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.

智力优点。胚胎生殖细胞最终被指定为精子和卵，因此是生命的连续性。 这些细胞属于一类有趣的干细胞，它们高度专业化，能够在卵和精子结合后产生所有细胞类型。 在许多生物体中，像旋转器和人类一样多样化，生殖细胞包含细胞器，称为生殖颗粒。尽管从100年前发现了细菌颗粒，但它们的组成，组装和功能尚未得到很好的了解。 由于某些确定的颗粒成分对于生殖细胞规范至关重要，因此假设这些细胞器在生殖线发育中起主要功能作用。在各种生物的细菌颗粒中已经鉴定出含有小结构模块的蛋白质（tudor结构域）。最近发现，这些结构域与果蝇（果蝇），青蛙和哺乳动物中PIWI家族蛋白的甲基化氨基酸相互作用，证明了这种相互作用机制的显着进化保护。需要Tudor Piwi复合物来保护下一代DNA免受由移动遗传元件引起的有害突变，从而突出了该复合物的基本生物学作用。 尽管都铎式结构域蛋白和PIWI在胚芽颗粒中相互作用，但该保守的都铎Piwi复合物及其单个成分的结构尚不清楚。 这是该项目的一部分的结构分析，将大大提高对这些组件组装及其在细菌颗粒中的功能排列的理解。 这些研究将提供这种大型大分子颗粒复合物的第一个结构快照。此外，该项目将测试Tudor域蛋白经常包含多个与Tudor目标相互作用的域的想法。 具体而言，该项目的目的是（1）确定与单粒子冷冻电子微观显微镜专家合作的果蝇铎王朝Piwi蛋白复合物的结构，而2）测试了以下假说：果蝇蛋白质的多个tudor蛋白识别胚胎细菌性细菌剂的多个Tudor域识别胚胎细菌剂中的新型靶标。 还将使用一种新型的体内化学交联方法来识别潜在的新都铎王朝靶标，以稳定都铎王朝及其相互作用的蛋白质在生存胚胎中，以及随后对结果纯化复合物的质谱分析。 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员以及研究生和本科生。 在首席研究员的监督下，博士后学者和研究生将为大学生和研究生设计并教授分子遗传学实验室课程。此外，高中实习生将在夏季积极招募以进行研究。 个人响应系统（PRS）将用于评估基于直接学生兴趣和动机，使用远程输入设备来衡量的研究主题的影响。博士后研究人员，学生和主要研究人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海报典范会议，在此期间，学生将其研究向州代表和普通公众展示。此外，学生和调查员将讨论他们在大学广播电台的研究，该广播在五个州和大学周刊上都有听众。默里州立大学的学生是60％的女性和35％的第一代大学生，其中包括科学代表性不足的小组的大部分，他们将被招募参加该项目。

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