Mitochondria, the organelles in which energy is harnessed from food into usable ATP form, are moved and shaped within specialized cell types to fulfill different energy needs. The focus of this proposal is to use the model system spermatogenesis in Drosophila melanogaster to elucidate molecular mechanisms by which mitochondrial morphogenesis, specifically mitochondrial movement, occurs. Drosophila spermatogenesis is an ideal context for the analysis of mitochondrial morphogenesis, since mitochondria aggregate, fuse, interwrap, unfurl, and elongate during meiosis and spermatid development; defects in these processes often lead to male sterility, and male sterile mutants can be generated, maintained, and characterized easily. The majority of the proposal centers on the nmd gene, its paralog CG4701, and other members of the AAA+ ATPase family to which nmd and CG4701 belong. The nmd gene is required for mitochondrial aggregation during Drosophila spermatogenesis and encodes an AAA+ ATPase homologous to a S. cerevisiae mitochondrial outer membrane protein of unknown function. Nmd and its paralog CG4701 are related, though not orthologous, to the AAA+ ATPases spastin and katanin 60, which sever microtubules. The overall goals are to determine the mechanisms by which these gene products influence mitochondrial movement and aggregation. The subcellular localization of Nmd and CG4701 will be determined through epitope tagging and analysis of transgenic flies. The function of CG4701, whose expression pattern suggests testis specificity, will be determined by traditional as well as RNAi mutant analysis. The roles of Nmd and CG4701 in microtubule dynamics will be assessed through visualization of microtubule structure in mutants as well as through assessment of Nmd and CG4701 binding to microtubules (both in their wild type and permanently ATP-bound forms). The roles of spastin and katanin 60 in spermatogenesis will be tested via phenotypic analysis of spastin male sterile alleles as well as mutant alleles of a testis specific katanin 60 paralog. Finally, characterization and cloning of the mitoshell gene, required for proper aggregation of mitochondria in spermatids, will elucidate from another angle molecular mechanisms of mitochondrial aggregation. The long term objectives of this study are to learn basic molecular mechanisms of mitochondrial biology and to set the stage for analysis of human orthologs of the genes in question. Previously, identification of mitochondrial fusion mediators in Drosophila and Saccharomyces cerevisiae led to deepened understanding of the genetic diseases optic atrophy and Charcot Marie Tooth syndrome, each associated with an ortholog of a mitochondrial fusion gene identified in model systems. Since many neurodegenerative diseases result from mitochondrial defects, the proposed work may in the long term uncover mechanisms underlying other such disorders. Mitochondrial defects underlie many neurodegenerative diseases and may be associated with premature aging as well as infertility. Elucidation of molecular mechanisms by which mitochondria are moved and shaped in different cell types will enable deeper understanding of mitochondria-related disorders, setting the stage for the design of treatments. ? ? ?
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