CAREER: Structure-Function Analysis of Germ Cell Organelles in Drosophila

智力上的优点。胚胎生殖细胞最终被指定为精子和卵子，因此负责生命的延续。 这些细胞属于一类有趣的干细胞，它们既高度特化，又能在卵子和精子结合产生胚胎后产生所有类型的细胞。 在许多生物中，如轮虫和人类，生殖细胞含有细胞器，称为生殖颗粒。虽然胚芽颗粒早在100多年前就被发现了，但它们的组成、组装和功能还没有得到很好的理解。 由于一些确定的颗粒成分是至关重要的生殖细胞的规格，它已被假设，这些细胞器在生殖细胞发育中发挥重要的功能作用。含有小结构模块（Tudor结构域）的蛋白质已在各种生物的胚芽颗粒中被鉴定。最近，这些结构域被发现直接相互作用的Piwi家族蛋白质的甲基化氨基酸在苍蝇（果蝇），青蛙，和哺乳动物，证明了显着的进化保守的这种相互作用机制。Tudor Piwi复合体被要求保护下一代DNA免受移动的遗传元件引起的有害突变，从而突出了这种复合体的基本生物学作用。 虽然Tudor结构域蛋白和Piwi在胚芽颗粒中相互作用，但这种保守的Tudor Piwi复合物及其单个组分的结构尚不清楚。 这种结构分析，这是该项目的一部分，将大大推进这些组件的组装和它们在胚芽颗粒内的功能安排的理解。 这些研究将提供这种大分子颗粒复合物的第一个结构快照。此外，该项目还将测试Tudor结构域蛋白经常包含与不同Tudor靶标相互作用的多个结构域的想法。 具体而言，该项目的目的是（1）与单粒子冷冻电子显微镜专家合作确定果蝇Tudor Piwi蛋白复合物的结构，以及2）测试果蝇Tudor蛋白的多个Tudor结构域识别胚胎生殖颗粒中的新靶点的假设。 还将进行实验，以确定潜在的新的Tudor目标，使用一种新的体内化学交联方法来稳定Tudor及其在活胚胎中的相互作用蛋白，并随后对所得纯化复合物进行质谱分析。 更广泛的影响。 这项研究将与教育活动相结合，包括培训和指导博士后研究人员以及研究生和本科生。 在主要研究者的监督下，博士后学者和研究生将为大学毕业生和研究生设计和教授分子遗传学实验室课程。此外，还将积极招募高中实习生在暑期开展研究。 一个个人反应系统（PRS）将被用来评估研究课题的影响纳入课程讲座的基础上，直接学生的兴趣和动机，通过计算机记录的响应使用远程输入设备测量。博士后研究员，学生和主要研究者将积极参加几个大学支持的外展活动，包括在肯塔基州国会大厦，在此期间，学生提出他们的研究，他们的国家代表和公众的海报在国会大厦会议。此外，学生和研究人员将在大学广播电台和大学周报上讨论他们的研究，该电台在五个州有听众。默里州立大学的学生中，60%是女性，35%是第一代大学生，其中很大一部分来自科学领域代表性不足的群体，他们将被招募参加这一项目。

Intellectual merit. Embryonic germ cells are eventually specified to become sperm and egg, and therefore are responsible for continuity of life. These cells belong to an intriguing class of stem cells, which are both highly specialized and able to give rise to all cell types after the union of egg and sperm produces an embryo. In many organisms, as diverse as rotifer and human, germ cells contain organelles, known as germ granules. Although germ granules were discovered more than 100 years ago, their composition, assembly, and function are not well understood. Since some of the identified granule components are crucial for germ cell specification, it has been hypothesized that these organelles play a major functional role in germline development. Proteins containing small structural modules (Tudor domains) have been identified in germ granules of various organisms. Recently these domains were found to directly interact with methylated amino acids of Piwi family proteins in flies (Drosophila), frogs, and mammals, demonstrating the remarkable evolutionary conservation of this interaction mechanism. The Tudor Piwi complex is required to safeguard the next generation of DNA against harmful mutations caused by mobile genetic elements, thus highlighting the fundamental biological role of this complex. Although Tudor domain proteins and Piwi interact in germ granules, the structure of this conserved Tudor Piwi complex and of its individual components is not known. This structural analysis, which is part of this project, will greatly advance the understanding of the assembly of these components and their functional arrangement within germ granules. The studies will provide the first structural snapshot of this large macromolecular complex of the granules. In addition, the project will test the idea that Tudor domain proteins frequently contain multiple domains that interact with different Tudor targets. Specifically, the aims of this project are (1) to determine the structure of the Drosophila Tudor Piwi protein complex in collaboration with an expert in single-particle cryo-electron microscopy, and 2) to test the hypothesis that the multiple Tudor domains of the Drosophila Tudor protein recognize novel targets in embryonic germ granules. Experiments will also be undertaken to identify potential new Tudor targets using a novel in vivo chemical crosslinking approach to stabilize Tudor and its interacting proteins in living embryos, and a subsequent mass spectrometry analysis of resulting purified complexes. Broader impacts. This research will be integrated with educational activities, including training and mentoring of a postdoctoral researcher and graduate and undergraduate students. Under the supervision of the principal investigator, the postdoctoral scholar and graduate students will design and teach a molecular genetics laboratory course for college seniors and graduate students. In addition, high school interns will be actively recruited to carry out research during the summer. A Personal Response System (PRS) will be used to evaluate the impact of research topics incorporated into course lectures based on immediate student interest and motivation as measured by computer-logged responses using remote input devices. The postdoctoral researcher, students, and the principal investigator will actively participate in several university-supported outreach activities, including the Posters-at-the-Capitol conference at the Kentucky state capitol, during which students present their research to their state representatives and the general public. Furthermore, the students and investigator will discuss their research on the university radio, which has a listening audience in five states, and in the university weekly newspaper. Students at Murray State University, who are 60% women and 35% first-generation college students, including a large proportion from groups underrepresented in science, will be recruited to participate in this project.