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Completed Project

Fluorescent Microscopy of Early-Stage Phagocytosis in Tetrahymena thermophila

Min Chen, Vassar College ’16 and Prof. J. William Straus

Phagocytosis is a complex process by which cells membrane-encapsulate and internalize extracellular particulate for digestion. In mammals, phagocytosis is seen in the immune system for defense while in ciliates like Tetrahymena the process is crucial to obtaining nutrients. It is much easier to study the process in ciliates like Tetrahymena rather than mammals because these protists perform phagocytosis constitutively and do not require particulate matter to initiate the process.This makes it possible to image phagosomes without the presence of solid materials. In order to follow uptake and protease activity, cells were labelled with various fluorgenic substrates and imaged with widefield fluorescent microscopy (after washing in ddH20 and fixing in formaldehyde.)  Nascent phagosomes of cells incubated with BODIPY TR casein and green fluorescent E.coli exhibited crescent migration pattern starting from the oral apparatus to the aboral side of the cell with vesicles budding off the terminal end. This consistent pattern suggests the existence of an underlying cytoskeletal structure which guides the forming phagosomes. Attempts to label the deep fiber with Tubulin Tracker and correlate the structure with the movement of vesicles proved inconclusive; future antibody labeling of tubulin will be explored. A mutant, non-phagosomal strain of Tetrahymena was also studied to compare patterns of uptake. A smaller, flatter morph of the mutants grown at grown at 38 °C was found to exclusively take up label. The dispersion of DAPI stain in this morph suggests that these cells were closer to death than the dominant morph.