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

PURPOSE: 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 (αA-crystallin) fused with green fluorescence protein (GFP) and the...

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Autores principales: Liu, Bing-Fen, Anbarasu, Kumarasamy, Liang, Jack J-N.
Formato: Texto
Lenguaje:English
Publicado: Molecular Vision 2007
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2045701/
https://www.ncbi.nlm.nih.gov/pubmed/17615546
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author Liu, Bing-Fen
Anbarasu, Kumarasamy
Liang, Jack J-N.
author_facet Liu, Bing-Fen
Anbarasu, Kumarasamy
Liang, Jack J-N.
author_sort Liu, Bing-Fen
collection PubMed
description PURPOSE: 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 (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 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 αA- and αB-crystallin and weak interactions between αA- and βB2- or γC-crystallin, which is consistent with our previous two-hybrid system study. The R120G αB-crystallin mutant, however, showed significantly less FRET than wild-type αB-crystallin. There are also more R120G αB-crystallin transfected cells with protein aggregates than wild-type αB-crystallin transfected cells. Cotransfection with αA-crystallin could not rescue R120G αB-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.
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spelling pubmed-20457012007-10-31 Confocal fluorescence resonance energy transfer microscopy study of protein-protein interactions of lens crystallins in living cells Liu, Bing-Fen Anbarasu, Kumarasamy Liang, Jack J-N. Mol Vis Research Article PURPOSE: 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 (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 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 αA- and αB-crystallin and weak interactions between αA- and βB2- or γC-crystallin, which is consistent with our previous two-hybrid system study. The R120G αB-crystallin mutant, however, showed significantly less FRET than wild-type αB-crystallin. There are also more R120G αB-crystallin transfected cells with protein aggregates than wild-type αB-crystallin transfected cells. Cotransfection with αA-crystallin could not rescue R120G αB-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. Molecular Vision 2007-06-14 /pmc/articles/PMC2045701/ /pubmed/17615546 Text en http://creativecommons.org/licenses/by/3.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Research Article
Liu, Bing-Fen
Anbarasu, Kumarasamy
Liang, Jack J-N.
Confocal fluorescence resonance energy transfer microscopy study of protein-protein interactions of lens crystallins in living cells
title Confocal fluorescence resonance energy transfer microscopy study of protein-protein interactions of lens crystallins in living cells
title_full Confocal fluorescence resonance energy transfer microscopy study of protein-protein interactions of lens crystallins in living cells
title_fullStr Confocal fluorescence resonance energy transfer microscopy study of protein-protein interactions of lens crystallins in living cells
title_full_unstemmed Confocal fluorescence resonance energy transfer microscopy study of protein-protein interactions of lens crystallins in living cells
title_short Confocal fluorescence resonance energy transfer microscopy study of protein-protein interactions of lens crystallins in living cells
title_sort confocal fluorescence resonance energy transfer microscopy study of protein-protein interactions of lens crystallins in living cells
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2045701/
https://www.ncbi.nlm.nih.gov/pubmed/17615546
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AT liangjackjn confocalfluorescenceresonanceenergytransfermicroscopystudyofproteinproteininteractionsoflenscrystallinsinlivingcells