FLASH/REASH LABELING OF TETRACYSTEINE-TAGGED PROTEINS IN ZEBRAFISH

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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.