Rational design of a genetically encoded infrared fluorescent protease reporter

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 DESCRIPTION (provided by applicant): Proteases play fundamental roles in almost every major biological process. Changes to proteolytic systems lead to many diseases including cancer, neurodegenerative and cardiovascular diseases. Several chemical dye-based protease reporters have been successful in imaging protease activity in animals. However, these reporters have limitations in imaging developing embryos since it is difficult to target chemical dyes to specific tissues and injection of chemical dyes may perturb development. A genetically encoded protease reporter is thus preferred. Furthermore, compared to fluorescence resonance energy transfer (FRET)-based reporters that suffers from weak signals and require image processing to obtain FRET signal, a fluorogenic protease reporter that becomes fluorescent upon protease activation will be ideal. Such genetically encoded fluorogenic protease reporters will enable us to directly visualize protease activity with spatiotemporal resolution in intact animals. Here we aim to: 1) Design and improve genetically encoded infrared fluorescent protease reporters. Such genetically encoded protease reporters become fluorescent upon protease activation and require no exogenous cofactor. 2): To visualize spatiotemporal dynamics of apoptosis during embryonic morphogenesis. Apoptosis plays an essential role in embryogenesis of Drosophila. Inhibition of apoptosis impairs many critical morphogenetic movements during embryo development, including segmentation, germ band retraction, dorsal closure and head involution. However, it is not clear whether apoptosis is spatiotemporally correlated to morphogenesis. Here, we will use a caspase reporter to visualize spatiotemporal dynamics of apoptosis during these morphogenetic movements. 3): To characterize dynamics of apoptosis during brain tumor development in Drosophila and mice. During cancer development, oncogene activation leads to cell overprolieration, which triggers apoptosis. Evading apoptosis clears this barrier for rapid tumor development, and is one of the hallmarks of cancer. Such dynamics of apoptosis during cancer development has previously been proposed. Here we will use the caspase reporter to characterize dynamics of apoptosis during glioblastoma development in Drosophila and mice.