Live-cell multiphoton fluorescence correlation spectroscopy with an improved large Stokes shift fluorescent protein
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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 |
| 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.
Note: Order does not necessarily reflect citation order of authors. |
| Citation: | Guan, Y., M. Meurer, S. Raghavan, A. Rebane, J. R. Lindquist, S. Santos, I. Kats, et al. 2015. “Live-cell multiphoton fluorescence correlation spectroscopy with an improved large Stokes shift fluorescent protein.” Molecular Biology of the Cell 26 (11): 2054-2066. doi:10.1091/mbc.E14-10-1473. http://dx.doi.org/10.1091/mbc.E14-10-1473. |
| Full Text & Related Files: |
4472016.pdf (1.387Mb; PDF) 
|
| 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. |
| Published Version: | doi:10.1091/mbc.E14-10-1473 |
| Other Sources: | http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4472016/pdf/ |
| Terms of Use: | This article is made available under the terms and conditions applicable to Other Posted Material, as set forth at http://nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of-use#LAA |
| Citable link to this page: | http://nrs.harvard.edu/urn-3:HUL.InstRepos:21459109 |
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