Internalization of particulates through phagocytosis is of fundamental importance to diverse cell types including professional phagocytes of the immune system and microorganisms that use the process for capturing food. Internalized particulates are contained within motile intracellular vesicles known as phagosomes. Fusion of phagosomes with lysosomes provides the chemical environment for digestion of the internalized particulates. In Tetrahymena, phagosomes appear to be vertically aligned, and the alignment is maintained as phagosomes move toward the posterior end of the cell. Motility of phagosomes either ceases or becomes random in a knockout of MYO1, a class XIV myosin that contains MyTH4/FERM domains. Inhibition of actin filament assembly induces random motility of phagosomes in wild type cells. Therefore, both Myo1 and actin filaments are required for maintaining directionality of phagosome motility. GFP fused to the FERM domain from Myo1 localizes to phagosomes, which appear to be attached to the longitudinal rows of cytoskeleton basal bodies. This project aims to investigate directionality of phagosome motility. The project will use motility of phagosomes in Tetrahymena to test the hypothesis that directed motility of phagosomes involves the interaction of the Myo1 FERM domain with basal body-associated actin filaments that are putative tracks for phagosome motility. The hypothesis will be tested in vivo and in vitro. For analysis of phagosome motility in vivo, the GFP-FERM strain will be challenged with fluorescent beads as markers for phagosomes. Time-lapse confocal microscopy will be used to monitor the velocity and direction of phagosome motility in relation to the longitudinal alignment of cytoskeleton basal bodies. It is expected that overexpressed FERM will compete with endogenous Myo1 for binding sites and interfere with directionality of phagosome motility. A prediction of the hypothesis is that disruptions in the longitudinal alignment of cytoskeleton basal bodies will induce random motility of phagosomes. To test this prediction, heat shock will be used to induce discontinuities in the alignment of basal bodies. Phagosome motility will be analyzed in the heat-shocked cells after they have resumed normal phagocytosis. To further demonstrate that phagosomes navigate along longitudinal rows of basal bodies, cytoskeletons will be isolated from the GFP-FERM strain. Nondenatured total cell lysate from cells challenged with fluorescent beads will be added to the cytoskeleton fraction, and phagosome motility will be analyzed. The outcomes of this project will significantly contribute to our understanding of how phagosomes maintain directionality.
Broader Impacts An education plan provides research training for undergraduates and science workshops for middle school students. Research students in the PI's laboratory learn more than how to use a specialized technique in an experiment. They are participants in all phases of the research experience including experimental design, data acquisition, data interpretation, and preparation of data for publication. Research students are required to attend biweekly departmental seminars that feature undergraduate research presentations.
