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.
References
[1]
Fields S, Song O (1989) A Novel Genetic System to Detect protein-protein interactions. Nature 340: 245–246.
[2]
Michnick SW, Ear PH, Manderson EN, Remy I, Stefan E (2007) Universal strategies in research and drug discovery based on protein-fragment complementation assays. Nat Rev Drug Discov 6: 569–582.
[3]
Ghosh I, Hamilton AD, Regan L (2000) Antiparallel leucine zipper-directed protein reassembly: Application to the green fluorescent protein. J Am Chem Soc 122: 5658–5659.
[4]
Hu CD, Chinenov Y, Kerppola TK (2002) Visualization of interactions among bZIP and Rel family proteins in living cells using bimolecular fluorescence complementation. Mol Cell 9: 789–98.
[5]
Hu CD, Kerppola TK (2003) Simultaneous visualization of multiple protein interactions in living cells using multicolor fluorescence complementation analysis. Nat Biotechnol 21: 539–545.
[6]
Grinberg AV, Hu CD, Kerppola TK (2004) Visualization of Myc/Max/Mad family dimers and the competition for dimerization in living cells. Mol Cell Biol 24: 4294–308.
[7]
Mervine SM, Yost EA, Sabo JL, Hynes TR, Berlot CH (2006) Analysis of G protein betagamma dimer formation in live cells using multicolor bimolecular fluorescence complementation demonstrates preferences of beta1 for particular gamma subunits. Mol Pharmacol 70: 194–205.
[8]
Magliery TJ, Wilson CG, Pan W, Mishler D, Ghosh I, et al. (2005) Detecting protein-protein interactions with a green fluorescent protein fragment reassembly trap: scope and mechanism. J Am Chem Soc 127: 146–157.
[9]
Massoud TF, Gambhir SS (2003) Molecular imaging in living subjects: seeing fundamental biological processes in a new light. Genes Dev 17: 545–580.
[10]
Fan F, Wood KV (2007) Bioluminescent assays for high-throughput screening. Assay Drug Dev Technol 5: 127–136.
[11]
Kaihara A, Kawai Y, Sato M, Ozawa T, Umezawa Y (2003) Locating a protein-protein interaction in living cells via split Renilla luciferase complementation. Anal Chem 75: 4176–4181.
[12]
Luker KE, Smith MC, Luker GD, Gammon ST, Piwnioca-Worms H, et al. (2004) Kinetics of regulated protein-protein interactions revealed with firefly luciferase complementation imaging in cells and living animals. Proc Natl Acad Sci U S A 101: 12288–12293.
[13]
Paulmurugan R, Umezawa Y, Gambhir SS (2002) Noninvasive imaging of protein-protein interactions in living subjects by using reporter protein complementation and reconstitution strategies. Proc Natl Acad Sci U S A 99: 15608–15613.
[14]
Remy I, Michnick SW (2006) A highly sensitive protein-protein interaction assay based on Gaussia luciferase. Nat Methods 3: 977–979.
[15]
Ozawa T, Kaihara A, Sato M, Tachihara K, Umezawa Y (2001) Split luciferase as an optical probe for detecting protein-protein interactions in mammalian cells based on protein splicing. Anal Chem 7 3: 2516–2521.
[16]
Saka Y, Hagemann AI, Piepenburg O, Smith JC (2007) Nuclear accumulation of Smad complexes occurs only after the midblastula transition in Xenopus. Development 134: 4209–18.
[17]
Conti E, Franks NP, Brick P (1996) Crystal structure of firefly luciferase throws light on a superfamily of adenylate-forming enzymes. Structure 4: 287–298.
[18]
Nakatsu T, Ichikawa S, Hiratake J, Saldanha A, Kobashi N, et al. (2006) Structural basis for the spectral difference in luciferase bioluminescence. Nature 440: 372–376.
[19]
Viviani VR, Ohmiya Y (2007) Beetle luciferases in measuring gene expression and imaging cell functions. Luciferases and Fluorescent Proteins. pp. 151–160.
[20]
Kim SB, Kanno A, Ozawa T, Tao H, Umezawa Y (2007) Nongenomic activity of ligands in the association of androgen receptor with SRC. ACS Chem Biol 2: 484–492.
[21]
Sawano A, Miyawaki A (2000) Directed evolution of green fluorescent protein by a new versatile PCR strategy for site-directed and semi-random mutagenesis. Nucleic Acids Res 28: E78.
[22]
Kurata T, Nakabayashi J, Yamamoto TS, Mochii M, Ueno N (2001) Visualization of endogenous BMP signaling during Xenopus development. Differentiation 67: 33–40.
[23]
Massagué J (1998) TGF-beta signal transduction. Annu Rev Biochem 67: 753–91.
[24]
Kawabata M, Inoue H, Hanyu A, Imamura T, Miyazono K (1998) Smad proteins exist as monomers in vivo and undergo homo- and hetero-oligomerization upon activation by serine/threonine kinase receptors. EMBO J 17: 4056–65.
[25]
Backer JM, Myers MG Jr, Shoelson SE, Chin DJ, Sun XJ, et al. (1992) Phosphatidylinositol 3′-kinase is activated by association with IRS-1 during insulin stimulation. EMBO J 11: 3469–79.
[26]
Sato M, Ozawa T, Inukai K, Asano T, Umezawa Y (2002) Fluorescent indicators for imaging protein phosphorylation in single living cells. Nat Biotechnol 20: 287–94.
[27]
Adams JM, Cory S (2007) The Bcl-2 apoptotic switch in cancer development and therapy. Oncogene 26: 1324–1337.
[28]
Nakajima Y, Kimura T, Sugata K, Enomoto T, Asakawa A, et al. (2005) Multicolor luciferase assay system: one-step monitoring of multiple gene expressions with a single substrate. BioTechniques 38: 891–894.
