This proposal aims to improve and exploit fluorescent proteins (FPs);quantum dots (QDs), methods for delivery of nanoparticles such as QDs to cytoplasmic targets, and techniques for correlative optical/electron microscopic (EM) imaging. Photostability of FPs will be improved by creating very large, diverse libraries of genetic variants and screening them in immobilized live cells for photophysical stability under conditions mimicking single molecule imaging. Particular emphasis will be placed on FPs emitting at long wavelengths where cellular autofluorescence is minimal, and FPs that share excitation maxima but emit at different wavelengths for simultaneous multicolor hyper-resolution localization. QDs with small size but long emission wavelengths, QDs with large gaps between excitation and emission maxima, and photoswitchable QDs will be optimized. To help deliver QDs to cytoplasmic targets, peptides that release nanoparticle cargoes from endosomes will be evolved by phage display then applied to QDs. Once QDs have entered the cytoplasm, they will bind to tagged proteins of interest either via biarsenical-tetracysteine pairing or hapten-single-chain antibody complexes. Correlative optical/EM imaging is extremely valuable for combining live cell dynamics with yet higher spatial resolution, including cellular context including cytoskeleton and organelles. Such correlative imaging will be advanced by developing FPs that generate singlet oxygen to trigger formation of EM-visible nanoprecipitates, by improving cathodoluminescence, i.e. detection of FPs and QDs by electron-beam-excited fluorescence, and by optimizing QDs of readily distinguishable sizes and shapes. To validate the new probes and technologies, functional dynamics of key subcellular processes will be studied using model systems currently under study in the labs of the Co-PI's. These include the """"""""Mediator complex"""""""" - a conserved multi- subunit complex which regulates the transcriptional machinery in yeast, mouse, and humans, and dynamics of molecular markers of organelles like the Golgi apparatus and centriole during mitosis. Public Health Relevance: Microscopic imaging is one of the best ways to integrate genetics, biochemistry, physiology, and microanatomy into a coherent picture of cell function, especially when key specific proteins of interest can be tagged to make them uniquely visible. The overall goal of this proposal is to improve the versatility, detection sensitivity, and spatial resolution of general methods to image the dynamic location and function of nearly any desired protein(s) inside cells, both under normal conditions and during disease processes.
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