Targetable and Ratiometric Fluorescent Sensors For Probing Brain Mobile Zinc

 描述(申请人提供)：在不同的大脑区域中发现了高浓度的易螯合(可移动的)锌离子，然而锌信号通路在中枢神经系统发育、学习和记忆形成以及运动和感觉功能等领域的细节仍然难以捉摸。这些动态的移动锌离子池可以用荧光锌传感器来跟踪，并成为许多旨在了解富锌神经元功能的研究的焦点；然而，很少有人试图阐明锌离子在神经发生过程中的分子机制或阐明锌在运动和感觉功能中的信号通路。 为了解决这些不足，我们建议开发一种锌选择性传感器，它结合了可调小分子传感器和可遗传编码标签的优点。我们将设计、制备和表征基于反应的小分子-蛋白质杂化荧光锌传感器，用于靶向亚细胞细胞器和细胞膜。具体地说，我们将制备一种基于反应的混合绿色荧光锌传感器，用于靶向增殖和分化的神经前体细胞(NPC)中的线粒体、细胞核、胞浆和内质网，并使用它来绘制不同地点神经前体细胞中可移动的锌离子池。杂交探针将使用纯化的蛋白质和功能化的小分子传感器进行体外表征，克隆的蛋白质标签表达基因包括亚细胞靶向序列，并在细胞成像中监测传感器的定位和相对内源性锌水平。我们还将开发一种基于红色发射反应的混合探针，用于定量NPC中的移动锌。这将通过设计、制备和表征融合到荧光蛋白的蛋白质标签来实现，然后在纤维荧光显微镜中进行校准，然后对内源性锌离子进行定量。最后，我们将开发一种新型的细胞-表面展示的融合蛋白来探测突触锌，因为高时空分辨率的锌传输成像需要能够定位到神经细胞表面的新型传感器。我们将使用融合到蛋白质标签上的跨膜蛋白，该标签可以与细胞外锌传感器结合在细胞培养中进行成像，并与合作者在与听觉功能相关的脑组织切片中进行成像。 这项研究将对生物无机化学和神经科学领域具有重要意义，因为它将提供新型传感器，用于直接探测各种神经生物学平台中的移动锌信号通路，并可在此基础上解决各种更广泛的生物学问题。

 DESCRIPTION (provided by applicant): High concentrations of readily chelatable (mobile) Zn2+ are found in various brain regions, yet the details of zinc signaling pathways in areas such as CNS development, learning and memory formation, and motor and sensory function, remain elusive. These dynamic pools of mobile Zn2+ may be tracked using fluorescent zinc sensors and are the focus of many studies aimed at understanding the function of zinc- enriched neurons; however, few have attempted to elucidate the molecular mechanisms of Zn2+ during neurogenesis or clarify zinc signaling pathways in motor and sensory functions. To address these deficiencies, we propose the development of a category of zinc-selective sensors that combine the advantages of tunable small molecule sensors and genetically encodable tags. We will design, prepare, and characterize reaction-based small molecule-protein hybrid fluorescent zinc sensors for targeting subcellular organelles and cell membranes. Specifically, we will prepare a reaction-based hybrid green fluorescent zinc sensor for targeting the mitochondria, nucleus, cytosol, and endoplasmic reticulum in proliferating and differentiated neural progenitor cells (NPCs) and use it to map mobile Zn2+ pools in NPCs at discrete locales. Hybrid probes will be characterized in vitro using purified proteins and functionalized small molecule sensors, protein tag-expressing genes cloned to include subcellular targeting sequences, and in cellulo imaging carried out to monitor sensor localization and relative endogenous zinc levels. We will also develop a red-emitting reaction-based hybrid probe for quantifying mobile zinc in NPCs. This will be approached through the design, preparation, and characterization of a protein tag fused to a fluorescent protein, followed by in cellulo fluorescence microscopy for calibration and then endogenous Zn2+ quantification. Finally, we will develop a novel cell-surface displayed hybrid fusion protein for probing synaptic zinc because imaging zinc transmission with high spatiotemporal resolution requires novel sensors that can be localized to neuronal cell surfaces. We will employ a transmembrane protein fused to a protein tag that can be coupled with an extracellular zinc sensor for imaging in cell culture, and with collaborators, in brain tissue slices of relevance to auditory function. This study will be of great importance to the fields of bioinorganic chemistry and neuroscience by providing novel sensors that will be utilized to directly probe mobile zinc signaling pathways in various neurobiological platforms and which can then be built upon to address various broader biological questions.