High-sensitivity real-time imaging of dual protein-protein interactions in living subjects using multicolor luciferases.
Networks of protein-protein interactions play key roles in numerous important biological processes in living subjects. An effective methodology to assess protein-protein interactions in living cells of interest is protein-fragment complement assay (PCA). Particularly the assays using fluorescent proteins are powerful techniques, but they do not directly track interactions because of its irreversibility or the time for chromophore formation. By contrast, PCAs using bioluminescent proteins can overcome these drawbacks. We herein describe an imaging method for real-time analysis of protein-protein interactions using multicolor luciferases with different spectral characteristics. The sensitivity and signal-to-background ratio were improved considerably by developing a carboxy-terminal fragment engineered from a click beetle luciferase. We demonstrate its utility in spatiotemporal characterization of Smad1-Smad4 and Smad2-Smad4 interactions in early developing stages of a single living Xenopus laevis embryo. We also describe the value of this method by application of specific protein-protein interactions in cell cultures and living mice. This technique supports quantitative analyses and imaging of versatile protein-protein interactions with a selective luminescence wavelength in opaque or strongly auto-fluorescent living subjects.
Hida
Naoki
N
Department of Chemistry, Graduate School of Science, The University of Tokyo, Bunkyo-Ku, Tokyo, Japan.
Awais
Muhammad
M
Takeuchi
Masaki
M
Ueno
Naoto
N
Tashiro
Mayuri
M
Takagi
Chiyo
C
Singh
Tanuja
T
Hayashi
Makoto
M
Ohmiya
Yoshihiro
Y
Ozawa
Takeaki
T
D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D018076:DNA, Complementary / Q000378:metabolism; D055504:Insulin Receptor Substrate Proteins / Q000378:metabolism; D008156:Luciferases / Q000378:metabolism*; D008163:Luminescent Measurements / Q000379:methods; D051379:Mice; D008856:Microscopy, Fluorescence / Q000295:instrumentation / Q000379:methods; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010766:Phosphorylation; D025941:Protein Interaction Mapping; D012680:Sensitivity and Specificity; D014982:Xenopus laevis / Q000196:embryology* / Q000378:metabolism*; D051025:bcl-Associated Death Protein / Q000378:metabolism
D018076:DNA, Complementary; D055504:Insulin Receptor Substrate Proteins; D008156:Luciferases; D019869:Phosphatidylinositol 3-Kinases; D051025:bcl-Associated Death Protein; C526295:Irs1 protein, mouse