QUANTIFYING ACTIVATION DYNAMICS OF SYNTHETIC MAP KINASE CASCADE BY 2P-FLIM

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 我们希望进行定量实时成像的激酶ERK在合成的哺乳动物MAP激酶级联，我们已经在酿酒酵母中构建。使用新的基因编码的FRET传感器，是特定的ERK，我们建议使用双光子荧光寿命成像，以量化其激活动力学，无论是暂时的和空间的。我们拥有的两个FRET传感器是基于荧光对EGFP-mRFP 1和Cerulean-Venus。我们打算遵循开发传感器的小组最近发表的方法。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. We wish to carry out quantitative real-time imaging of the kinase ERK in synthetic mammalian MAP kinase cascade that we have constructed in saccharomyces cerevisiae. Using novel genetically encoded FRET sensors that are specific for ERK, we propose to use 2-photon fluorescence lifetime imaging to quantify its activation dynamics, both temporarily and spatially. The two FRET sensors that we have are based on the fluorescent pairs EGFP-mRFP1 and Cerulean-Venus. We intend to follow the method that was published recently by the group that developed the sensors.