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.

项目总结/摘要 不孕症和生育能力受损影响着数百万美国人;研究正常配子形成对 开发新的治疗方法细胞间桥是一种高度保守的结构，在哺乳动物中对生育力至关重要。 从昆虫到哺乳动物，它们允许协调行为和细胞质共享 相邻单元格之间的内容。尽管细胞间桥的结构已经研究了50多年， 多年来，关于它们的形成和调节，仍有许多东西需要了解。该项目的长期目标是 以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，细胞间桥的最佳研究实例是在发育中的果蝇卵中 室，这引起了成熟的鸡蛋。这些细胞间的桥，或称环形通道， 为发育中的卵母细胞提供营养细胞。它们富含肌动蛋白和肌动蛋白结合蛋白， 它们的尺寸扩大到最终直径约10微米。肌动蛋白成核Arp 2/3复合物与 在环道生长;然而，分子机制，该复合物是本地化和激活， 环运河是未知的。初步研究已经在生殖细胞环管中发现了两种新的蛋白质 发育中的卵室-Ste 20家族激酶，Misshapen（Msn）和SH 2/SH 3衔接蛋白， Dreadlocks（码头）。改变Msn或Dock的水平会导致环道结构的缺陷和散装的失败 细胞质从滋养细胞转移到卵母细胞。本建议的目的是确定以下方面的作用： Msn，码头，和Arp 2/3复合物在环管生长。由于MSN和Dock功能相同， 在其他发育环境中，这两种途径都与Arp 2/3基因或生物化学相关。 复合物或其激活剂，这导致了中心假设，即MSN和Dock合作促进 通过Arp 2/3复合物的直接或间接调节环管生长。目标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 生殖系环管的生长。目的2将利用克隆突变分析、RNAi、遗传上位性、 免疫荧光和生物化学来测试Msn是否在环管期间调节Arp 2/3复合物。 增长在目标3中，将使用克隆突变体分析、RNAi、上位性和免疫荧光来确定 Dock在环管生长中的作用。这项工作将提供重要的洞察这些高度的作用 调节细胞间桥生长的保守蛋白。识别其他网桥组件， 果蝇可能会影响我们对正常人类配子发生和不育的理解。两个Msn 和Arp 2/3同源物与癌症转移有关，因此表征了它们的基本细胞特征 这些功能将使我们深入了解它们在其他疾病中的作用。这项工作将暴露大学生 研究人员尖端技术和强大的遗传模式生物，同时回答重要的 发育生物学的问题。