An ultrasensitive biosensor for high-resolution kinase activity imaging in awake mice
| Autor: | Mikhail Drobizhev, Brian Tenner, Bian Liu, Ingie Hong, Rosana S. Molina, Thomas E. Hughes, Lin Tian, Jin Fan Zhang, Albert Mo, Richard L. Huganir, Wei Lin, Jason Z. Zhang, Sohum Mehta, Richard H. Roth, Jin Zhang |
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| Rok vydání: | 2020 |
| Předmět: |
Primary Cell Culture
Gene Expression Biosensing Techniques Hippocampus Dinoprostone Article 03 medical and health sciences Mice In vivo Genes Reporter Glucagon-Like Peptide 1 Animals Humans Myocytes Cardiac Kinase activity Alprostadil Protein kinase A Molecular Biology 030304 developmental biology Fluorescent Dyes Gene Library Neurons 0303 health sciences Chemistry Kinase 030302 biochemistry & molecular biology HEK 293 cells Optical Imaging Cell Biology Cell sorting Cyclic AMP-Dependent Protein Kinases Cell biology Dihydroxyphenylalanine High-Throughput Screening Assays HEK293 Cells Microscopy Fluorescence Multiphoton Signal transduction Function (biology) HeLa Cells Signal Transduction |
| Zdroj: | Nat Chem Biol |
| ISSN: | 1552-4469 |
| Popis: | 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, in vivo visual cortex imaging in awake mice reveals highly dynamic neuronal PKA activity rapidly recruited by forced locomotion. REPORTING SUMMARY. Further information on research design is available in the Nature Research Reporting Summary linked to this article. |
| Databáze: | OpenAIRE |
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