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Imaging protein interactions with bioluminescence resonance energy transfer (BRET) in plant and mammalian cells and tissues.

ABSTRACT: FRET is a well established method for cellular and subcellular imaging of protein interactions. However, FRET obligatorily necessitates fluorescence excitation with its concomitant problems of photobleaching, autofluorescence, phototoxicity, and undesirable stimulation of photobiological processes. A sister technique, bioluminescence resonance energy transfer (BRET), avoids these problems because it uses enzyme-catalyzed luminescence; however, BRET signals usually have been too dim to image effectively in the past. Using a new generation electron bombardment-charge-coupled device camera coupled to an image splitter, we demonstrate that BRET can be used to image protein interactions in plant and animal cells and in tissues; even subcellular imaging is possible. We have applied this technology to image two different protein interactions: (i) dimerization of the developmental regulator, COP1, in plant seedlings; and (ii) CCAAT/enhancer binding protein alpha (C/EBPalpha) in the mammalian nucleus. This advance heralds a host of applications for imaging without fluorescent excitation and its consequent limitations.

SUBMITTER: Xu X

PROVIDER: S-EPMC1891211 | biostudies-literature | 2007 Jun

REPOSITORIES: biostudies-literature

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Imaging protein interactions with bioluminescence resonance energy transfer (BRET) in plant and mammalian cells and tissues.

Xu Xiaodong X Soutto Mohammed M Xie Qiguang Q Servick Stein S Subramanian Chitra C von Arnim Albrecht G AG Johnson Carl Hirschie CH

Proceedings of the National Academy of Sciences of the United States of America 20070605 24

FRET is a well established method for cellular and subcellular imaging of protein interactions. However, FRET obligatorily necessitates fluorescence excitation with its concomitant problems of photobleaching, autofluorescence, phototoxicity, and undesirable stimulation of photobiological processes. A sister technique, bioluminescence resonance energy transfer (BRET), avoids these problems because it uses enzyme-catalyzed luminescence; however, BRET signals usually have been too dim to image effe ...[more]

PMID: 17551013

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Project description:BACKGROUND: Nuclear receptors (NR) regulate transcription of genes involved in many biological processes such as development, cell proliferation, differentiation and cell death. Amongst them, PPARG2 and THR control tissue glucose and lipid homeostasis which are deregulated in severe pathophysiological conditions such as metabolic syndromes. METHODOLOGY/PRINCIPAL FINDINGS: Here, we describe a real time BRET approach to monitor heterodimerization between RXR and PPARG2 or THR in vitro or in living cells. The presence of a specific DNA target was required to induce in vitro a BRET shift reflecting heterodimerization of RXR/PPARG2 or RXR/THR. As in electrophoretic mobility shift assay (EMSA), the stringency and specificity of the BRET shift assay depended upon assay condition optimization including MgCl2 concentration. For the nuclear receptors, we found by mutagenesis analysis that each heterodimer partner must harbor an intact DNA binding domain to induce BRET and heterodimerization on a DNA target. Moreover the interaction between the PPARG2 ligand binding domain and the RXR DNA binding domain stabilized the heterodimer on its DNA target. BRET microscopy in living cells highlighted the heterodimerization of RXR/PPARG2 within the nucleus clustered in discrete foci that may represent active target gene transcription regulation regions. BRET imaging also suggested that heterodimerization between RXR and PPARG2 required the DNA binding of PPARG2. CONCLUSIONS/SIGNIFICANCE: The BRET approach described here allowed us to study the dynamic interactions which exist between NR in vitro or in living cells and can provide important information on heterodimerization modes, affinity with a given RE and subcellular localization of the heterodimers. This method could be used to study real time changes of NR heterodimers occurring on DNA depending upon cell activation, chromatin state and help to define the mechanisms of ligands or drug action designed to target NRs.

Dataset Information

Imaging protein interactions with bioluminescence resonance energy transfer (BRET) in plant and mammalian cells and tissues.

Publications

Imaging protein interactions with bioluminescence resonance energy transfer (BRET) in plant and mammalian cells and tissues.

Similar Datasets

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