Confocal fluorescence resonance energy transfer microscopy study of protein-protein interactions of lens crystallins in living cells.
OBJECTIVE
To determine protein-protein interactions among lens crystallins in living cells.


METHODS
Fluorescence resonance energy transfer (FRET) microscopy was used to visualize interactions in living cells directly. Two genes, one (alphaA-crystallin) fused with green fluorescence protein (GFP) and the other (each of the following genes: alphaB-, betaB2-, gammaC-crystallin, and R120G alphaB-crystallin mutant) fused with GFP variant red fluorescence protein (RED), were cotransfected into HeLa cells. After culture, confocal microscopy images were taken and FRET values were calculated.


RESULTS
FRET occurs when the two proteins interact. The data show strong interactions between alphaA- and alphaB-crystallin and weak interactions between alphaA- and betaB2- or gammaC-crystallin, which is consistent with our previous two-hybrid system study. The R120G alphaB-crystallin mutant, however, showed significantly less FRET than wild-type alphaB-crystallin. There are also more R120G alphaB-crystallin transfected cells with protein aggregates than wild-type alphaB-crystallin transfected cells. Cotransfection with alphaA-crystallin could not rescue R120G alphaB-crystallin from aggregation.


CONCLUSIONS
FRET microscopy gave excellent results on the protein-protein interactions among crystallins. It supports many previous studies and provides a novel technique for further study of protein-protein interactions among lens proteins including membrane and cytoskeletal proteins.
Liu
Bing-Fen
BF
Ophthalmic Research/Surgery, Brigham and Women's Hospital, Boston, MA 02115, USA. bfliu@rics.bwh.harvard.edu <bfliu@rics.bwh.harvard.edu>
Anbarasu
Kumarasamy
K
Liang
Jack J-N
JJ
D002470:Cell Survival; D003459:Crystallins / Q000235:genetics / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007908:Lens, Crystalline / Q000378:metabolism*; D018613:Microscopy, Confocal; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D014162:Transfection
D003459:Crystallins; D049452:Green Fluorescent Proteins