单一荧光探针对细胞内活性氧簇调控磷酸酶机制的影像分析

Reactive oxygen species (ROS) and phosphatase are very important to cellular signal transduction. Meanwhile, ROS play a key role to regulate the activity of phosphatase in the process of signal transduction. However, the multi-parallel assays of several signal substances with multiple probes are often limited by the discrepant biological locations together with large invasive effects and difficult quantification. In this project, we conceive a strategy of using single fluorescent probe with dual-response ability for simultaneous visualization of ROS and phosphatase, and further reveal the correlation between ROS and phosphatase activities. The investigations in this project mainly include: (1) selecting two fluorophores with the discriminative responses to ROS and phosphatase, then to construct them in to a single molecular switch for the two targeted analytes; (2) illustrating the fluorescent signal readout mechanism on the basis of spectra profiles, and building a multi-channel method for simultaneous imaging of cellular ROS and phosphatase; (3) real-time monitoring ROS and phosphatase, and revealing the regulation mechanism of phosphatase by ROS. The successful performance of this project is expected to provide a new visual measuring technique for evaluating the correlated multiple analytes in vivo, and open a new venue in the field of cellular signaling.

活性氧簇和磷酸酶是重要的细胞内信号传导物质，同时活性氧簇对磷酸酶活性的调控在信号通路中起着至关重要的作用。对细胞信号通路中多组分的监测一直是影像分析的难题，多重荧光探针策略存在细胞内定位差异、入侵效应大、定量化困难等问题。本项目拟开发细胞内活性氧簇和磷酸酶双响应的单一荧光探针，利用多通道荧光成像策略，实现对活性氧簇及其调控磷酸酶活性过程的同步成像分析，并揭示两者之间的关联性：（1）筛选对活性氧簇和磷酸酶产生具有可区分光谱响应的荧光团，设计合成对活性氧簇和磷酸酶具有双重荧光响应的单一分子开关；（2）阐明探针分子的荧光信号读出与光谱选择性机制，建立多通道荧光成像方法，实现对细胞内活性氧簇和磷酸酶活性的同步影像分析；（3）实时监测细胞内活性氧簇对磷酸酶活性的影响，揭示活性氧簇对磷酸酶活性调控的分子机制。本项目的成果将为生物体内关联性多组分检测和细胞内信号传导的影像分析提供新的思路和手段。

细胞内信号小分子对生物大分子的调节作用对细胞信号传导至关重要，但是同时对信号小分子和大分子进行同步监测仍然是生物影像分析的难题。为了克服多重荧光探针对细胞正常生理条件的干扰等问题，本项目开发了一系列对细胞多重生物分子具有区分响应的单分子荧光探针，通过超分辨成像，实现对不同生物分子关联性的同步成像分析，揭示两者之间的调控机制。主要发现点和研究成果如下：1）设计对信号分子和磷酸酶具有双响应的单分子荧光探针，实时追踪二者在活细胞内水平和活性的变化，揭示了细胞内活性氧和硫化氢水平的变化对磷酸酶活性的影响；2）通过构建对活性氧、蛋白质和核酸三响应的线粒体定位荧光探针，实现了对三种物质动力学的同步实时追踪，发现了活性氧对调节线粒体核蛋白的解离起着重要的作用；3）分别合成对脂滴、线粒体和自噬溶酶体标记的荧光探针，监测活性氧水平升高时多重细胞器之间的相互作用。上述研究工作取得了多项具有创新性和系统性的研究成果，部分研究工作已经在Angew. Chem.和Anal. Chem.等高影响力的国际期刊上发表SCI论文12篇。这些研究成果为生物分子影像分析和分子生物学的研究提供新的研究思路和手段。