Targetable and Ratiometric Fluorescent Sensors For Probing Brain Mobile Zinc

 描述（由申请人提供）：高浓度的易螯合（移动的）Zn 2+存在于不同的大脑区域，但在CNS发育、学习和记忆形成以及运动和感觉功能等领域的锌信号传导途径的细节仍然难以捉摸。这些动态的移动的Zn 2+池可以使用荧光锌传感器来跟踪，并且是旨在理解富锌神经元的功能的许多研究的焦点;然而，很少有人试图阐明Zn 2+在神经发生期间的分子机制或阐明运动和感觉功能中的锌信号传导途径。 为了解决这些缺陷，我们提出了一类锌选择性传感器的发展，联合收割机的可调小分子传感器和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，用于靶向亚细胞器和细胞膜。具体来说，我们将准备一个基于反应的混合绿色荧光锌传感器，针对线粒体，细胞核，胞质溶胶，和内质网在增殖和分化的神经祖细胞（NPC），并使用它来映射移动的锌池在NPC在离散的位置。将使用纯化的蛋白质和功能化的小分子传感器、克隆以包括亚细胞靶向序列的蛋白质标签表达基因在体外表征杂交探针，并在细胞成像中进行以监测传感器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中的移动的锌。这将通过融合到荧光蛋白的蛋白质标签的设计、制备和表征来实现，然后通过在纤维素荧光显微镜中进行校准，然后进行内源性Zn 2+定量。最后，我们将开发一种新的细胞表面展示的杂交融合蛋白探测突触锌，因为成像锌传输与高时空分辨率需要新的传感器，可以定位到神经元细胞表面。我们将采用一种融合到蛋白质标签的跨膜蛋白，该蛋白质标签可以与细胞外锌传感器偶联，用于在细胞培养中成像，并与合作者一起在与听觉功能相关的脑组织切片中成像。 这项研究将是非常重要的生物无机化学和神经科学领域提供新的传感器，将用于直接探测移动的锌信号通路在各种神经生物学平台，然后可以建立在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.