Genetic control of mitochondrial aggregation in Drosophila spermatogenesis

描述(由申请人提供)：线粒体是一种细胞器，将食物中的能量转化为可用的三磷酸腺苷形式，在特定的细胞类型中移动和塑造，以满足不同的能量需求。这项建议的重点是利用果蝇精子发生的模型系统来阐明线粒体形态发生，特别是线粒体运动发生的分子机制。果蝇精子发生是分析线粒体形态发生的理想环境，因为线粒体在减数分裂和精子细胞发育过程中聚集、融合、包裹、展开和伸长；这些过程中的缺陷通常会导致雄性不育，并且雄性不育突变很容易产生、维持和表征。大多数提案都集中在NMD基因及其同源基因CG4701，以及NMD和CG4701所属的AAA+ATPase家族的其他成员上。NMD基因是果蝇精子发生过程中线粒体聚集所必需的，它编码一个与酿酒酵母线粒体外膜蛋白同源的AAA+ATPase，功能未知。NMD及其类似的CG4701与切断微管的AAA+ATPase spastin和katanin 60相关，但不是同源的。总体目标是确定这些基因产物影响线粒体运动和聚集的机制。NMD和CG4701的亚细胞定位将通过表位标记和转基因果蝇的分析来确定。CG4701的功能将通过传统的和RNAi突变分析来确定，其表达模式表明了睾丸的特异性。NMD和CG4701在微管动力学中的作用将通过对突变体中微管结构的可视化以及通过评估NMD和CG4701与微管(野生型和永久结合ATP的形式)的结合来评估。Spastin和katanin 60在精子发生中的作用将通过对spastin雄性不育等位基因和睾丸特异katanin 60 paralog突变等位基因的表型分析来测试。最后，线粒体在精子细胞中适当聚集所必需的线粒体基因的鉴定和克隆，将从另一个角度阐明线粒体聚集的分子机制。这项研究的长期目标是了解线粒体生物学的基本分子机制，并为分析人类相关基因的同源基因奠定基础。此前，在果蝇和酿酒酵母中鉴定线粒体融合介体有助于加深对遗传性疾病视神经萎缩和夏科玛丽-牙齿综合征的理解，每种遗传病都与模型系统中确定的线粒体融合基因的同源基因有关。由于许多神经退行性疾病是由线粒体缺陷引起的，从长远来看，拟议的工作可能会揭示其他此类疾病的潜在机制。线粒体缺陷是许多神经退行性疾病的基础，可能与过早衰老和不孕不育有关。阐明线粒体在不同细胞类型中移动和塑造的分子机制将使人们能够更深入地了解线粒体相关疾病，为治疗方案的设计奠定基础。

DESCRIPTION (provided by applicant): 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.