(L5431) An Ultrasensitive PKA Biosensor for Multi‐modal Kinase Activity Detection and High-Resolution Imaging in Awake Mice

Bian Liu (Johns Hopkins University School of Medicine)| Jin-Fan Zhang (University of California, San Diego)| Ingie Hong (Johns Hopkins University School of Medicine)| Albert Mo (University of California, San Diego)| Richard Roth (Johns Hopkins University School of Medicine)| Brian Tenner (University of California, San Diego)| Wei Lin (University of California, San Diego)| Jason Zhang (University of California, San Diego)| Rosana Molina (Montana State University)| Mikhail Drobizhev (Montana State University)| Thomas Hughes (Montana State University)| Tian Lin (University of California, Davis)| Richard Huganir (Johns Hopkins University School of Medicine)| Sohum Mehta (University of California, San Diego)| Jin Zhang (University of California, San Diego)

Protein kinases control nearly every facet of cellular function. These key signaling nodes integrate diverse pathway inputs to regulate complex physiological processes, and aberrant kinase signaling is linked to numerous pathologies. While fluorescent protein-based biosensors have revolutionized the study of kinase signaling by allowing direct, spatiotemporally precise kinase activity measurements in living cells, powerful new molecular tools capable of robustly tracking kinase activity dynamics across diverse experimental contexts are needed to fully dissect the role of kinase signaling in physiology and disease. Here, we report the development of an ultrasensitive, second-generation excitation-ratiometric protein kinase A (PKA) activity reporter (ExRai-AKAR2), obtained via high-throughput linker library screening, that enables sensitive and rapid monitoring of live-cell PKA activity across multiple fluorescence detection modalities, including plate reading, cell sorting and one- or two-photon imaging. Notably, live imaging in cultured hippocampal neurons shows local PKA activity increase following individual calcium transients upon chemical stimulation. And in vivo visual cortex imaging in awake mice reveals highly dynamic neuronal PKA activity rapidly recruited by forced locomotion.