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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...

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Detalles Bibliográficos
Autores principales: 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.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: The American Society for Cell Biology 2015
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
Descripción
Sumario: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.