The growth of the germline ring canals during Drosophila melanogaster oogenesis

项目摘要/摘要 不孕不育和生育能力受损影响着数以百万计的美国人；研究正常的配子形成对于 开发新的治疗方法。细胞间桥是高度保守的结构，对植物的生育能力至关重要 从昆虫到哺乳动物，它们允许细胞质的协调行为和共享 相邻单元格之间的内容。尽管细胞间桥的结构已经被研究了50多年 几年来，关于它们的形成和调控仍有许多需要了解的地方。这个项目的长期目标是 以确定驱动细胞间桥形成、稳定和生长的分子机制。一 在配子发生中研究得最好的例子之一是发育中的果蝇卵。 小室，产生成熟的卵子。这些细胞间桥，或环形运河，连接着15个 支持哺乳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，使 它们的大小扩大到最终直径约10微米。 在环状渠道的生长中；然而，复合体定位和激活的分子机制 环状运河尚不为人所知。初步研究已经在胚系环管中发现了两种新的蛋白质 发育中的卵室-Ste20家族激酶、畸形(MSN)和SH2/SH3接头蛋白， 长发(Dock)。改变MSN或Dock水平会导致环管结构缺陷和散装失效 从哺育细胞到卵母细胞的细胞质转移。这项提案的目标是确定 MSN、Dock和Arp2/3复合体在环管生长中的作用。因为MSN和Dock在相同的 在其他发育环境中，这两种途径都与Arp2/3有遗传或生化联系 复合体或其激活剂，这导致了核心假设，即MSN和Dock协同行动以促进 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、过度表达、分子上位性和免疫荧光来确定MSN在 胚系环管的生长。AIM 2将使用克隆突变分析、RNAi、遗传上位性、 免疫荧光和生化检测MSN是否调节环管内Arp2/3复合体 成长。在目标3中，将使用克隆突变分析、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力，以了解这些高度 调控细胞间桥生长的保守蛋白。在中标识其他网桥组件 果蝇可能会影响我们对正常人类配子发生和不孕不育的理解。两者都是MSN 而Arp2/3同系物与癌症转移有关，因此描述了它们的基本细胞 这些功能将提供对它们在其他疾病中的作用的洞察。这项工作将使本科生 研究人员以尖端技术和强大的遗传模式生物同时回答重要 发育生物学中的问题。

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.