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

TRTech-PGR：使用下一代串联荧光计时器绘制植物细胞信号传导、运输和发育中蛋白质生命的时空图谱

• 批准号: 2049642
• 负责人: Hisashi Koiwa
• 金额: $ 150万
• 依托单位: Texas A&M AgriLife Research
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-07-01 至 2025-06-30
• 项目状态: 未结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2049642&HistoricalAwards=false
• 关键词: TRTech; PGR; Spatiotemporal; Mapping; Protein

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.

根据其特性可视化单个蛋白质是研究细胞蛋白功能的基本分析过程。 生物发生后，蛋白质会经历一系列成熟和运输过程，并具有独特的速率，通常受发展和环境线索的调节。 蛋白质的寿命通常确定输出信号的幅度，这决定了生物学结果。 因为蛋白质成熟，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进行，因此将年龄信息和蛋白质的位置信息联系起来，促进了理解观察结果。 但是，迄今为止，还没有可视化活植物组织中蛋白质年龄的标准工具。该项目将开发一系列植物优化的，遗传编码的传感器蛋白，即串联荧光定时蛋白（TDFT），这些蛋白（TDFT）会根据蛋白质年龄来改变颜色。 TDFT的操作原理很简单，依赖于连接的绿色和红色荧光蛋白之间的差异成熟时间，并且仅需要设置常规的荧光显微镜。 难以识别每个应用程序特定的最佳配置，这对于TDFT技术在植物中的广泛应用而言是令人难以置信的。 该项目将生成一个用于工厂用途的经过验证的，用户友好的TDFT向量的工具箱。 开发的TDFT将被部署以解决植物细胞生物学中的基本问题，包括在气孔开发过程中运输和传输生物合成蛋白和免疫信号传导蛋白和免疫信号传导蛋白和细胞谱系跟踪。该项目旨在开发分析管道来开发非限制性可视化植物中的空间蛋白质寿命和静脉内的动力学动力学的动力学/转移量的动态。 在活细胞中，新生和老化蛋白的动态时空分布是细胞信号的组成部分。普遍用于研究界的常规荧光标记策略通常提供蛋白质分布的终点概况，但缺乏蛋白质成熟和运输的时间动态的分辨率。归因于蛋白质成熟和各种翻译后修饰的基本调节过程只能通过时间敏感的荧光标签来成像。 TDFTS基于GFP和RFP的差异成熟时间产生特异性荧光特征。 这些是荧光蛋白的内在特性，除常规落叶或共聚焦显微镜外，不需要特殊的仪器。为了使TDFT技术广泛用于植物研究，该项目将开发一系列具有不同时间范围的TDFT。 每个TDFT将使用拟南芥和其他植物物种中的瞬态表达系统进行验证。 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.

项目成果

Functional diversity of Medicago truncatula RNA polymerase II CTD phosphatase isoforms produced in the Arabidopsis thaliana superexpression platform

拟南芥超表达平台产生的蒺藜苜蓿 RNA 聚合酶 II CTD 磷酸酶亚型的功能多样性

• DOI: 10.1016/j.plantsci.2022.111309
• 发表时间: 2022
• 期刊: Plant Science
• 影响因子: 5.2
• 作者: Fukudome, Akihito;Ishiga, Yasuhiro;Nagashima, Yukihiro;Davidson, Katherine H.;Chou, Hsiu-An;Mysore, Kirankumar S.;Koiwa, Hisashi
• 通讯作者: Koiwa, Hisashi