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

根据生物体中的单个蛋白质的性质将其可视化是研究细胞蛋白质功能的基本分析过程。在生物发生过程中，蛋白质经历了一系列成熟、运输和周转的过程，并以独特的速度发生，这些过程往往受到发育和环境线索的调节。蛋白质的寿命通常决定了输出信号的幅度，而输出信号又决定了生物结果。由于蛋白质的成熟、运输和周转可以以细胞特有的方式和在特定的亚细胞区域发生，因此将蛋白质的年龄信息和位置信息联系起来有助于理解观察到的情况。然而，到目前为止，还没有标准的工具来可视化活的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、遗传编码的传感器蛋白，即串联荧光计时器蛋白(TdFT)，它根据蛋白质年龄改变颜色。TdFTs的工作原理很简单，依赖于串联的绿色和红色荧光蛋白之间的不同成熟时间，并且只需要建立传统的荧光显微镜。对于tdft技术在工厂中的广泛应用来说，很难确定针对每种应用的最佳配置。该项目将为工厂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中的基本问题，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跟踪。该项目旨在开发分析管道，用于非破坏性地显示植物中的时空蛋白生命，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，新生蛋白和衰老蛋白的动态时空分布是细胞信号的组成部分。在研究界普遍使用的传统荧光标记策略，通常提供了蛋白质分布的终点轮廓，但缺乏对蛋白质成熟和运输的时间动力学的分辨率。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记进行成像。TdFT根据GFP和RFP的不同成熟时间产生特定时间的荧光信号。这些都是荧光蛋白质的固有特性，除了传统的荧光或共聚焦显微镜外，不需要特殊的仪器。为了使tdFT技术广泛应用于植物研究，该项目将开发一系列不同时间范围的tdFT。每个tdFT都将在拟南芥和其他植物中使用瞬时表达系统进行验证。特色化的tdFT将用于解决植物生物学中的基本问题，如在植物免疫反应期间复杂形成时的蛋白质稳定性，分泌系统中的膜蛋白质周转，以及气孔发育期间的细胞谱系分析。该奖项反映了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。

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.