Localization and functional analysis of Nmd and CG4701 AAA proteins in mitochondrial and microtubule dynamics in Drosophila spermatogenesis. Bethany L. Wagner, Lindsay A. Regruto, Melissa Lorenzo, Jessica Gerard, Sarah C. Pyfrom, Karen G. Hales. Department of Biology, Davidson College, Davidson, NC.

   CG4701 and nmd are paralogous genes in Drosophila melanogaster that are associated with mitochondrial shaping defects during the early stages of spermatogenesis, resulting in recessive male sterility. Mutations in testis-enriched CG4701 cause polynucleated spermatids, suggestive of cytokinesis defects during meiosis, and vacuolated Nebenkerne (mitochondrial aggregates). The broadly-expressed and essential gene nmd has hypomorphic alleles with differing phenotypes. In males mutant for one allele, mitochondria fail to aggregate, preventing Nebenkern formation; in contrast, males with another allele display cytokinesis defects similar to CG4701. Preliminary results from nmd knockdown using RNAi show mutant phenotypes similar to the nmd allele with mitochondrial aggregation defects. Nmd localizes to mitochondria and centrosomes/basal bodies, and recent localization of a tagged version of CG4701 suggest that it colocalizes with Nmd. CG4701-RFP with a point mutation in the predicted transmembrane domain shows protein mislocalization and does not fully rescue the mutant phenotype. Both Nmd and CG4701 belong to the AAA ATPase family of proteins and are closely related to known microtubule severing proteins spastin and katanin. Therefore, we hypothesized that both Nmd and CG4701 interact with microtubules; we investigated the localization and expression of -tubulin in nmd and CG4701 mutants using GFP-tagged versions of the proteins. -tubulin-GFP partially rescued the nmd and CG4701 mutant phenotype providing support for a functional connection between Nmd and CG4701 and microtubules. Alternative detection techniques in testes from mutants will answer whether microtubule dynamics are altered in the mutants.