Remorin膜微区的超分辨率成像及其响应逆境胁迫的分子机制研究

质膜微区(microdomain)是调节质膜功能特化的基本单位，而微区蛋白则是细胞响应逆境胁迫重要组分。Remorin是植物中最早鉴定的微区蛋白，由于缺乏超时空分辨率成像及分析技术，不同Remorins形成膜微区的时空尺度的差异及其对胁迫响应的机制尚不明确。本项目拟在我们过去工作的基础上，通过运用光激活荧光蛋白标记、改制超分辨率成像和开发超分辨单分子追踪分析算法等，开展如下研究：1）分析6类Remorins的定位、运动规律和聚合状态；2）比较不同Remorins形成的不同尺度的膜微区的动力学和结构差异；3）Remorins和膜微区在胁迫响应中的动态观测和比较;4）膜微区和Remorins及其互作蛋白之间的互作网络对早期事件的响应规律。本项目旨在拓展单分子检测技术的综合联用，阐释Remorin膜微区对逆境响应的信号激发机制及其时空特异性，为质膜动态和膜微区活性调节的研究提供关键的技术手段。

Membrane microdomains are basic units for regulating structural and functional specialization of plasma membrane, while the functional proteins resided in membrane microdomains are important components for responding to biotic and abiotic stimuli. Remorins are the first definitely identified proteins in membrane microdomain, however, the underlying molecular and cellular mechanisms for the different spatiotemporal scale microdomains formed by Remorins are still largely unclear, mainly due to the lack of super resolution imaging and analytic techniques. Based on our previous studies, we will apply photoactivatable fluorescent protein (mEos3.2) to label six kinds of Remorins, reform the super-resolution imaging technology and develop single-molecule analysis algorithm to investigate microdomain dynamics and their early response to enviromental stimuli. First, we will analyze the localization, dynamics and sub-stoichiometric subunit status of the 6 different Remorin proteins. Then we will compare the dynamics and structure difference of membrane microdomains formed by different Remorins at different spatiotemporal scales. Moreover, we are going to investigate the early response of Remorins and microdomain during different enviromental stimuli. Finally, we will further explore the interaction networks involved in the responses to early stress stimuli among the microdomains, Remorins and their interaction proteins. This project will expand the single molecule imaging and analytical techniques to reveal the signal activation mechanism and spatiotemporal dynamics for early stress response of Remorins and their formed membrane microdomains. Surely, the results of this investigation will provide foundation techniques for the better studies of the membrane dynamics and the regulation of the activity of membrane microdomains.