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.
