Project Summary/Abstract Motility is critical to the life cycle and pathogenicity of many parasites. While targeting motility is successful in the treatment of multiple bacterial diseases, the motility and motile structures of eukaryotic pathogens remain understudied and underexploited as treatment targets. Kinetoplastids, which are eukaryotic parasites that cause multiple neglected tropical diseases, exhibit unique flagellar motility. Their flagella beat with a bending wave that propagates from the tip to the base of their flagellum. This is unlike nearly all other eukaryotes, which beat from the base to the tip. Because kinetoplastid flagellum bending wave propagation direction switches under certain chemical and environmental conditions, and because the motile elements of kinetoplastid the flagellum are nearly identical to all other eukaryotes, it is likely that unique regulation mechanisms innate to axonemal dyneins, the molecular motors that drive flagellar motility, tune this tip-to-base motility. Testing this hypothesis requires quantitative single-molecule biophysical characterization of kinetoplastid dynein regulation mechanisms. The broad goal of this research program is to enable the development of novel treatments for kinetoplastid- associated diseases that target the tip-to-base motility of kinetoplastid flagella. The specific aims of this project focus on quantifying axonemal dynein regulation mechanisms from Trypanosoma brucei brucei, which will be used as a model for kinetoplastid flagella. The aims include characterizing how force regulates the motility of inner arm axonemal dyneins and how dynein-associated light chains and posttranslational modification to tubulin regulate outer arm axonemal dyneins. This interdisciplinary project will take molecular biological (CRISPR/Cas9, cloning, protein tagging), biochemical (in vitro reconstitutions, ATPase assays), genomic and proteomic (RNA- Seq, mass spec), and biophysical (ultrafast dual-trap optical tweezers, total internal reflectance fluorescence microscopy) experimental approaches. The collected data will be integrated and understood by making quantitative biophysical models of axonemal dynein motility mechanisms. The expected outcome will be a framework from which to develop pan-kinetoplastid drugs that target parasite motility. Successful completion of the project will ultimately lead to a greater understanding of the fundamental mechanisms of pathogenic parasite motility and could lead to novel treatments for African sleeping sickness, Chagas disease, and leishmaniasis.