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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...
Autores principales: | , , |
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Formato: | Texto |
Lenguaje: | English |
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Molecular Vision
2007
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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. |
format | Text |
id | pubmed-2045701 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2007 |
publisher | Molecular Vision |
record_format | MEDLINE/PubMed |
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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