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Live-cell multiphoton fluorescence correlation spectroscopy with an improved large Stokes shift fluorescent protein
We report an improved variant of mKeima, a monomeric long Stokes shift red fluorescent protein, hmKeima8.5. The increased intracellular brightness and large Stokes shift (∼180 nm) make it an excellent partner with teal fluorescent protein (mTFP1) for multiphoton, multicolor applications. Excitation...
Autores principales: | , , , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
The American Society for Cell Biology
2015
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4472016/ https://www.ncbi.nlm.nih.gov/pubmed/25877871 http://dx.doi.org/10.1091/mbc.E14-10-1473 |
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author | Guan, Yinghua Meurer, Matthias Raghavan, Sarada Rebane, Aleksander Lindquist, Jake R. Santos, Sofia Kats, Ilia Davidson, Michael W. Mazitschek, Ralph Hughes, Thomas E. Drobizhev, Mikhail Knop, Michael Shah, Jagesh V. |
author_facet | Guan, Yinghua Meurer, Matthias Raghavan, Sarada Rebane, Aleksander Lindquist, Jake R. Santos, Sofia Kats, Ilia Davidson, Michael W. Mazitschek, Ralph Hughes, Thomas E. Drobizhev, Mikhail Knop, Michael Shah, Jagesh V. |
author_sort | Guan, Yinghua |
collection | PubMed |
description | We report an improved variant of mKeima, a monomeric long Stokes shift red fluorescent protein, hmKeima8.5. The increased intracellular brightness and large Stokes shift (∼180 nm) make it an excellent partner with teal fluorescent protein (mTFP1) for multiphoton, multicolor applications. Excitation of this pair by a single multiphoton excitation wavelength (MPE, 850 nm) yields well-separable emission peaks (∼120-nm separation). Using this pair, we measure homo- and hetero-oligomerization interactions in living cells via multiphoton excitation fluorescence correlation spectroscopy (MPE-FCS). Using tandem dimer proteins and small-molecule inducible dimerization domains, we demonstrate robust and quantitative detection of intracellular protein–protein interactions. We also use MPE-FCCS to detect drug–protein interactions in the intracellular environment using a Coumarin 343 (C343)-conjugated drug and hmKeima8.5 as a fluorescence pair. The mTFP1/hmKeima8.5 and C343/hmKeima8.5 combinations, together with our calibration constructs, provide a practical and broadly applicable toolbox for the investigation of molecular interactions in the cytoplasm of living cells. |
format | Online Article Text |
id | pubmed-4472016 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2015 |
publisher | The American Society for Cell Biology |
record_format | MEDLINE/PubMed |
spelling | pubmed-44720162015-08-16 Live-cell multiphoton fluorescence correlation spectroscopy with an improved large Stokes shift fluorescent protein Guan, Yinghua Meurer, Matthias Raghavan, Sarada Rebane, Aleksander Lindquist, Jake R. Santos, Sofia Kats, Ilia Davidson, Michael W. Mazitschek, Ralph Hughes, Thomas E. Drobizhev, Mikhail Knop, Michael Shah, Jagesh V. Mol Biol Cell Articles We report an improved variant of mKeima, a monomeric long Stokes shift red fluorescent protein, hmKeima8.5. The increased intracellular brightness and large Stokes shift (∼180 nm) make it an excellent partner with teal fluorescent protein (mTFP1) for multiphoton, multicolor applications. Excitation of this pair by a single multiphoton excitation wavelength (MPE, 850 nm) yields well-separable emission peaks (∼120-nm separation). Using this pair, we measure homo- and hetero-oligomerization interactions in living cells via multiphoton excitation fluorescence correlation spectroscopy (MPE-FCS). Using tandem dimer proteins and small-molecule inducible dimerization domains, we demonstrate robust and quantitative detection of intracellular protein–protein interactions. We also use MPE-FCCS to detect drug–protein interactions in the intracellular environment using a Coumarin 343 (C343)-conjugated drug and hmKeima8.5 as a fluorescence pair. The mTFP1/hmKeima8.5 and C343/hmKeima8.5 combinations, together with our calibration constructs, provide a practical and broadly applicable toolbox for the investigation of molecular interactions in the cytoplasm of living cells. The American Society for Cell Biology 2015-06-01 /pmc/articles/PMC4472016/ /pubmed/25877871 http://dx.doi.org/10.1091/mbc.E14-10-1473 Text en © 2015 Guan 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 for Cell Biology. |
spellingShingle | Articles Guan, Yinghua Meurer, Matthias Raghavan, Sarada Rebane, Aleksander Lindquist, Jake R. Santos, Sofia Kats, Ilia Davidson, Michael W. Mazitschek, Ralph Hughes, Thomas E. Drobizhev, Mikhail Knop, Michael Shah, Jagesh V. Live-cell multiphoton fluorescence correlation spectroscopy with an improved large Stokes shift fluorescent protein |
title | Live-cell multiphoton fluorescence correlation spectroscopy with an improved large Stokes shift fluorescent protein |
title_full | Live-cell multiphoton fluorescence correlation spectroscopy with an improved large Stokes shift fluorescent protein |
title_fullStr | Live-cell multiphoton fluorescence correlation spectroscopy with an improved large Stokes shift fluorescent protein |
title_full_unstemmed | Live-cell multiphoton fluorescence correlation spectroscopy with an improved large Stokes shift fluorescent protein |
title_short | Live-cell multiphoton fluorescence correlation spectroscopy with an improved large Stokes shift fluorescent protein |
title_sort | live-cell multiphoton fluorescence correlation spectroscopy with an improved large stokes shift fluorescent protein |
topic | Articles |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4472016/ https://www.ncbi.nlm.nih.gov/pubmed/25877871 http://dx.doi.org/10.1091/mbc.E14-10-1473 |
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