Genetic control of mitochondrial aggregation in Drosophila spermatogenesis

果蝇精子发生中线粒体聚集的遗传控制

• 批准号: 7252912
• 负责人: KAREN G HALES
• 金额: $ 21.18万
• 依托单位: DAVIDSON COLLEGE
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-06-01 至 2011-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7252912
• 关键词: ATP phosphohydrolase; Affect; Alleles; Binding; Biological Models; Biology; Cells; Cellular biology; Charcot-Marie-Tooth Disease; Chemicals; Cloning; DNA Transposable Elements; Defect; Depth; Development; Disease; Drosophila genus; Drosophila melanogaster; Dyes; Epitopes; Eukaryotic Cell; Family; Fertility; Food; Gene Expression; Gene Proteins; Genes; Genetic; Genetic Transcription; Genome; Goals; Green Fluorescent Proteins; Hereditary Disease; Homologous Gene; Human; Imagery; Infertility; Lead; Learning; Life; Location; Male Sterility; Mediating; Mediator of activation protein; Meiosis; Membrane Proteins; Microtubules; Mitochondria; Molecular; Morphogenesis; Movement; Mutagenesis; Mutation; Nature; Neurodegenerative Disorders; Optic Atrophy; Organelles; Orthologous Gene; Pattern; Phosphorus; Play; Polymerase Chain Reaction; Premature aging syndrome; Preparation; Process; Proteins; RNA Interference; Research; Role; Saccharomyces cerevisiae; Shapes; Specificity; Spermatids; Spermatocytes; Spermatogenesis; Staging; Sterility; Structure; Syndrome; Testing; Testis; Tissues; Transgenes; Transgenic Organisms; Work; alpha Tubulin; base; cell type; fly; fusion gene; genetic analysis; katanin; male; male health; member; mutant; paralogous gene; polyclonal antibody; positional cloning; promoter; protein expression; relating to nervous system; research study; spastin; therapy design

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.

描述（由申请人提供）：线粒体是一种细胞器，其将食物中的能量转化为可用的 ATP 形式，在特定的细胞类型内移动和成形，以满足不同的能量需求。该提案的重点是使用果蝇的精子发生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背景，因为线粒体在减数分裂和精子细胞发育过程中聚集、融合、缠绕、展开和伸长；这些过程中的缺陷通常会导致雄性不育，并且雄性不育突变体可以很容易地产生、维持和表征。该提案的大部分内容集中在 nmd 基因、其旁系同源物 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，它编码与功能未知的酿酒酵母线粒体外膜蛋白同源的 AAA+ ATP 酶。 Nmd 及其旁系同源物 CG4701 与切断微管的 AAA+ ATP 酶 spastin 和 katanin 60 相关，但不是直系同源的。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转基因果蝇的表位标记和分析来确定。 CG4701 的表达模式表明睾丸特异性，其功能将通过传统分析和 RNAi 突变分析来确定。 Nmd 和 CG4701 在微管动力学中的作用将通过突变体中微管结构的可视化以及通过评估 Nmd 和 CG4701 与微管的结合（野生型和永久 ATP 结合形式）来评估。 spastin和katanin 60在精子发生中的作用将通过spastin雄性不育等位基因以及睾丸特异性katanin 60旁系同源物的突变等位基因的表型分析来测试。最后，精子细胞中线粒体适当聚集所需的线粒体壳基因的表征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，并为分析相关基因的人类直向同源物奠定基础。此前，果蝇和酿酒酵母中线粒体融合介质的鉴定加深了对遗传病视神经萎缩和夏科玛丽图斯综合征的了解，每种疾病都与模型系统中鉴定的线粒体融合基因的直系同源物相关。由于许多神经退行性疾病是由线粒体缺陷引起的，因此从长远来看，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形成的分子机制将有助于更深入地了解线粒体相关疾病，为治疗设计奠定基础。