Designing a new class of fluorescent reporters for imaging dynamic cell signaling in live animals

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Abstract Proteases and kinases control a broad range of cellular processes. Dysregulation of protease and kinase activities causes many diseases including cancer. Furthermore, temporal dynamics of kinase signaling can elicit distinct cellular responses. To image dynamics of protease and kinase activity in live animals, genetically encoded fluorescent reporters are ideal because they require no exogenous molecule and are non-invasive. Although green fluorescent protein (GFP)-based Förster resonance energy transfer (FRET) reporters can be used to image dynamics of kinase signaling in cultured cells where they can achieve subcellular resolution and compartmentalized kinase signaling, in vivo use of the FRET-based kinase (and protease) reporters is difficult because of small magnitude of the fluorescence changes of the fluorophores. We seek to design, demonstrate and apply new classes of fluorescent reporters including the GFP-based reporters that, upon kinase activation, phase separate and form highly concentrated droplets via multivalent protein-protein interactions. Our approach is inspired by recent work showing that multivalent interactions drive protein phase separation to form protein droplets that concentrate the protein ~10 fold. The phase separation-based kinase reporter has both large dynamic range and high brightness because fluorophores concentrate in discrete punctae. This punctal signal pattern is distinctive and easily detectable in whole animals and robust for in vivo detection of kinase activity. The reporter also achieves fast kinetics at the second-to-minute timescale and is reversible, with no apparent toxicity in transgenic animals. To further demonstrate the new design principle, we aim to design and apply phase separation-based kinase reporters to visualize dynamics of several key kinases during animal development and disease. We will also develop fluorogenic protease reporters to visualize dynamic apoptosis signaling in live animals. Furthermore, we will combine the green fluorescent kinase reporters with the infrared fluorogenic caspase reporters to simultaneously visualize kinase signaling and apoptosis and investigate how they are correlated to morphogenetic processes during embryogenesis and tissue homeostasis. We will also apply these reporters to investigate dynamic cell signaling network during brain tumorigenesis.