TRTech-PGR: Spatiotemporal Mapping of Protein Life in Plant Cell Signaling, Trafficking, and Development with the Next-Generation Tandem Fluorescent Timers

根据单个蛋白质的性质可视化生物体中的单个蛋白质是研究细胞蛋白质功能的重要分析过程。在生物发生过程中，蛋白质以独特的速率经历一系列成熟和转运过程，这些过程通常受发育和环境因素的调节。蛋白质的寿命往往决定了输出信号的振幅，从而决定了生物学结果。由于蛋白质的成熟、转运和周转可能以细胞特异性的方式和特定的亚细胞结构域发生，将蛋白质的年龄信息和位置信息联系起来有助于理解这一观察结果。然而，到目前为止，还没有标准的工具来可视化活植物组织中蛋白质的年龄。该项目将开发一系列植物优化的遗传编码传感器蛋白，即串联荧光计时器蛋白（tdFT），它会根据蛋白质的年龄改变颜色。tdft的工作原理简单，依赖于串联连接的绿色和红色荧光蛋白之间的成熟时间差异，只需要设置常规荧光显微镜。确定适合每种应用的最佳配置的困难阻碍了tdFT技术在工厂的广泛应用。该项目将为植物目的生成一个经过验证的、用户友好的tdFT载体工具箱。开发的tdFT将用于解决植物细胞生物学的基本问题，包括细胞壁生物合成蛋白和免疫信号蛋白的运输和周转以及气孔发育过程中的细胞谱系追踪。本项目旨在开发植物蛋白质时空非破坏性可视化分析管道，并研究体内关键交通/信号蛋白的细胞内运输/周转动力学。在活细胞中，新生和衰老蛋白质的动态时空分布是细胞信号传导的组成部分。传统的荧光标记策略，普遍用于研究界，通常提供蛋白质分布的端点概况，但缺乏蛋白质成熟和运输的时间动态解决方案。蛋白质成熟和各种翻译后修饰的基本调控过程只能通过时间敏感的荧光标签成像。tdft根据GFP和RFP的不同成熟时间产生时间特异性荧光特征。这是荧光蛋白的固有特性，不需要特殊的仪器，而不是传统的聚光或共聚焦显微镜。为了使tdFT技术广泛应用于植物研究，本项目将开发一系列不同时间范围的tdFT。每个tdFT将使用拟南芥和其他植物物种的瞬时表达系统进行验证。表征tdFT将用于解决植物生物学的基本问题，如植物免疫反应中复合物形成时的蛋白质稳定性，分泌系统中的膜蛋白周转，以及气孔发育过程中的细胞谱系分析。该奖项反映了美国国家科学基金会的法定使命，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，被认为值得支持。

Visualizing individual proteins in organisms based on their property is an essential analytical process for studying the function of cellular proteins. Upon biogenesis, proteins undergo a series of maturation and transport processes as well as turnover with a unique rate, which are often regulated by developmental and environmental cues. The longevity of proteins often determines the amplitude of output signals, which determines the biological outcomes. Because protein maturation, transport, and turnover could occur in cell-specific manner and at specific subcellular domains, linking age information and location information of proteins facilitate understanding the observation. However, to date, no standard tools to visualize the age of proteins in living plant tissues have been available. This project will develop a series of plant-optimized, genetically coded sensor proteins, i.e., tandem fluorescent timer proteins (tdFT), which change colors based on the protein ages. The operation principle of tdFTs is simple and relies on differential maturation time between green and red fluorescent proteins connected in tandem, and requires only conventional fluorescent microscopy set up. Difficulty in identifying optimum configurations specific to each application has been prohibitive for the broad application of tdFT technology in plants. The project will generate a toolbox of validated, user-friendly tdFT vectors for plant purposes. The developed tdFT will be deployed to address fundamental questions in plant cell biology, including transport and turnover of a cell wall biosynthesis protein and immunity signaling proteins and cell lineage tracking during stomatal development.This project aims to develop analysis pipelines for non-destructive visualization of spatiotemporal protein life in plants and examine intracellular transport/turnover dynamics of key traffic/signaling proteins in vivo. In living cells, dynamic spatiotemporal distributions of nascent and aged proteins are integral parts of cellular signaling. Conventional fluorescence-tagging strategies, which are prevalently used for the research community, typically provide an endpoint profile of protein distribution but lack the resolution in temporal dynamics of protein maturation and trafficking. Essential regulatory processes attributed to protein maturation and various post-translational modifications can only be imaged by time-sensitive fluorescent tags. tdFTs produce time-specific fluorescent signatures based on differential maturation times of GFP and RFP. These are an intrinsic property of fluorescent proteins, which do not require special instruments other than conventional epifluorescence or confocal microscopes. To make tdFT technology widely available for plant research, this project will develop a series of tdFT with various time ranges. Each tdFT will be validated using a transient expression system in Arabidopsis and other plant species. Characterized tdFT will be used to address fundamental questions in plant biology, such as protein stability upon complex formation during plant immune response, membrane protein turnover in the secretory system, and cell lineage analysis during stomatal development.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.