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An Ultrasensitive PKA Biosensor for Multi‐modal Kinase Activity Detection and High‐Resolution Imaging in Awake Mice
Author(s) -
Liu Bian,
Zhang JinFan,
Hong Ingie,
Mo Albert,
Roth Richard,
Tenner Brian,
Lin Wei,
Zhang Jason,
Molina Rosana,
Drobizhev Mikhail,
Hughes Thomas,
Lin Tian,
Huganir Richard,
Mehta Sohum,
Zhang Jin
Publication year - 2021
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2021.35.s1.05431
Subject(s) - protein kinase a , kinase , microbiology and biotechnology , biology , signal transduction , live cell imaging , chemistry , neuroscience , cell , biochemistry
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.