Imaging protein interactions with bioluminescence resonance energy transfer (BRET) in plant and mammalian cells and tissues.
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.
Xu
Xiaodong
X
Department of Biological Sciences, Box 1634-B, Vanderbilt University, Nashville, TN 37235, USA.
Soutto
Mohammed
M
Xie
Qiguang
Q
Servick
Stein
S
Subramanian
Chitra
C
von Arnim
Albrecht G
AG
Johnson
Carl Hirschie
CH
D000818:Animals; D017360:Arabidopsis / Q000737:chemistry / Q000166:cytology*; D029681:Arabidopsis Proteins / Q000032:analysis; D022762:CCAAT-Enhancer-Binding Proteins / Q000032:analysis / Q000378:metabolism; D018929:Cell Culture Techniques; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D019281:Dimerization; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D006801:Humans; D008163:Luminescent Measurements; D008164:Luminescent Proteins / Q000032:analysis* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008856:Microscopy, Fluorescence; D010902:Pituitary Gland / Q000166:cytology / Q000378:metabolism; D010940:Plant Proteins / Q000032:analysis*; D011485:Protein Binding; D011506:Proteins / Q000032:analysis* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D036226:Seedlings / Q000737:chemistry / Q000166:cytology; D013050:Spectrometry, Fluorescence; D014026:Nicotiana / Q000737:chemistry / Q000166:cytology*
D029681:Arabidopsis Proteins; D022762:CCAAT-Enhancer-Binding Proteins; D008164:Luminescent Proteins; D010940:Plant Proteins; D011506:Proteins; D011993:Recombinant Fusion Proteins