Defective Drosophila spermatogenesis in CG4701 and nmd mutants possibly connected to faulty protein transport and peroxisome biogenesis
Mitochondria are shape-changing, energy harnessing organelles. Their morphogenesis can be studied in D. melanogaster spermatogenesis. The male sterile strains nmdP[ry4], nmdN162I, and CG4701 exhibit aberrant mitochondrial phenotypes. nmdP[ry4] mutants display aggregation failure, nmdN162I and CG4701 mutants show failed meiotic cytokinesis, and CG4701 mutants show vacuolated nebenkerns. Msp1, the S. cerevisiae ortholog of CG4701 and Nmd, traffics Peroxin proteins (PEX) from mitochondria to peroxisomes, and we suspect that CG4701 and Nmd may have related roles. Pex proteins regulate biogenesis of peroxisomes which metabolize very-long-chain-fatty-acids (VLCFA). We visualized peroxisomal organization at each stage of spermatogenesis in WT and CG4701-/-flies. We observed that peroxisomes normally demonstrate stage-specific patterns of organization, including being positioned near mitochondria and microtubule organizing centers (MTOCs), sites associated with Nmd localization. Co-visualization of MTOCs and peroxisomes confirmed co-localization. CG4701 males sometimes demonstrated reduced stage-specific peroxisomal clustering and smaller peroxisomes at the basal body from onion to elongation stage. In line with our hypothesis that defective peroxisome biogenesis is associated with defects in the nmd and CG4701 mutants, pex2 and pex13 mutants phenocopy the cytokinesis failure of nmd and CG4701 mutants. To test for a peroxisomal function of nmd and CG4701, we developed protocols for overloading peroxisomes via VLCFA food supplementation; preliminary results show shriveled testes and significant defects in the onion stage in pex13 mutants as compared to LCFA controls. Finally, to test whether pex mutations compromise nmd and CG4701 functions, we are comparing localization of Nmd-GFP and CG4701-RFP in pex mutants to their localization in WT. Our results thus far support the protein transport hypothesis.