EAGER: Developing a Fluorescent Biosensor to Enable Real-Time Analysis of MAP Kinase Signaling Pathways in Living Arabidopsis Plants

知识价值。了解信号转导的分子机制是生物学家的重要研究目标。对模式植物拟南芥的研究表明，给定的信号分子具有处理信息的潜力，以响应各种不同的刺激。例如，已知来自拟南芥的MAP激酶蛋白MPK6参与与生物胁迫、非生物胁迫、细胞命运决定和细胞分裂相关的信号通路。这一观察结果提出了一个重要的实验问题，即一种蛋白质如何正确地解释和处理来自各种不同输入的信息。实现这种通路特异性的一种被提出的机制涉及空间分区的概念。在这个模型中，被生物应激源激活的MPK6激酶分子将被限制在一个亚细胞位置，而响应细胞分裂信号的MPK6分子将位于另一个亚细胞位置。为了直接测试空间调控在MAP激酶信号传导中的作用，有必要使用某种工具来可视化活细胞内MAP激酶活性的时空动态。监测MAP激酶活性的荧光生物传感器分子为这一挑战提供了一种解决方案，它们最近被开发用于监测人类MAP激酶。在这个项目中，将开发一种荧光生物传感器，能够测量拟南芥MAP激酶MPK6的活性。对于拟南芥，可以用共聚焦显微镜观察到一个完整的、活的有机体。此外，有了拟南芥，基因分析的全部力量可以用于涉及将要开发的激酶活性传感器类型的未来实验。MPK6生物传感器将首次为研究人员提供一种在亚细胞水平上监测活的拟南芥植物中蛋白激酶活性的时空动态的工具。由于MPK6在大量信号通路中发挥作用，因此这种工具将引起科学界的广泛兴趣。更广泛的影响。计划生产的MPK6生物传感器具有广泛应用于植物信号转导界成员的潜力。此外，该项目还将提供2名博士后、1名博士生和1名本科生的培训机会。本项目聘用的博士后将作为本科生的导师，为博士后提供宝贵的指导经验。

Intellectual Merit. Understanding the molecular mechanisms of signal transduction constitutes an important research goal for biological scientists. Work with the model plant Arabidopsis thaliana has revealed that a given signaling molecule has the potential to process information in response to a wide range of different stimuli. For example, the MAP kinase protein MPK6 from Arabidopsis is known to be involved in signaling pathways related to biotic stress, abiotic stress, cell fate determination, and cell division. This observation raises the important experimental question of how one protein can properly interpret and process information from a variety of different inputs. One proposed mechanism for achieving this type of pathway specificity invokes the concept of spatial compartmentalization. In this model, molecules of the MPK6 kinase that are activated by a biotic stressor would be restricted to one subcellular location, while MPK6 molecules responding to cell division cues would be located in another. To directly test the role that spatial regulation plays in MAP kinase signaling, it is necessary to have some type of tool for visualizing the spatial and temporal dynamics of MAP kinase activity within living cells. Fluorescent biosensor molecules that monitor MAP kinase activity provide one solution to this challenge, and they have recently been developed for monitoring human MAP kinases. For this project a fluorescent biosensor that will be able to measure the activity of the Arabidopsis MAP kinase MPK6 will be developed. With Arabidopsis, it is possible to observe with a confocal microscope an intact, living organism. In addition, with Arabidopsis the full power of genetic analysis can brought to bear on future experiments involving the type of kinase activity sensor that will be developed. An MPK6 biosensor will, for the first time, provide researchers with a tool for monitoring at the subcellular level the spatial and temporal dynamics of protein kinase activity in a living Arabidopsis plant. Because MPK6 plays a role in a large number of signaling pathways, such a tool would be of wide interest to the scientific community.Broader Impacts. The MPK6 biosensor that it is planned to produce has the potential to be widely used by members of the plant signal transduction community. In addition, this project will provide training opportunities for two post-doctoral fellows, one PhD student, and one undergraduate. The postdocs hired to work on this project will serve as the mentors for the undergraduate student, which will provide the postdocs with valuable mentoring experience.