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B.-F. Liu, J. J. Liang; Confocal Fluorescence Resonance Energy Transfer (FRET) Cell Imaging Study of Protein-Protein Interactions Between Lens Membrane Protein and Crystallins. Invest. Ophthalmol. Vis. Sci. 2007;48(13):3633.
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© ARVO (1962-2015); The Authors (2016-present)
To investigated protein-protein interactions of lens proteins in living cells.
Confocal fluorescence resonance energy transfer (FRET) microscope imaging was used to determine protein-protein interactions. Initially, we used GFP-αA as the donor and DsRED-crystallin (αA, αB, ßB2, and γC) as the acceptor. GFP-αA gene and each DsRED-crystallin gene were either transfected alone or co-transfected to HeLa cells. From confocal images of GFP, RED, and FRET channels, net FRET was calculated. Subsequently, the FRET study was extended to MIP26/AQP0 and crystallins. Either wild-type MIP26 or its C-terminal segment F4 (amino acid sequence 225-263) was used as the donor (GFP fusion protein) and αA-, or αB-, or ßB2-, or γC-crystallin was used as the acceptor (DsRED fusion protein).
Confocal FRET imaging confirms the presence of protein-protein interactions among crystallins, which provides feasibility of extending such study to MIP26. FRET microscope images showed that distribution of the wild-type MIP26 was restricted mostly around the membrane, but the MIP26 fragment was seen all over the cells. The results demonstrate interactions between MIP26/AQP0 and crystallins and the extent of interactions with F4 decreased in the order of αA>αB>ßB2>γC.
Our results demonstrate that protein-protein interactions among lens crystallins can be seen in the living cells and confirm that MIP26/AQP0 interacts with crystallins. One interaction site was the cytoplasm C-terminal segment, which has been reported to be an active site for many biological functions of MIP26/AQP0.
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