Development and Characterization of a Red Fluorescent Protein-Based Sensor RZnP1 for the Detection of Cytosolic Zn2.
Development and Characterization of a Red Fluorescent Protein-Based Sensor RZnP1 for the Detection of Cytosolic Zn2.
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DOI：
10.1021/acssensors.2c01774
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发表时间：
2022-12
期刊：
影响因子：
8.9
通讯作者：
Anna Dischler;Drew Maslar;Chen Zhang;Yan Qin
中科院分区：
文献类型：
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作者：
Anna Dischler;Drew Maslar;Chen Zhang;Yan Qin
荧光传感器已开发用于记录Zn2+动态和测量Zn2+浓度在细胞内。以往对单波长传感器的研究主要集中在绿色传感器上。在这里，我们设计了一个基因编码的，基于单一红色荧光蛋白的Zn2+传感器，红锌探针（RZnP1），它可以检测细胞内的Zn2+浓度。RZnP1表现出对胞浆Zn2+的敏感响应（Kd = 438 pM），良好的亮度（量子产率(QY) = 0.15），良好的原位动态范围（Fmax/Fmin = 4.0），以及对Zn2+的特异性优于其他生物相关的金属阳离子。RZnP1提供了一种用多个细胞内离子串联成像Zn2+的方法。我们展示了在HeLa细胞中使用RZnP1和Ca2+传感器GCaMP5G同时记录Zn2+和Ca2+。我们还使用RZnP1和mitto - gznp2（一种基于绿色荧光蛋白（GFP）的线粒体Zn2+传感器）同时跟踪大鼠原代神经元培养中细胞质和线粒体中Zn2+的动态。我们的工作不仅扩展了Zn2+传感器的工具箱，而且还演示了同时成像Zn2+与其他阳离子以及多个亚细胞区室之间动态的技术。
Fluorescent sensors have been developed to record Zn2+ dynamics and measure Zn2+ concentrations within the cell. Most previous efforts on developing single-wavelength sensors are focused on green sensors. Here, we engineer a genetically encoded, single red fluorescent protein-based Zn2+ sensor, Red Zinc Probe (RZnP1), which can detect intracellular concentrations of Zn2+. RZnP1 demonstrates a sensitive response to cytosolic Zn2+ (Kd = 438 pM), decent brightness (quantum yield (QY) = 0.15), good in situ dynamic range (Fmax/Fmin = 4.0), and specificity for Zn2+ over other biologically relevant metal cations. RZnP1 offers a way to image Zn2+ with multiple intracellular ions in tandem. We demonstrate the simultaneous recording of Zn2+ and Ca2+ using RZnP1 alongside the Ca2+ sensor GCaMP5G in HeLa cells. We also use RZnP1 with mito-GZnP2, a green fluorescent protein (GFP)-based mitochondrial Zn2+ sensor, to track Zn2+ dynamics in the cytosol and mitochondria concurrently in rat primary neuron culture. Our work not only expands the toolbox of Zn2+ sensors but also demonstrates techniques for imaging Zn2+ dynamics along with other cations and between multiple subcellular compartments simultaneously.