The growth of the germline ring canals during Drosophila melanogaster oogenesis

果蝇卵子发生过程中种系环管的生长

• 批准号: 10046938
• 负责人: Lindsay Kyle Lewellyn
• 金额: $ 41.31万
• 依托单位: BUTLER UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-01 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10046938
• 关键词: Actin-Binding Protein; Actins; Adaptor Signaling Protein; Adherens Junction; Affect; American; Animals; Authorship; Behavior; Biochemical; Biochemistry; Biological Models; Caliber; Cell Adhesion; Cells; Cellular biology; Complex; Cytokinesis; Cytoskeleton; Data; Data Analyses; Defect; Development; Developmental Biology; Diagnosis; Disease; Docking; Drosophila genus; Drosophila melanogaster; Dynamin; E-Cadherin; Elements; Endocytosis; Equipment; Experimental Designs; Funding; Gametogenesis; Genetic; Genetic Epistasis; Germ Cells; Goals; Growth; Guanosine Triphosphate Phosphohydrolases; Human; Image Analysis; Immunoprecipitation; Infertility; Label; Lead; Measures; Membrane; Mentors; Messenger RNA; Modeling; Modernization; Molecular; Monitor; Morphogenesis; Mutation; Myosin ATPase; Myosin S-2; Normal Statistical Distribution; Nurses; Oocytes; Oogenesis; Organelles; Pathway interactions; Phosphotransferases; Positioning Attribute; Process; Protein Kinase; Proteins; Publishing; Quantitative Microscopy; RNA Interference; Reagent; Regulation; Research; Research Personnel; Role; STEM field; Sampling; Somatic Cell; Structural Protein; Structure; Students; Subfecundity; System; Techniques; Testing; Time; Training; Wasps; Western Blotting; Work; base; beta catenin; egg; experience; experimental study; fluorescence imaging; genetic analysis; genetic regulatory protein; improved; infertility treatment; insight; interest; overexpression; quantitative imaging; recruit; sperm cell; trafficking; undergraduate student

Project Summary Infertility and impaired fecundity affect millions of people each year; therefore, understanding the pathways and mechanisms that regulate normal gamete formation are necessary. Intercellular bridges are essential structures found in developing sperm and eggs across the animal kingdom; they connect neighboring cells and allow the sharing of materials and coordination of behaviors. The long-term goal of this project is to understand how intercellular bridges are formed, stabilized, and undergo expansion. The germline intercellular bridges, or ring canals, found in the developing fruit fly egg chamber have emerged as the premier model system. Formed after incomplete cytokinesis, the germline ring canals undergo a 20-fold expansion to facilitate the transfer of materials from the supporting nurse cells to the developing oocyte. Mutations that affect ring canal formation, stability, or expansion lead to infertility. Many structural and regulatory proteins localize to the ring canals and/or regulate aspects of their structure; however, an integrated model connecting the proteins and pathways is lacking. The PI’s lab characterized a role for the Ste20 kinase, Misshapen (Msn), the SH2/SH3 adaptor protein, Dreadlocks (Dock), and the coordinated activity of two actin nucleators, the Arp2/3 complex and the formin, Diaphanous (Dia), in the regulation of ring canal size and stability; however the precise mechanisms underlying their contribution and the connections between these regulators is not known. The objective of this proposal is to determine how these proteins contribute to ring canal formation, stability, and expansion, and how their activity is integrated with each other and with other known ring canal proteins. The central hypothesis is that Msn, Dock, Dia, and the Arp2/3 complex spatially and temporally coordinate endocytosis of adherens junction proteins, myosin activity, and changes to the actin cytoskeleton to promote ring canal expansion and maintain stability. Aim 1 will test the hypothesis that regulated endocytosis of the adherens junction protein, E-Cadherin, regulates ring canals size and stability. Aim 2 will determine whether the Arp2/3 complex and/or Dia indirectly regulate ring canal expansion through effects on adherens junctions or myosin activity. Aim 3 will use a candidate-based approach to identify Dock-interacting proteins that coordinate changes in the actin cytoskeleton and cell adhesion in the germline. A combination of undergraduate-appropriate techniques will be used, including fluorescence imaging of live and fixed samples, quantitative image analysis, epistasis experiments, and basic biochemistry. The PI’s strong track record of mentoring 25 undergraduate student researchers, her expertise in cell and developmental biology, and the power and accessibility of the fruit fly model system make her well-positioned to complete the proposed aims. Through their involvement in these aims, students will gain valuable training and experience that will increase their interest in STEM fields. The proteins, structures, and processes being studied are also utilized during normal morphogenesis and can be mis-regulated in disease; therefore, insight from this work will improve our understanding of both normal development and disease in humans.

项目概要 因此，不孕不育和生育力受损每年都会影响数百万人； 调节正常配子形成的机制是必要的结构。 发现在动物界的精子和卵子中发育；它们连接相邻的细胞并允许 该项目的长期目标是了解如何共享材料和协调行为。 细胞间桥形成、稳定并经历扩张。种系细胞间桥或环。 在发育中的果蝇卵室中发现的运河已成为第一个模型系统。 胞质分裂不完全，种系环管扩张20倍以促进物质转移 从支持护士细胞到发育中的卵母细胞的突变，影响环管的形成、稳定性或。 许多结构和调节蛋白定位于环管和/或调节。 它们结构的各个方面；然而，缺乏连接蛋白质和途径的集成模型。 PI 的实验室描述了 Ste20 激酶、Misshapen (Msn)、SH2/SH3 接头蛋白、辫子的作用 (Dock)，以及两个肌动蛋白成核剂、Arp2/3 复合物和福明 (Diaphanous) 的协调活动 (Dia)，在调节环管尺寸和稳定性方面；然而其背后的精确机制； 这些监管机构之间的贡献和联系尚不清楚。该提案的目的是 确定这些蛋白质如何促进环管形成、稳定性和扩张，以及它们的活性如何 相互整合并与其他已知的环管蛋白整合 中心假设是 Msn、Dock、 Dia 和 Arp2/3 复合体在空间和时间上协调粘附连接蛋白的内吞作用， 肌球蛋白活性，以及​​肌动蛋白细胞骨架的变化，以促进环管扩张并保持稳定性。 目标 1 将检验以下假设：调节粘附连接蛋白 E-钙粘蛋白的内吞作用，调节 环管的大小和稳定性将决定 Arp2/3 复合体和/或 Dia 是否间接调节环。 通过影响粘附连接或肌球蛋白活性来进行运河扩张，目标 3 将使用基于候选的方法。 识别协调肌动蛋白细胞骨架和细胞粘附变化的对接相互作用蛋白的方法 将使用适合本科生的技术组合，包括荧光。 活体和固定样品成像、定量图像分析、上位实验和基础生物化学。 该 PI 在指导 25 名本科生研究人员方面拥有良好的记录，她在细胞和 发育生物学以及果蝇模型系统的强大功能和可访问性使她能够很好地 通过参与这些目标，学生将获得宝贵的培训和帮助。 经验将增加他们对 STEM 领域正在研究的蛋白质、结构和过程的兴趣。 在正常形态发生过程中也被利用，并且在疾病中可能被错误调节；因此，由此可见 这项工作将增进我们对人类正常发育和疾病的理解。