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.

项目概要/摘要 不孕症和生育力受损影响着数百万美国人；研究正常配子的形成至关重要； 开发新的治疗方法细胞间桥是高度保守的结构，对于生育能力至关重要。 从昆虫到哺乳动物的生物体，它们允许协调行为和共享细胞质 尽管细胞间桥的结构已被研究了 50 多个。 多年来，关于它们的形成和监管还有很多东西需要了解。该项目的长期目标是。 确定驱动细胞间桥形成、稳定和生长的分子机制。 配子发生过程中细胞间桥研究最充分的例子之一是正在发育的果蝇卵 这些细胞间桥或环管连接着 15 个细胞。 它们富含 f-肌动蛋白和肌动蛋白结合蛋白，为发育中的卵母细胞提供支持。 它们的尺寸扩大到最终直径约 10 µm，这与肌动蛋白成核 Arp2/3 复合物有关。 然而，在环管生长中，复合物定位并激活的分子机制 初步研究已经在种系环管中发现了两种新的蛋白质。 卵室发育的过程 – Ste20 家族激酶、Misshapen (Msn) 和 SH2/SH3 衔接蛋白， 辫子 (Dock) 改变 Msn 或 Dock 的水平会导致环管结构缺陷和散装失败。 该提案的目的是确定从护士细胞到卵母细胞的细胞质转移的作用。 Msn、Dock和Arp2/3复合体在环管内生长，因为Msn和Dock内部的功能相同。 其他发育环境中的途径，并且两者都在遗传或生物化学上与 Arp2/3 相关 复合体或其激活剂，这导致了中心假设：Msn 和 Dock 合作促进 通过直接或间接调节 Arp2/3 复合体的环管生长，Aim 1 将使用克隆突变体。 分析、RNAi、过表达、分子上位性和免疫荧光以确定 Msn 在 目标 2 将使用克隆突变分析、RNAi、遗传上位性、 免疫荧光和生物化学测试 Msn 是否在环管过程中调节 Arp2/3 复合物 在目标 3 中，将使用克隆突变体分析、RNAi、上位性和免疫荧光来确定生长。 这项工作将为了解这些高度的作用提供重要的见解。 鉴定细胞间桥生长调节中的保守蛋白。 果蝇可能会影响我们对正常人类配子发生以及不育症的理解。 和 Arp2/3 同源物与癌症转移有关，因此表征它们的基本细胞 这项工作将让本科生了解它们的功能。 研究人员利用尖端技术和强大的遗传模型生物体，同时回答重要的问题 发育生物学问题。