9724105 LeBowitz The paraflagellar rod (PFR) is essential for the normal motility of Leishmania promastigotes, yet the molecular structure, assembly and function of this unique cytoskeletal structure are not well understood. The PFR extends the length of the axoneme after it emerges from the flagellar pocket and is built up from discrete filaments organized in lattice-like arrays. The major protein components of the PFR do not reveal significant homology to other cytoskeletal proteins. This lack of homology together with the elaborate and unusual structure of the PFR suggests a novel type of cytoskeletal filament and organization. Based on the characterization of L. mexicana lines lacking the major PFR proteins, PFR-1 and PFR-2, a simple model for the organization of the PFR is proposed that has a number of testable features. First, PFR-1 protein is an essential constituent of the proximal domain of the PFR and PFR-2 is absent from the proximal domain. Second, PFR-1 protein can assemble into filaments in the absence of PFR-2 either forming a homopolymer or polymerizing with as yet unidentified partners. Third, PFR-2 protein is a primary constituent of the distal domain of the PFR. Fourth, assembly of the distal domain of the PFR requires prior assembly of the proximal domain of the PFR whose assembly in turn is coordinated with assembly of the axoneme. This ordering of PFR assembly explains the observed inability of PFR-2 protein to assemble into a stable structure in the absence of PFR-1, and it explains the presence of a properly positioned structure that contains PFR-1 in the absence of PFR-2. Furthermore, it accounts for the puzzling observation that in dividing cells examined by immunofluorescence with anti-PFR antibodies, the daughter flagellum often stains more weakly than the parental flagellum. This would be expected if the PFR was assembled stepwise from the proximal to the distal zone. Genetic and cytological experiments are proposed t o test and extend this model of the organization of the PFR in Leishmania mexicana. These specific objectives will be accomplished: 1. Ultrastructural characterization of PFR-1 and PFR-1/PFR-2 null mutants. The objective of this set of experiments is to determine whether or not substructures of the PFR are retained in the mutant lines that lack PFR-1 2. Determination of the location within the PFR of the PFR-1 and PFR-2 proteins. The objective of this set of experiments is to separately localize PFR-1 and PFR-2 proteins by immunogold labeling of thin sections and whole mount cytoskeletons. 3. Test of the order of assembly of the domains of the PFR. The objective of these experiments is to determine whether the nascent daughter flagella of dividing L. mexicana assemble the proximal domain of the PFR prior to the distal domain. Among unicellular organisms there is an immense diversity of differences in physiology and structure that reflects the vast experimentation in form and function that has occurred during evolution. The means by which unicellular organisms move using flagella and cilia as basis has been elaborated and altered as among diverse unicellular organisms. In this award Dr. LeBowitz will study the function and form of the paraflagellar rod of Leishmania. Understanding how the paraflagellar rod is assembled will yield important information on the diversity of motility mechanisms of unicellular organisms. Members of Leishmania species include pathogens that are causitive agents of human disease. Potential applications of this information includes finding compounds that impede the formation of paraflagellar rod preventing motility and spread of the organisms. ***
