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

知识价值。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，但人们对它们的组成、组装和功能还不太了解。由于一些已鉴定的颗粒成分对生殖细胞的分化至关重要，因此人们假设这些细胞器在生殖细胞发育中起着重要的功能作用。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出来。最近，这些结构域在果蝇、青蛙和哺乳动物中被发现与Piwi家族蛋白甲基化氨基酸直接相互作用，表明这种相互作用机制具有显著的进化保守性。都铎Piwi复合体是保护下一代DNA免受由移动遗传元件引起的有害突变所必需的，从而突出了该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。作为该项目的一部分，这种结构分析将极大地促进对这些成分的组装及其在胚芽颗粒中的功能安排的理解。这些研究将提供这种颗粒大分子复合物的第一个结构快照。此外，该项目将测试都铎结构域蛋白经常包含与不同都铎目标相互作用的多个结构域的想法。具体来说，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)验证果蝇Tudor蛋白的多个Tudor结构域识别胚胎胚芽颗粒中的新靶点的假设。实验还将采用一种新的体内化学交联方法来确定潜在的新的Tudor靶点，以稳定Tudor及其在活胚胎中的相互作用蛋白，并随后对纯化的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，包括博士后研究员、研究生和本科生的培训和指导。在首席研究员的指导下，博士后和研究生将为大学高年级学生和研究生设计和教授一门分子遗传学实验课程。此外，将积极招募高中实习生在暑期进行研究。个人回应系统（PRS）将用于评估纳入课程讲座的研究课题的影响，根据学生的直接兴趣和动机，通过使用远程输入设备的计算机记录的回应来衡量。博士后研究员、学生和首席研究员将积极参加几项大学支持的外展活动，包括在肯塔基州议会大厦举行的国会大厦海报会议，在此期间，学生向州代表和公众展示他们的研究成果。此外，学生和调查人员将在大学广播和大学周报上讨论他们的研究，这些广播在五个州都有听众。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界代表性不足的群体，将被招募参加这个项目。

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.