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

职业：果蝇生殖细胞器的结构功能分析

• 批准号: 1054962
• 负责人: Alexey Arkov
• 金额: $ 74.66万
• 依托单位: Murray State University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-05-01 至 2017-04-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1054962&HistoricalAwards=false
• 关键词: CAREER; Structure; Function; Analysis; Germ

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.

智力上的优点。胚胎生殖细胞最终被指定为精子和卵子，因此负责生命的连续性。 这些细胞属于一类有趣的干细胞，它们高度特化，并且能够在卵子和精子结合产生胚胎后产生所有细胞类型。 在许多生物体中，从轮虫到人类，生殖细胞都含有细胞器，称为生殖颗粒。尽管细菌颗粒早在 100 多年前就已被发现，但它们的组成、组装和功能尚不清楚。 由于一些已鉴定的颗粒成分对于生殖细胞的规范至关重要，因此推测这些细胞器在生殖细胞发育中发挥着重要的功能作用。已在各种生物体的胚芽颗粒中鉴定出含有小结构模块（Tudor 结构域）的蛋白质。最近发现这些结构域与果蝇、青蛙和哺乳动物中 Piwi 家族蛋白的甲基化氨基酸直接相互作用，证明了这种相互作用机制的显着进化保守性。 Tudor Piwi 复合体需要保护下一代 DNA 免受移动遗传元件引起的有害突变，从而凸显了该复合体的基本生物学作用。 尽管 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 复合物及其各个成分的结构尚不清楚。 该结构分析是该项目的一部分，将极大地促进对这些组件的组装及其在胚芽颗粒内的功能排列的理解。 这些研究将提供这种大颗粒大分子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。 具体来说，该项目的目标是 (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，2) 检验果蝇 Tudor Piwi 蛋白复合物的多个 Tudor 结构域的假设果蝇 Tudor 蛋白识别胚胎胚芽颗粒中的新靶标。 还将进行实验，以确定潜在的新 Tudor 靶标，使用新型体内化学交联方法稳定活体胚胎中的 Tudor 及其相互作用蛋白，并对所得纯化复合物进行后续质谱分析。 更广泛的影响。 这项研究将与教育活动相结合，包括对博士后研究员以及研究生和本科生的培训和指导。 在首席研究员的监督下，博士后学者和研究生将为大学高年级学生和研究生设计和讲授分子遗传学实验课程。此外，暑期还将积极招募高中实习生开展研究。 个人响应系统（PRS）将用于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响应来衡量）来评估纳入课程讲座的研究主题的影响。博士后研究员、学生和首席研究员将积极参加几项由大学支持的外展活动，包括在肯塔基州议会大厦举行的“Posters-at-the-Capitol”会议，在此期间，学生向州代表和公众展示他们的研究成果民众。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，该广播电台在五个州都有听众。默里州立大学的学生将被招募参与该项目，其中 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。

项目成果

Protein components of ribonucleoprotein granules from Drosophila germ cells oligomerize and show distinct spatial organization during germline development

• DOI: 10.1038/s41598-019-55747-x
• 发表时间: 2019-12
• 期刊: Scientific Reports
• 影响因子: 4.6
• 作者: Hieu D. L. Vo;Wahiduzzaman;Samuel J. Tindell;Jimiao Zheng;Ming Gao;A. Arkov