The role of Tubulin-binding cofactor E (TBCE)-Like, the protein product of the mu

微管蛋白结合辅因子 E (TBCE)-Like 的作用，mu 的蛋白质产物

• 批准号: 8750519
• 负责人: JAMES J FABRIZIO
• 金额: $ 32.18万
• 依托单位: COLLEGE OF MOUNT ST. VINCENT
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-15 至 2018-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8750519
• 关键词: Activities of Daily Living; Address; Animal Model; Animals; Architecture; Binding; Biogenesis; Biological Models; Cell Nucleus; Cell membrane; Cells; Complex; Cone; Confocal Microscopy; Contraceptive Agents; Contraceptive methods; Cyst; Cytoplasm; Cytoskeleton; Defect; Development; Drosophila genus; Dyes; Ectopic Expression; Electron Microscopy; Excision; F-Actin; Failure; Fertility; Flagella; Fluorescence Microscopy; Genes; Genetic; Giant Cells; Haploidy; Human; Immunofluorescence Immunologic; In Situ Hybridization; Individual; Infertility; Investments; Length; Male Contraceptions; Male Infertility; Mammals; Maps; Meiosis; Membrane; Messenger RNA; Microtubule Depolymerization; Microtubules; Molecular; Molecular Chaperones; Movement; Nuclear; Phenocopy; Phenotype; Process; Proteins; RNA Interference; Resolution; Role; Sperm Head; Sperm Tail; Spermatids; Spermatogenesis; Spermiogenesis; Stretching; System; Tail; Testis; Transcript; Travel; Tubulin; Work; base; cofactor; fly; human male; insight; male; man; molecular marker; mutant; neuronal cell body; novel; overexpression; paralogous gene; public health relevance; research study; spastin; sperm cell; time use

DESCRIPTION (provided by applicant): Since the mechanisms of spermatogenesis are highly conserved, insights into the causes of human male infertility, as well as insights into novel forms of male contraception, may come from studies on model organisms. Spermatogenesis in animals occurs in a syncytium, and individual spermatozoa are resolved from this syncytium during a post-meiotic step of spermatogenesis known as spermatid individualization. Individualization in Drosophila begins when a membrane-cytoskeleton complex known as the individualization complex (IC) assembles around the sperm heads and travels down the tails, squeezing out excess cytoplasm from between the sperm tails and reorganizes membrane around each of the spermatids as it travels. After individualization, each syncytial spermatid has been encased within its own plasma membrane and is thus transformed into an individual spermatozoon. ICs are, in part, composed of 64 F-actin-based investment cones, and each spermatid is individualized by one investment cone. While the investment cones of wild-type ICs travel in a coordinated ensemble, ICs from mulet mutant testes become severely discoordinated upon departure from the nuclei and fail to individualize, indicating that mulet is required for individualization. Since the mulet gene encodes Tubulin-Binding Cofactor E (TBCE)-like, a protein required for microtubule depolymerization, microtubule removal may be necessary for spermatid individualization. Indeed, a network of parallel inter-flagellar microtubules, normally removed from between the sperm tails prior to individualization, persists in mulet mutant testes. Thus, the removal of these microtubules appears to be necessary for the coordinated travel of the investment cones and spermatid individualization. The proposed work seeks to characterize the role of TBCE-like in Drosophila individualization. First, the mulet mutant phenotype will be fully characterized by fluorescence and confocal microscopy using known markers of individualization in order to determine if the observed individualization defect is caused by a defect in the IC itself or by the persistence of the inter-flagellar microtubule network. In order o determine where TBCE-like protein is required during spermatogenesis, in situ hybridization and immunofluorescence analyses will be employed to localize TBCE- like mRNA and protein, respectively. Rescue and RNAi experiments will also be conducted using the UAS/GAL4 system in order to distinguish between requirements for TBCE-like in the germline and the soma. Finally, the role of the inter-flagellar microtubule network in spermatid individualization will be elucidated using the UAS/GAL4 system to ectopically stabilize or depolymerize the network and examining the subsequent effects on spermatid individualization. The relationship between the investment cones and the microtubule network will also be examined using standard and spinning disk confocal microscopy. Ultimately, since TBCE- like is conserved in mammals and is expressed in mammalian testes, this work may uncover a novel aspect of individualization in flies and mammals, and thus pave the way for novel fertility treatments and contraception.

描述（申请人提供）：由于精子发生机制高度保守，因此对人类男性不育原因的见解以及对新形式的见解 男性避孕的方法，可能来自于对模式生物的研究。动物的精子发生发生在合胞体中，在精子发生的减数分裂后步骤中，单个精子从合胞体中分离出来，称为精子细胞个体化。果蝇的个体化开始于一种被称为个体化复合物（IC）的膜-细胞骨架复合物在精子头部周围组装并沿着尾部向下移动，从精子尾部之间挤出多余的细胞质，并在其移动过程中重组每个精子细胞周围的膜。个体化后，每个合胞体精子细胞被包裹在自己的质膜内，从而转化为单个精子。IC部分由64个基于F-肌动蛋白的包埋锥组成，每个精子细胞由一个包埋锥个性化。虽然野生型IC的投资锥旅行在一个协调的合奏，从mulet突变体睾丸的IC成为严重不协调后，离开细胞核和未能个性化，表明mulet是需要个性化。由于mulet基因编码微管蛋白结合辅因子E（TBCE）样蛋白，微管解聚所需的蛋白质，微管去除可能是精子细胞个体化所必需的。事实上，一个平行的鞭毛间微管网络，通常从精子尾部之间的个性化之前，持续存在于mulet突变睾丸。因此，这些微管的去除似乎是必要的投资锥和精子细胞个性化的协调旅行。拟开展的工作旨在描述TBCE样在果蝇个体化中的作用。首先，使用已知的个体化标记物通过荧光和共聚焦显微镜对mulet突变体表型进行充分表征，以确定观察到的个体化缺陷是由IC本身的缺陷还是由鞭毛间微管网络的持续性引起的。为了确定TBCE样蛋白在精子发生过程中所需的位置，将分别采用原位杂交和免疫荧光分析来定位TBCE样mRNA和蛋白。还将使用UAS/GAL 4系统进行拯救和RNAi实验，以区分种系和索马中对TBCE样的要求。最后，鞭毛间微管网络在精子细胞个体化中的作用将被讨论。 阐明了使用UAS/GAL 4系统异位稳定或去乙酰化的网络，并检查随后对精子细胞个体化的影响。包埋锥和微管网络之间的关系也将使用标准和旋转圆盘共聚焦显微镜检查。最终，由于TBCE样在哺乳动物中是保守的，并在哺乳动物睾丸中表达，这项工作可能会揭示苍蝇和哺乳动物个体化的一个新方面，从而为新的生育治疗和避孕铺平道路。