BTT EAGER: Cell type-specific profiling 2.0: Capturing subpopulations of cells undergoing a response

BTT EAGER：细胞类型特异性分析 2.0：捕获正在发生反应的细胞亚群

• 批准号: 1844803
• 负责人: Roger Deal
• 金额: $ 29.69万
• 依托单位: Emory University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-03-01 至 2023-02-28
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1844803&HistoricalAwards=false
• 关键词: BTT; EAGER; Cell; type; specific

Understanding how plants or animals function requires the ability to study molecular processes within specific cell types or even in single cells of the body. It is well known that cells within organisms respond to stimuli, such as light, gravity, hormones or any number of environmental triggers; however, when and how cells respond remains a mystery in part because it is technologically challenging to identify which cells are responding. Often responsive cells are not easily distinguished from non-responsive cells. In this high-risk, high reward project, the research team solves this problem by developing a molecular tool to identify and capture when and where cells respond to a stimulus, such as a hormone signal, in plant roots. The method will be broadly useful to detect other responses, and in many different plants and plant organs, due to its universal and modular design. The new technology will allow scientists to test new environments or treatments and identify how plant cells respond rapidly and precisely. The outcomes will impact researchers' ability to understand growth responses of diverse plants ranging from crops to model systems. Undergraduate students will have in-depth research training by contributing to the project during a summer research intensive. Recent advances in molecular profiling of specific plant cell types have generated important breakthroughs in understanding cell differentiation and cell type-specific stress responses. The research team has played a key role in this through the development and application of our INTACT (Isolation of Nuclei TAgged specific Cell Types) and TRAP (Translating Ribosome Affinity Purification) and TRAP (Translating Ribosome Affinity Purification) methods in crop plants. Despite the utility of INTACT and TRAP, there remains a pressing need to be able to capture the subsets of cells of a given cell type as they respond to a stimulus. Cells undergoing a response to such a stimulus are a subset of a population and cannot be easily captured by available methods. This limited resolution hinders the ability to address the molecular details of plant-microbe interactions, responses to hormone signaling, and spatially non-uniform abiotic stresses. The research team will address this problem by developing a system for selective purification of nuclei and translating ribosomes from cell subpopulations that are actively undergoing a specific response, such as drought sensing or interaction with a microbe. This will be achieved by producing two-component INTACT and TRAP systems in which one component (the biotinylatable nuclear envelope- or ribosomal-tagging protein) is expressed from a cell type-specific promoter, while Biotin ligase is driven from a stimulus-inducible promoter. In this way, the entire population of a cell type can be analyzed by purification of organelles using anti-GFP beads, while the subset of responsive cells can be analyzed by Streptavidin bead purification.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

了解植物或动物的功能需要能够研究特定细胞类型甚至身体单细胞内的分子过程。众所周知，生物体内的细胞会对刺激做出反应，如光、重力、激素或任何数量的环境触发因素;然而，细胞何时以及如何做出反应仍然是一个谜，部分原因是确定哪些细胞正在做出反应在技术上具有挑战性。反应性细胞通常不容易与非反应性细胞区分开。在这个高风险、高回报的项目中，研究小组通过开发一种分子工具来解决这个问题，该工具可以识别和捕获细胞何时何地对植物根部的刺激（如激素信号）做出反应。该方法将广泛用于检测其他响应，并在许多不同的植物和植物器官，由于其通用和模块化的设计。这项新技术将使科学家能够测试新的环境或治疗方法，并确定植物细胞如何快速准确地做出反应。这些结果将影响研究人员理解从作物到模型系统等各种植物的生长反应的能力。本科生将通过在夏季研究密集期间为该项目做出贡献来进行深入的研究培训。特定植物细胞类型分子谱的最新进展在理解细胞分化和细胞类型特定的应激反应方面取得了重要突破。该研究团队通过在作物中开发和应用我们的INTACT（分离核标记的特定细胞类型）和TRAP（翻译核糖体亲和纯化）和TRAP（翻译核糖体亲和纯化）方法，在这方面发挥了关键作用。尽管INTACT和TRAP具有实用性，但仍然迫切需要能够在给定细胞类型的细胞亚群响应刺激时捕获它们。经历对这种刺激的响应的细胞是群体的子集，并且不能通过可用的方法容易地捕获。这种有限的分辨率阻碍了解决植物-微生物相互作用的分子细节、对激素信号的响应以及空间非均匀的非生物胁迫的能力。研究小组将通过开发一种系统来解决这个问题，该系统用于选择性纯化细胞核并翻译来自细胞亚群的核糖体，这些细胞亚群正在积极地进行特定的反应，例如干旱感应或与微生物的相互作用。这将通过产生双组分INTACT和TRAP系统来实现，其中一种组分（可生物素化的核膜或核糖体标记蛋白）从细胞类型特异性启动子表达，而生物素连接酶从刺激诱导型启动子驱动。通过这种方式，可以通过使用抗GFP珠纯化细胞器来分析细胞类型的整个群体，而响应细胞的子集可以通过链霉亲和素珠纯化来分析。该奖项反映了NSF的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估而被认为值得支持。