基本生物学问题的新方法。这一进化距离让研究人员获得了广阔的视角，了解生命是如何解决其许多功能困境的。参与这一研究项目的研究人员将利用这种生物在生命树上的独特地位及其巨大的可获得性生物学，来探索与精子与卵子相遇时在“高等”生物体中触发的事件的细胞机制：细胞如何相互通信以触发允许细胞-细胞融合的变化；细胞如何在交配过程中相互附着；以及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核。这项工作的结果将有助于理解与高等生物体的这些过程相关的细胞生物学。四膜虫的可及性使其成为训练本科生分子和细胞生物学艺术以及良好科学实践的模式系统。研究人员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，通过质谱学鉴定这些蛋白质，利用产生的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，以检查交配相关活动中细胞成分(蛋白质、细胞骨架)的定位。随着学生接受培训，他们将被鼓励进行“交叉培训”，在课堂和实验室环境中学习多种类型的研究专业知识，这项研究的正式问题是：1)四膜虫中介导配对形成的细胞黏附蛋白能否被识别出来，它们是否与交配型基因座的产物有关？2)四膜虫细胞在交配前接触时，细胞内钙的水平是否高于后生动物配子中伴随受精反应的水平？3)减数分裂后核与交配接合部的膜系统的物理联系如何保护这些核不受触发宏观自噬的信号的影响，以及4)什么膜运输途径导致微泡的产生和释放到交配细胞之间的细胞外间隙，这些释放的微泡是否起信号功能？这项研究最终探索了通过表面蛋白的细胞间信号，并可能释放微泡和由这些信号触发的程序化细胞反应。该项目的工作将由接受正式科学培训的本科生完成。

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.