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: 9138214
• 负责人: Lindsay Kyle Lewellyn
• 金额: $ 41.07万
• 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 2020-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9138214
• 关键词: Actin-Binding Protein; Actins; Adaptor Signaling Protein; Affect; American; Animal Model; Behavior; Biochemistry; Biological Assay; Biological Models; Biological Process; Caliber; Cell division; Cell physiology; Cells; Chemicals; Clustered Regularly Interspaced Short Palindromic Repeats; Complex; Couples; Cyst; Cytokinesis; Cytoplasm; Data; Defect; Development; Developmental Biology; Developmental Process; Diagnosis; Disease; Drosophila genus; Drosophila melanogaster; Epithelial Cells; Evolution; Failure; Family; Female; Fertility; Gametogenesis; Genetic Epistasis; Genetic Models; Germ Cells; Goals; Growth; Homologous Gene; Human; Immunofluorescence Immunologic; In Vitro; Infertility; Insecta; Lead; Learning; Link; Mammalian Cell; Mammals; Membrane; Molecular; Morphogenesis; Mus; Mutation; Neoplasm Metastasis; Nurses; Oocytes; Oogenesis; Organ; Organism; Pathway interactions; Phenotype; Phosphotransferases; Process; Proteins; RNA Interference; Reagent; Regulation; Research; Research Personnel; Role; Somatic Cell; Staging; Structure; Subfecundity; Supporting Cell; System; Techniques; Testing; United States; Western Blotting; Work; base; egg; fluorescence imaging; genome editing; in vivo; insight; loss of function; loss of function mutation; mutant; novel; overexpression; prevent; research study; sperm cell

PROJECT SUMMARY/ABSTRACT Infertility and impaired fecundity affect millions of Americans; studying normal gamete formation is essential to developing new treatments. Intercellular bridges are highly conserved structures that are essential for fertility in organisms from insects to mammals, and they allow for the coordinated behavior and sharing of cytoplasmic contents between neighboring cells. Although the structure of intercellular bridges has been studied for over 50 years, there is still much to be learned about their formation and regulation. The long-term goal of this project is to determine the molecular mechanisms that drive intercellular bridge formation, stabilization, and growth. One of the best-studied examples of intercellular bridges in gametogenesis is in the developing fruit fly egg chamber, which gives rise to the mature egg. These intercellular bridges, or ring canals, connect the 15 supporting nurse cells to the developing oocyte. They are rich in f-actin and actin binding proteins which allow them to expand in size to a final diameter of ~10 µm. The actin-nucleating Arp2/3 complex has been implicated in ring canals growth; however, the molecular mechanism by which the complex is localized to and activated at the ring canals is not known. Preliminary studies have identified two novel proteins at the germline ring canals of the developing egg chamber – the Ste20 family kinase, Misshapen (Msn), and the SH2/SH3 adaptor protein, Dreadlocks (Dock). Altering the levels of Msn or Dock leads to defects in ring canal structure and failure of bulk cytoplasmic transfer from the nurse cells to the oocyte. The objective of this proposal is to determine the role of Msn, Dock, and the Arp2/3 complex in ring canal growth. Because Msn and Dock function within the same pathway in other developmental contexts, and both have been linked genetically or biochemically to the Arp2/3 complex or its activators, this has led to the central hypothesis that Msn and Dock act cooperatively to promote ring canal growth through direct or indirect regulation of the Arp2/3 complex. Aim 1 will use clonal mutant analysis, RNAi, over-expression, molecular epistasis, and immunofluorescence to determine the role for Msn in growth of the germline ring canals. Aim 2 will use clonal mutant analysis, RNAi, genetic epistasis, immunofluorescence, and biochemistry to test whether Msn regulates the Arp2/3 complex during ring canal growth. In Aim 3, clonal mutant analysis, RNAi, epistasis, and immunofluorescence will be used to determine the role for Dock in ring canal growth. This work will provide important insight into the role for these highly conserved proteins in regulation of intercellular bridge growth. Identifying additional bridge components in Drosophila could impact our understanding of normal human gametogenesis as well as infertility. Both Msn and Arp2/3 homologs have been implicated in cancer metastasis, so characterizing their basic cellular functions would provide insight into their role in other diseases. This work will expose undergraduate researchers to cutting edge techniques and a powerful genetic model organism while answering important questions in developmental biology.

项目摘要/摘要 不孕症和繁殖力受损会影响数百万美国人；研究正常的配子形成对于 开发新的治疗方法。细胞间桥是高度组成的结构，对于生育至关重要 从昆虫到哺乳动物的生物，它们允许进行协调的行为和胞质共享 相邻细胞之间的内容。尽管细胞间桥的结构已经研究了50多个 多年来，关于它们的形成和监管仍然有很多需要了解的东西。这个项目的长期目标是 确定驱动细胞间桥形成，稳定和生长的分子机制。一 在配子发生中，在果蝇中最有研究的细胞间桥的例子是 腔室，产生成熟的鸡蛋。这些细胞间的桥或环管连接15 支持护士细胞到发育中的卵母细胞。它们富含F-肌动蛋白和肌动蛋白结合蛋白，允许 它们的尺寸扩大到最终直径约为10 µm。肌动蛋白 - 核ARP2/3复合物已与 在环管生长中；但是，该复合物被定位为在 环管尚不清楚。初步研究已经确定了种系环管处的两种新型蛋白质 发育中的卵室 - Ste20家族激酶，畸形（MSN）和SH2/SH3适配器蛋白 辫子（码头）。改变MSN或码头的水平会导致环管结构的缺陷和散装失败 细胞质从护士细胞转移到卵母细胞。该提议的目的是确定 MSN，码头和环管生长中的ARP2/3复合物。因为MSN和Dock在同一功能中功能 在其他发育环境中的途径，并且两者在ARP2/3上都一般或生化链接 复合物或其激活剂，这导致了一个中心假设，即MSN和Dock合作起来促进 通过直接或间接调节ARP2/3复合物的环管生长。 AIM 1将使用克隆突变体 分析，RNAi，过表达，分子位术和免疫荧光，以确定MSN在 AIM 2将使用克隆突变体分析，RNAi，遗传性遗传学， 免疫荧光和生物化学测试MSN是否调节环管过程中的ARP2/3复合物 生长。在AIM 3中，将使用克隆突变体分析，RNAi，上毒和免疫荧光来确定 码头在环管生长中的作用。这项工作将为这些角色提供重要的见解 保守的蛋白质在调节细胞间桥生长中。确定其他桥梁组件 果蝇可能会影响我们对正常人配子发生以及不育的理解。两个MSN 和ARP2/3同源物已在癌症转移中暗示，因此表征其基本细胞 功能将为他们在其他疾病中的作用提供洞察力。这项工作将揭露本科 研究人员对尖端技术和强大的遗传模型有机体，同时回答重要 发育生物学中的问题。