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.

项目摘要 不孕症和生育力受损每年影响数百万人;因此，了解途径和 调节正常配子形成的机制是必需的。细胞间桥是细胞的基本结构 在整个动物王国的精子和卵子发育中发现;它们连接相邻的细胞， 材料共享和行为协调。这个项目的长期目标是了解如何 细胞间桥形成、稳定并经历扩张。生殖细胞间的桥或环 在发育中的果蝇卵室中发现的管道已经成为首要的模型系统。后形成 不完全的胞质分裂，生殖细胞环管经历了20倍的扩张，以促进材料的转移 从滋养细胞到卵母细胞影响环管形成、稳定性或 扩张导致不孕。许多结构和调节蛋白定位于环管和/或调节环管的结构。 它们的结构方面;然而，缺乏连接蛋白质和途径的综合模型。的 PI的实验室表征了Ste 20激酶Misshapen（Msn）、SH 2/SH 3衔接蛋白Dreadlocks （Dock），以及两种肌动蛋白成核剂Arp 2/3复合物和Arp， (Dia)，在环管的大小和稳定性的调节;然而，其背后的确切机制， 这些监管机构之间的贡献和联系尚不清楚。这项建议的目的是 确定这些蛋白质如何有助于环管的形成，稳定性和扩张，以及它们的活性如何 彼此整合并与其他已知的环管蛋白整合。核心假设是，MSN，Dock， Dia和Arp 2/3复合物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，并改变肌动蛋白细胞骨架，以促进环管扩张和维持稳定性。 目的1将检验粘附连接蛋白E-Cadherin的调节性内吞作用， 环道的大小和稳定性。目的2将确定Arp 2/3复合物和/或Dia是否间接调节环 通过对粘附连接或肌球蛋白活性的影响来扩张椎管。Aim 3将使用基于候选人的 一种鉴定Dock相互作用蛋白的方法，该蛋白协调肌动蛋白细胞骨架和细胞粘附的变化 在生殖细胞中。将使用适合本科生的技术组合，包括荧光 活体和固定样品的成像、定量图像分析、上位性实验和基础生物化学。 PI在指导25名本科生研究人员方面的良好记录，她在细胞和 发育生物学，以及果蝇模型系统的功能和可访问性，使她处于有利地位， 完成提出的目标。通过参与这些目标，学生将获得宝贵的培训， 这将增加他们对STEM领域的兴趣。正在研究的蛋白质、结构和过程 在正常形态发生期间也被利用，并且在疾病中可能被错误调节;因此， 这项工作将提高我们对人类正常发育和疾病的理解。