Elucidating the mechanism of incomplete cytokinesis and intercellular bridge formation in animal germline cells

阐明动物生殖细胞不完全胞质分裂和细胞间桥形成的机制

• 批准号: 9908472
• 负责人: Kari L Price
• 金额: $ 6.49万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-02-01 至 2022-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9908472
• 关键词: Address; Affinity Chromatography; Alleles; Animals; Antibodies; Appearance; Behavior; Biochemical; Biogenesis; Biology; Biotinylation; Cardiac Myocytes; Cell division; Cell membrane; Cells; Characteristics; Clustered Regularly Interspaced Short Palindromic Repeats; Complex; Coupled; Cytokinesis; Cytology; Cytoplasm; Cytoskeleton; Data; Development; Disease; Drosophila genus; Drosophila melanogaster; Electron Microscopy; Electrons; Enzymes; Event; Excision; Female; Fertility; Gametogenesis; Germ Cells; Goals; Health; Hodgkin Disease; Human; Image; Imaging Techniques; Label; Lead; Light; Lymphoma; Lymphoproliferative Disorders; Mass Spectrum Analysis; Mediating; Membrane; Membrane Proteins; Mitosis; Modeling; Modification; Molecular; Mutagenesis; Mutation; Nature; Normal tissue morphology; Ovary; Pathway interactions; Phenotype; Prevalence; Process; Proteins; Proteome; Proteomics; Publishing; Reagent; Reed-Sternberg-like Cell; Regulation; Research Personnel; Role; Sterility; Structure; Testing; Testis; Tissue Extracts; Tissues; Transgenic Organisms; Urothelial Cell; Validation; daughter cell; density; design; electron density; electron tomography; experimental study; fly; genetic analysis; insight; interest; live cell imaging; loss of function; male; novel; physical separation; sex; spatiotemporal; temporal measurement

Proposal Summary The goal of this project is to investigate the mechanism of incomplete cytokinesis that results in the formation of intercellular bridges in animal germline cells. These bridges, called ring canals in Drosophila, are integral to the formation of gametes as disruption of these bridges results in sterility. Incomplete cytokinesis events are widely observed in not only the germline but in some somatic tissues and are a hallmark of classical Hodgkin lymphoma providing evidence that incomplete cytokinesis events are relevant to non-germline biology. Using the Drosophila germline as a model for incomplete cytokinesis, the experiments proposed are designed to characterize the formation of ring canals in male and female germlines, elucidate the composition of proteins at the ring canal membrane via biochemical screens, and characterize novel ring canal components identified from these screens. The three integrated aims of this proposal will provide a comprehensive understanding of the cytoskeletal and cellular events that mediate ring canal formation, identify proteins that contribute to ring canal formation, and assess the function of novel proteins required for ring canal formation in the male and female germlines. Experiments proposed in Aim 1 will define the cellular events that mediate ring canal formation in the Drosophila germline. The mechanism of ring canal formation is unknown as there are no published studies examining ring canal formation with high-temporal resolution in the developing ovary or testis. Furthermore, the composition of ring canals differs between males and females suggesting there are varying strategies in the formation of ring canals between the sexes. Ultrastructural studies of ring canals reveal they are marked by an electron density at the membrane, however the components of this density are unknown. Using a combination of live cell imaging and electron tomography, ring canal formation and the accompanying changes in membrane electron density will be characterized. These experiments will be of great interest to researchers studying germline biology. Experiments proposed in Aim 2 will identify ring canal proteins that may facilitate ring canal formation. A proximity-dependent biotinylation proteomic approach coupled with isolation of intact ring canal complexes will reveal the substructure of ring canal complexes and identify proteins for further validation and characterization. The proteomes of males and females will be compared, allowing further insight into the shared features of ring canal formation between the germlines. In Aim 3, candidate proteins identified in either screen will be validated and characterized to determine whether they contribute to the mechanism of ring canal formation. Protein localization will first be assessed using antibodies or fluorescent tags, followed by genetic analyses to test for function. Identification of proteins that contribute to the inhibition of membrane abscission will be of interest to researchers studying incomplete cytokinesis in both germline and non-germline contexts.

提案摘要 该项目的目标是研究导致形成不完全胞质分裂的机制 动物生殖细胞中的细胞间桥。这些桥梁在果蝇中被称为环管，是形成的组成部分 配子的破坏，因为这些桥梁的破坏导致不育。不完全胞质分裂事件不仅在 种系，但在某些体细胞组织中，是经典霍奇金淋巴瘤的标志，提供证据表明 不完整的胞质分裂事件与非种系生物学相关。使用果蝇种系作为模型 不完全胞质分裂，提出的实验旨在表征男性和女性环管的形成 雌性种系，通过生化筛选阐明环管膜上的蛋白质组成，以及 表征从这些屏幕中识别出的新型环管组件。该提案的三个综合目标将 提供对介导环管形成的细胞骨架和细胞事件的全面了解，识别 有助于环管形成的蛋白质，并评估环管形成所需的新型蛋白质的功能 在雄性和雌性种系中。 目标 1 中提出的实验将定义介导果蝇种系中环管形成的细胞事件。 环管形成的机制尚不清楚，因为没有已发表的研究检查环管形成 发育中的卵巢或睾丸的高时间分辨率。此外，环管的组成也不同 男性和女性表明两性之间环管的形成有不同的策略。超微结构 对环管的研究表明，它们以膜上的电子密度为标志，但其组成部分 密度未知。结合活细胞成像和电子断层扫描，环管形成和 膜电子密度的伴随变化将被表征。这些实验将会引起人们极大的兴趣 研究种系生物学的研究人员。目标 2 中提出的实验将鉴定环管蛋白，这些蛋白可能有助于 环管形成。邻近依赖性生物素化蛋白质组学方法与完整环管的分离相结合 复合物将揭示环管复合物的子结构并识别蛋白质以进行进一步验证和 表征。将比较男性和女性的蛋白质组，以便进一步深入了解男性和女性的共同特征 种系之间形成环管。在目标 3 中，将验证在任一筛选中鉴定的候选蛋白质并 表征以确定它们是否有助于环管形成的机制。蛋白质定位首先 使用抗体或荧光标签进行评估，然后进行遗传分析以测试功能。蛋白质鉴定 研究不完全胞质分裂的研究人员将对有助于抑制膜脱落的物质感兴趣 在种系和非种系环境中。