Imaging protein interactions with bioluminescence resonance energy transfer (BRET) in plant and mammalian cells and tissues
- Xiaodong Xu*,
- Mohammed Soutto*,
- Qiguang Xie*,
- Stein Servick*,
- Chitra Subramanian†,
- Albrecht G. von Arnim†, and
- Carl Hirschie Johnson*,‡
- *Department of Biological Sciences, Box 1634-B, Vanderbilt University, Nashville, TN 37235; and
- †Department of Biochemistry and Cellular and Molecular Biology, University of Tennessee, Knoxville, TN 37996
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Edited by J. Woodland Hastings, Harvard University, Cambridge, MA, and approved April 16, 2007 (received for review March 3, 2007)
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 α (C/EBPα) in the mammalian nucleus. This advance heralds a host of applications for imaging without fluorescent excitation and its consequent limitations.
Footnotes
- ‡To whom correspondence should be addressed. E-mail: carl.h.johnson{at}vanderbilt.edu
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Author contributions: X.X. and M.S. contributed equally to this work. X.X., M.S., and C.H.J. designed research; X.X., M.S., Q.X., and S.S. performed research; C.S. and A.G.v.A. contributed new reagents/analytic tools; X.X., M.S., Q.X., and C.H.J. analyzed data; and C.H.J. wrote the paper.
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The authors declare no conflict of interest.
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This article is a PNAS Direct Submission.
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See Commentary on page 9917.
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This article contains supporting information online at www.pnas.org/cgi/content/full/0701987104/DC1.
- Abbreviations:
- BRET,
- bioluminescence resonance energy transfer;
- CCD,
- charge-coupled device;
- COP1,
- constitutive photomorphogenesis 1;
- EB-CCD,
- electron bombardment-CCD;
- RLUC,
- Renilla luciferase;
- EYFP,
- enhanced yellow fluorescent protein;
- hRLUC,
- “humanized” RLUC;
- NA,
- numerical aperture.
- © 2007 by The National Academy of Sciences of the USA
