PROJECT SUMMARY Whether its photons or fries eating is fundamental for life. From this basic principle, living organisms have evolved innumerable strategies to capture energy and nutrients from their environment, leading, in turn, to the incredible ecological diversity spanning the gamut from light eating photosynthetic autotrophs to predatory heterotrophs. As part of the world’s aquatic ecosystems, the expansive family of heterotrophic protozoan predators play a critical role in environmental carbon and nutrient cycling as they consume 75% of primary producing planktonic autotrophs daily. The vast majority of these flagellated phagotrophs use self-generated currents to funnel their prokaryotic prey into an ancient and highly enigmatic feeding apparatus prior to digestion. This feeding structure begins as a plasma membrane surface opening (cytostome), descends into an internal tubular invagination (cytopharynx) and ends with prey being enveloped within budding vesicles destined for lysosome fusion. Here we refer to this organelle as the cytostome/cytopharynx complex or SPC and, despite its near ubiquitous presence in protozoans, next to nothing is known mechanistically about how this structure is formed or functions. Intriguingly, a class of these phagotrophic predators known as the kinetoplastids, gave rise to a lineage of parasitic protozoa that can infect a wide variety of organisms ranging from plants to humans. Curiously, one species in particular, Trypanosoma cruzi, retained this ancestral organelle much like its free-living relatives (e.g. bodonids) and continues to use it as its primary route of endocytosis. Due to the fact that T. cruzi is easily culturable, genetically tractable and not reliant on SPC mediated endocytosis for viability in vitro, we have been able to conduct the first ever in-dept molecular analyses of this ubiquitous feeding organelle. Our initial published work on this structure described the first known proteins targeted to the SPC and was followed by a report on the identification of a family of SPC targeted myosin motors that we show contribute directly to the endocytic process. As a continuation of these studies, this proposal seeks to generate a holistic understanding of how SPC mediated endocytosis fundamentally functions. We will begin by dismantling the unified activity of endocytosis into its constituent processes; cargo capture through surface receptors (Aim1), receptor signal transduction and activation of endocytic machinery (Aim2) and finally active transport of phagocytosed cargo along the SPC for digestion (Aim3). Each of these aims will address important basic aspects of protozoan biology that continue to remain poorly understood. Critically, this proposal will combine both a broad approach to identify cytostomal surface receptors and SPC specific signaling components with a focused analysis of the role of the Act2 isoform in the endocytic process. By combining this model organism with a broad range of ...