Confocal fluorescence resonance energy transfer microscopy studyof protein-protein interactions of lens crystallins in living cells

[摘要] Purpose: To determine protein-protein interactions among lenscrystallins in living cells.Methods: Fluorescence resonance energy transfer (FRET) microscopywas used to visualize interactions in living cells directly. Two genes,one (αA-crystallin) fused with green fluorescence protein (GFP)and the other (each of the following genes: αB-, βB2-, γC-crystallin, and R120G αB-crystallin mutant) fused with GFPvariant red fluorescence protein (RED), were cotransfected into HeLacells. After culture, confocal microscopy images were taken and FRETvalues were calculated.Results: FRET occurs when the two proteins interact. The data showstrong interactions between αA- and αB-crystallin and weakinteractions between αA- and βB2- or γC-crystallin,which is consistent with our previous two-hybrid system study. The R120GαB-crystallin mutant, however, showed significantly less FRET thanwild-type αB-crystallin. There are also more R120G αB-crystallin transfected cells with protein aggregates than wild-typeαB-crystallin transfected cells. Cotransfection with αA-crystallin could not rescue R120G αB-crystallin fromaggregation.Conclusions: FRET microscopy gave excellent results on theprotein-protein interactions among crystallins. It supports manyprevious studies and provides a novel technique for further study ofprotein-protein interactions among lens proteins including membrane andcytoskeletal proteins.

[学科分类] 生物化学/生物物理