FLASH/REASH LABELING OF TETRACYSTEINE-TAGGED PROTEINS IN ZEBRAFISH

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 这个项目的目标是熟悉标记斑马鱼胚胎的技术方面，这些斑马鱼胚胎表达的蛋白质经过基因改造，含有四半胱氨酸标签。该标签被商业上可获得的荧光团Flash-EDT2和ReAsH-EDT2所识别。任何一个荧光团与四半胱氨酸标记物的结合都会产生荧光信号，该信号可以将二氨基联苯胺光转化为嗜奥斯酸的沉淀物。与电子断层扫描相结合，这种方法将使我们能够以亚细胞分辨率定位任何感兴趣的蛋白质，而不会产生抗体。斑马鱼是一种特别有吸引力的模型，可以用基因编码的通用标签标记蛋白质：首先，斑马鱼的发育速度比高等脊椎动物快得多；因此，大多数器官和细胞类型都可以在受精后几天内观察到。其次，通过显微注射胚胎就可以产生瞬时或稳定表达标记蛋白的动物。第三，斑马鱼胚胎是透明的，很容易被光穿透，这是荧光检测和光转化的先决条件。最后，小斑马鱼胚胎可以在高压下冷冻和冷冻替代，从而忠实地保存在电子显微镜下。为了在斑马鱼中测试通用标签方法，我们首先在HEK细胞系和斑马鱼中创建了一个在强病毒启动子控制下编码带有线粒体靶向信号的黄色荧光蛋白(YFP)的表达载体。初步实验表明，MITO-YFP融合蛋白可以靶向线粒体，并在斑马鱼胚胎中显微注射该结构后获得强烈的嵌合体表达。我们现在正在将优化的四环素标签(FLNCCPGCCMEP)添加到融合蛋白中。YFP和ReAsH的荧光光谱没有明显重叠，它们的最大激发/发射峰分别位于512/529和593/608 nm。在NCMIR，我们建议检测显微注射斑马鱼胚胎中的ReAsH荧光团与mito-YFP-TetraCys蛋白或mito-YFP阴性对照的结合。首先，我们将改变ReAsH的浓度和暴露时间，以优化信噪比。如果我们观察到荧光显微镜所确定的与mito-YFP-TetraCys蛋白的特异性结合，我们将继续进行ReAsH介导的二氨基联苯的光转化，以便进行透射电子显微镜成像。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The goal of this project is to become familiar with the technical aspects of labeling zebrafish embryos that express proteins genetically altered to contain a tetracysteine tag. This tag is recognized by the commercially available fluorophores FlAsH-EDT2 and ReAsH-EDT2. Binding of either fluorophore to the tetracysteine tag results in a fluorescent signal that can photoconvert diaminobenzidine into an osmiophilic precipitate. In combination with electron tomography, this method will enable us to localize any protein of interest with sub-cellular resolution and without raising antibodies. The zebrafish is a particularly attractive model for labeling proteins with a genetically-encoded universal tag: First, zebrafish develop much faster than higher vertebrates; as a result, most organs and cell types can be observed within a few days after fertilization. Second, animals that transiently or stably express tagged proteins can be generated simply by microinjecting embryos. Third, zebrafish embryos are transparent and easily penetrated by light, a prerequisite for fluorescence detection and photoconversion. Finally, the small zebrafish embryos can be frozen at high pressure and freeze-substituted, resulting in faithful preservation for electron microscopy. To test the universal-tag approach in zebrafish, we first created an expression construct that encodes yellow fluorescent protein (YFP) with a mitochondrial targeting signal under the control of a strong viral promoter both in HEK cell lines and zebrafish. Pilot experiments have demonstrated targeting of the mito-YFP fusion protein to mitochondria and strong mosaic expression upon microinjection of this construct into zebrafish embryos. We are now adding an optimized tetracystein tag (FLNCCPGCCMEP) to the fusion protein. The fluorescence spectra of YFP and ReAsH do not overlap significatnly, as their excitation/emission maxima lie at 512/529 and 593/608 nm, respectively. At the NCMIR, we propose to examine the binding of the ReAsH fluorophore in microinjected zebrafish embryos to the mito-YFP-TetraCys protein or to the mito-YFP negative control. First, we will vary the ReAsH concentration and the duration of exposure to optimize the signal-to-noise ratio. If we observe specific binding to the mito-YFP-TetraCys protein, as determined by fluorescence microscopy, we will proceed to the ReAsH-mediated photoconversion of diaminobenzidin in order to perform TEM imaging.