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

要了解植物或动物的功能，需要有能力研究特定细胞类型甚至身体单个细胞内的分子过程。众所周知，生物体内的细胞对刺激做出反应，如光、重力、激素或任何数量的环境触发因素；然而，细胞何时以及如何反应仍然是一个谜，部分原因是识别哪些细胞做出反应在技术上具有挑战性。通常，响应性细胞和非响应性细胞不容易区分。在这个高风险、高回报的项目中，研究团队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捉细胞对植物根中的刺激(如激素信号)做出反应的时间和地点。由于其通用性和模块化设计，该方法将广泛用于检测其他反应，以及在许多不同的植物和植物器官中。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而准确地做出反应。这些结果将影响研究人员理解从农作物到模型系统等不同植物的生长反应的能力。本科生将通过在暑期密集研究期间为该项目做出贡献来进行深入的研究培训。最近在特定植物细胞类型的分子图谱方面的进展在理解细胞分化和特定细胞类型的逆境反应方面取得了重要突破。研究小组通过在作物中开发和应用我们的INTERNAL(核标记特定细胞类型的分离)、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。尽管完整和陷阱技术很有用，但仍然迫切需要能够在特定细胞类型的细胞对刺激做出反应时捕获它们的子集。对这种刺激做出反应的细胞是种群的一个子集，不容易用现有的方法捕获。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、对激素信号的反应以及空间非均匀非生物胁迫的能力。研究小组将通过开发一种选择性纯化细胞核的系统来解决这个问题，并翻译活跃在经历特定反应的细胞亚群中的核糖体，例如干旱感应或与微生物的相互作用。这将通过生产双组分完整和TRAP系统来实现，其中一个组分(可生物素化的核膜或核糖体标记蛋白)从细胞类型特定的启动子表达，而生物素连接酶由刺激诱导的启动子驱动。这样，一种细胞类型的整个种群可以通过使用抗GFP珠子纯化细胞器来分析，而响应细胞的子集可以通过链霉亲和素珠子纯化来分析。这一奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

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.