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Cassandra Barnes, Sungsu Lee, Peter D Calvert, Rajib Pramanik; Multi-photon FLIM for the study of protein-protein interactions in live rod photoreceptors. Invest. Ophthalmol. Vis. Sci. 2016;57(12):4819. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
The study of protein-protein interactions in photoreceptors has mainly been carried out in vitro. Here we showed that multi-photon FLIM (fluorescence lifetime imaging microscopy) is a useful tool for studying protein-protein interactions in live rod photoreceptors of transgenic Xenopus laevis.
Live retinas from transgenic X. laevis tadpoles expressing either enhanced green fluorescent protein (EGFP), EGFP fused to mCherry with a 6 amino acid (aa) linker, or EGFP fused to mCherry with a 25aa linker under control of the Xenopus opsin (XOP) promoter were dissected, sliced, and placed into an imaging chamber containing frog Ringers. EGFP was excited by two photon absorption using an 80MHz Ti-sapphire laser producing ~ 90 fs pulses, tuned to 850nm wavelength and focused to the diffraction limit. Scanning FLIM was achieved using a time correlated single photon counting (TCSPC) system (Becker and Hickl). Greater than 1000 events per voxel were collected, analyzed to yield the average lifetime of EGFP and assembled into 3D maps of EGFP lifetime in photoreceptors. Similar experiments were performed with a Clover – mRuby2 FRET (Förster resonance energy transfer) pair.
The average lifetime of excited EGFP in X. laevis rods was 2.302± 0.033ns (mean ± SD, n=24). Interestingly, the lifetime of EGFP varied amongst the major rod compartments with the mean lifetime in the outer segment averaging ~0.1ns shorter than in the inner segment. The average lifetime of EGFP decreased to 1.96 ± 0.53ns (n=3) when fused to a FRET acceptor, mCherry, with a 6aa linker, and to 2.015 ± 0.092ns (n=19) when fused to mCherry with a 25aa linker. Clover-mRuby2 has a much larger Forster radius and higher FRET efficiency, and thus offers the possibility of examining more distant protein interactions.
Our results show that multi-photon FLIM-FRET is a powerful tool in quantitatively analyzing direct and indirect protein-protein interactions in living retinal neurons at high spatial resolution. Choosing appropriate FLIM-FRET pairs allow high fidelity measures of distances between fluorophores. Finally, local variations in EGFP lifetime in the major rod compartments suggests differences in the cytoplasmic environments that may have physiological relevance.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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