Live-cell multiphoton fluorescence correlation spectroscopy with an improved large Stokes shift fluorescent protein.
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Abstract | 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. |
Year of Publication | 2015
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Journal | Mol Biol Cell
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Volume | 26
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Issue | 11
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Pages | 2054-66
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Date Published | 2015 Jun 01
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ISSN | 1939-4586
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DOI | 10.1091/mbc.E14-10-1473
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PubMed ID | 25877871
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PubMed Central ID | PMC4472016
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Grant list | NS083875 / NS / NINDS NIH HHS / United States
GM098083 / GM / NIGMS NIH HHS / United States
R01 NS083875 / NS / NINDS NIH HHS / United States
GM77238 / GM / NIGMS NIH HHS / United States
R01 GM077238 / GM / NIGMS NIH HHS / United States
R01 GM098083 / GM / NIGMS NIH HHS / United States
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