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Development of an optimized backbone of FRET biosensors for kinases and GTPases

Biosensors based on the principle of Förster (or fluorescence) resonance energy transfer (FRET) have shed new light on the spatiotemporal dynamics of signaling molecules. Among them, intramolecular FRET biosensors have been increasingly used due to their high sensitivity and user-friendliness. Time-...

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Autores principales: Komatsu, Naoki, Aoki, Kazuhiro, Yamada, Masashi, Yukinaga, Hiroko, Fujita, Yoshihisa, Kamioka, Yuji, Matsuda, Michiyuki
Formato: Online Artículo Texto
Lenguaje:English
Publicado: The American Society for Cell Biology 2011
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3226481/
https://www.ncbi.nlm.nih.gov/pubmed/21976697
http://dx.doi.org/10.1091/mbc.E11-01-0072
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author Komatsu, Naoki
Aoki, Kazuhiro
Yamada, Masashi
Yukinaga, Hiroko
Fujita, Yoshihisa
Kamioka, Yuji
Matsuda, Michiyuki
author_facet Komatsu, Naoki
Aoki, Kazuhiro
Yamada, Masashi
Yukinaga, Hiroko
Fujita, Yoshihisa
Kamioka, Yuji
Matsuda, Michiyuki
author_sort Komatsu, Naoki
collection PubMed
description Biosensors based on the principle of Förster (or fluorescence) resonance energy transfer (FRET) have shed new light on the spatiotemporal dynamics of signaling molecules. Among them, intramolecular FRET biosensors have been increasingly used due to their high sensitivity and user-friendliness. Time-consuming optimizations by trial and error, however, obstructed the development of intramolecular FRET biosensors. Here we report an optimized backbone for rapid development of highly sensitive intramolecular FRET biosensors. The key concept is to exclude the “orientation-dependent” FRET and to render the biosensors completely “distance-dependent” with a long, flexible linker. We optimized a pair of fluorescent proteins for distance-dependent biosensors, and then developed a long, flexible linker ranging from 116 to 244 amino acids in length, which reduced the basal FRET signal and thereby increased the gain of the FRET biosensors. Computational simulations provided insight into the mechanisms by which this optimized system was the rational strategy for intramolecular FRET biosensors. With this backbone system, we improved previously reported FRET biosensors of PKA, ERK, JNK, EGFR/Abl, Ras, and Rac1. Furthermore, this backbone enabled us to develop novel FRET biosensors for several kinases of RSK, S6K, Akt, and PKC and to perform quantitative evaluation of kinase inhibitors in living cells.
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spelling pubmed-32264812012-02-16 Development of an optimized backbone of FRET biosensors for kinases and GTPases Komatsu, Naoki Aoki, Kazuhiro Yamada, Masashi Yukinaga, Hiroko Fujita, Yoshihisa Kamioka, Yuji Matsuda, Michiyuki Mol Biol Cell Articles Biosensors based on the principle of Förster (or fluorescence) resonance energy transfer (FRET) have shed new light on the spatiotemporal dynamics of signaling molecules. Among them, intramolecular FRET biosensors have been increasingly used due to their high sensitivity and user-friendliness. Time-consuming optimizations by trial and error, however, obstructed the development of intramolecular FRET biosensors. Here we report an optimized backbone for rapid development of highly sensitive intramolecular FRET biosensors. The key concept is to exclude the “orientation-dependent” FRET and to render the biosensors completely “distance-dependent” with a long, flexible linker. We optimized a pair of fluorescent proteins for distance-dependent biosensors, and then developed a long, flexible linker ranging from 116 to 244 amino acids in length, which reduced the basal FRET signal and thereby increased the gain of the FRET biosensors. Computational simulations provided insight into the mechanisms by which this optimized system was the rational strategy for intramolecular FRET biosensors. With this backbone system, we improved previously reported FRET biosensors of PKA, ERK, JNK, EGFR/Abl, Ras, and Rac1. Furthermore, this backbone enabled us to develop novel FRET biosensors for several kinases of RSK, S6K, Akt, and PKC and to perform quantitative evaluation of kinase inhibitors in living cells. The American Society for Cell Biology 2011-12-01 /pmc/articles/PMC3226481/ /pubmed/21976697 http://dx.doi.org/10.1091/mbc.E11-01-0072 Text en © 2011 Komatsu et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0). “ASCB®,” “The American Society for Cell Biology®,” and “Molecular Biology of the Cell®” are registered trademarks of The American Society of Cell Biology.
spellingShingle Articles
Komatsu, Naoki
Aoki, Kazuhiro
Yamada, Masashi
Yukinaga, Hiroko
Fujita, Yoshihisa
Kamioka, Yuji
Matsuda, Michiyuki
Development of an optimized backbone of FRET biosensors for kinases and GTPases
title Development of an optimized backbone of FRET biosensors for kinases and GTPases
title_full Development of an optimized backbone of FRET biosensors for kinases and GTPases
title_fullStr Development of an optimized backbone of FRET biosensors for kinases and GTPases
title_full_unstemmed Development of an optimized backbone of FRET biosensors for kinases and GTPases
title_short Development of an optimized backbone of FRET biosensors for kinases and GTPases
title_sort development of an optimized backbone of fret biosensors for kinases and gtpases
topic Articles
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3226481/
https://www.ncbi.nlm.nih.gov/pubmed/21976697
http://dx.doi.org/10.1091/mbc.E11-01-0072
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