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

描述(申请人提供)：由于精子发生的机制是高度保守的，对人类男性不育原因的洞察以及对新形式的洞察 关于男性避孕的研究，可能来自对模型生物的研究。动物的精子发生在一个合胞体中，在精子发生的减数分裂后阶段，单个精子从这个合胞体中分离出来，称为精子细胞个体化。果蝇的个体化开始时，一个被称为个体化复合体(IC)的膜-细胞骨架复合体聚集在精子头部周围，沿着精子尾部向下移动，从精子尾部之间挤出多余的细胞质，并在移动过程中重组每个精子细胞周围的膜。在个体化后，每个合体精子细胞都被包裹在自己的质膜中，从而转化为单独的精子。ICs部分由F-肌动蛋白为基础的包被锥组成，每个精子细胞由一个包被锥个体化。野生型ICs的投资锥体在协调的整体中运行，而MULET突变体睾丸的ICs在离开细胞核后变得严重不协调，无法个体化，表明MULET是个体化所必需的。由于MULET基因编码微管解聚所需的微管结合辅因子E(Tbce)样蛋白，因此去除微管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，通常在个体化之前从精子尾巴之间移除，在Mulet突变的睾丸中持续存在。因此，去除这些微管对于投资锥体的协调运动和精子细胞的个体化是必要的。这项拟议的工作试图表征类TBCE在果蝇个体化中的作用。首先，为了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存在引起的，将使用已知的个体化标记通过荧光和共聚焦显微镜对MULET突变表型进行充分表征。为了确定在精子发生过程中需要TBCE样蛋白的位置，将分别使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/GAL4系统进行救援和RNAi实验，以区分种系和体细胞中对类似TBCE的需求。最后，鞭毛间微管网络在精子细胞个体化中的作用将是 阐明了使用UAS/GAL4系统异位稳定或解聚该网络，并检测随后对精子细胞个体化的影响。还将使用标准和旋转圆盘共聚焦显微镜检查包被锥和微管网络之间的关系。最终，由于类TBCE在哺乳动物中保守，并在哺乳动物的睾丸中表达，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。

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.