| Three-color spectral FRET microscopy localizes three interacting proteins in living cells. | |
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MedLine Citation:
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PMID: 20713013 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
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FRET technologies are now routinely used to establish the spatial relationships between two cellular components (A and B). Adding a third target component (C) increases the complexity of the analysis between interactions AB/BC/AC. Here, we describe a novel method for analyzing a three-color (ABC) FRET system called three-color spectral FRET (3sFRET) microscopy, which is fully corrected for spectral bleedthrough. The approach quantifies FRET signals and calculates the apparent energy transfer efficiencies (Es). The method was validated by measurement of a genetic (FRET standard) construct consisting of three different fluorescent proteins (FPs), mTFP, mVenus, and tdTomato, linked sequentially to one another. In addition, three 2-FP reference constructs, tethered in the same way as the 3-FP construct, were used to characterize the energy transfer pathways. Fluorescence lifetime measurements were employed to compare the relative relationships between the FPs in cells producing the 3-FP and 2-FP fusion proteins. The 3sFRET microscopy method was then applied to study the interactions of the dimeric transcription factor C/EBPalpha (expressing mTFP or mVenus) with the heterochromatin protein 1alpha (HP1alpha, expressing tdTomato) in live-mouse pituitary cells. We show how the 3sFRET microscopy method represents a promising live-cell imaging technique to monitor the interactions between three labeled cellular components. |
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Authors:
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Yuansheng Sun; Horst Wallrabe; Cynthia F Booker; Richard N Day; Ammasi Periasamy |
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Publication Detail:
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Type: Journal Article; Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't |
Journal Detail:
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Title: Biophysical journal Volume: 99 ISSN: 1542-0086 ISO Abbreviation: Biophys. J. Publication Date: 2010 Aug |
Date Detail:
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Created Date: 2010-08-17 Completed Date: 2010-12-01 Revised Date: 2011-08-25 |
Medline Journal Info:
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Nlm Unique ID: 0370626 Medline TA: Biophys J Country: United States |
Other Details:
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Languages: eng Pagination: 1274-83 Citation Subset: IM |
Copyright Information:
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2010 Biophysical Society. Published by Elsevier Inc. All rights reserved. |
Affiliation:
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W. M. Keck Center for Cellular Imaging, Departments of Biology and Biomedical Engineering, University of Virginia, Charlottesville, Virginia, USA. |
Export Citation:
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| MeSH Terms | |
Descriptor/Qualifier:
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Algorithms Animals CCAAT-Enhancer-Binding Proteins / metabolism* Cell Line Cell Survival Chromosomal Proteins, Non-Histone / metabolism* Fluorescence Resonance Energy Transfer / methods* Luminescent Proteins / metabolism* Mice Microscopy, Fluorescence / methods* Protein Binding Protein Multimerization Protein Transport Reference Standards |
| Grant Support | |
ID/Acronym/Agency:
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DK47301/DK/NIDDK NIH HHS |
| Chemical | |
Reg. No./Substance:
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0/CCAAT-Enhancer-Binding Proteins; 0/CEBPA protein, mouse; 0/Chromosomal Proteins, Non-Histone; 0/Luminescent Proteins; 107283-02-3/heterochromatin-specific nonhistone chromosomal protein HP-1 |
| Comments/Corrections | |
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