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

EAGER：开发荧光生物传感器以实时分析活体拟南芥植物中的 MAP 激酶信号通路

• 批准号: 1137950
• 负责人: Patrick Krysan
• 金额: $ 29.97万
• 依托单位: University of Wisconsin-Madison
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-03-15 至 2015-02-28
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1137950&HistoricalAwards=false
• 关键词: EAGER; Developing; Fluorescent; Biosensor; Enable

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.

智力优势。 了解信号转导的分子机制是生物科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以响应广泛的不同刺激的潜力。 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传导途径。 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。 一种用于实现这种类型的途径特异性的建议机制调用空间区室化的概念。 在这个模型中，被生物应激物激活的MPK6激酶分子将被限制在一个亚细胞位置，而对细胞分裂信号作出反应的MPK6分子将位于另一个亚细胞位置。 为了直接测试空间调节在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激酶活性的空间和时间动态。 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，并且它们最近被开发用于监测人类MAP激酶。 对于这个项目的荧光生物传感器，将能够测量拟南芥MAP激酶MPK6的活性将被开发。 对于拟南芥，可以用共聚焦显微镜观察完整的活体。 此外，与拟南芥的全部权力的遗传分析可以带来承担未来的实验，涉及类型的激酶活性传感器，将开发。 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拟南芥植物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。计划生产的MPK6生物传感器具有被植物信号转导社区成员广泛使用的潜力。此外，本项目还将为两名博士后研究员、一名博士生和一名本科生提供培训机会。受聘从事该项目的博士后将担任本科生的导师，这将为博士后提供宝贵的指导经验。