Abstract
Protein kinases are key modulators of intracellular signal transduction cascades, which determine various events in neuronal cells such as replication and differentiation. For many years, protein kinases were analyzed mostly by biochemical methods, which could handle the cells only en masse. For a better understanding of the role of kinases in neuronal cells, one would like to know the subcellular distribution of kinase activities and to follow a particular kinase activity for a specific period in a single cell. Genetically encoded biosensors based on the principle of Förster (or fluorescence) resonance energy transfer (FRET) and fluorescent proteins have been developed to accommodate such requirements. The method involves expression of the FRET biosensors in neuronal cells, time-lapse imaging under fluorescence microscopes, image processing, and quantification of FRET. This technique could be applicable to living organisms ranging from Caenorhabditis elegans to mouse, permitting visualization of spatio-temporal regulation of kinase activities and systemic understanding of the signaling networks in living animals.
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Acknowledgments
We thank Dr. J. Zhang for the plasmids. Y. Inaoka, K. Hirano, R. Sakai, and N. Nonaka are also to be thanked for their technical assistance. We are grateful to the members of the Matsuda Laboratory for their helpful discussions. K.A. was supported by a Grant-in-Aid for Scientific Research on Priority Areas and by the JST PRESTO program. M.M. was supported by the Research Program of Innovative Cell Biology by Innovative Technology (Cell Innovation) from the Ministry of Education, Culture, Sports, and Science (MEXT), Japan.
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Aoki, K., Komatsu, N., Goto, A., Matsuda, M. (2012). Visualization of Protein Kinase Activities in Living Cells. In: Mukai, H. (eds) Protein Kinase Technologies. Neuromethods, vol 68. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-824-5_1
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DOI: https://doi.org/10.1007/978-1-61779-824-5_1
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