High-throughput sensing and noninvasive imaging of protein nuclear transport by using reconstitution of split Renilla luciferase.
Nucleocytoplasmic trafficking of functional proteins plays a key role in regulating gene expressions in response to extracellular signals. We developed a genetically encoded bioluminescent indicator for monitoring the nuclear trafficking of target proteins in vitro and in vivo. The principle is based on reconstitution of split fragments of Renilla reniformis (Rluc) by protein splicing with a DnaE intein (a catalytic subunit of DNA polymerase III). A target cytosolic protein fused to the N-terminal half of Rluc is expressed in mammalian cells. If the protein translocates into the nucleus, the Rluc moiety meets the C-terminal half of Rluc, and full-length Rluc is reconstituted by protein splicing. We demonstrated quantitative cell-based in vitro sensing of ligand-induced translocation of androgen receptor, which allowed high-throughput screening of exo- and endogenous agonists and antagonists. Furthermore, the indicator enabled noninvasive in vivo imaging of the androgen receptor translocation in the brains of living mice with a charge-coupled device imaging system. These rapid and quantitative analyses in vitro and in vivo provide a wide variety of applications for screening pharmacological or toxicological compounds and testing them in living animals.
Department of Chemistry, School of Science, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
Sung Bae
SB
Kim
Takeaki
T
Ozawa
Shigeaki
S
Watanabe
Yoshio
Y
Umezawa
D021581:Active Transport, Cell Nucleus; D000818:Animals; D001921:Brain / Q000378:metabolism; D002460:Cell Line; D004258:DNA Polymerase III / Q000235:genetics; D003952:Diagnostic Imaging / Q000379:methods*; D017930:Genes, Reporter; D051379:Mice; D015202:Protein Engineering; D019154:Protein Splicing; D021381:Protein Transport; D011944:Receptors, Androgen / Q000378:metabolism*; D015238:Sulfotransferases / Q000235:genetics; D014162:Transfection
D011944:Receptors, Androgen; C074904:DNA polymerase III, alpha subunit; D004258:DNA Polymerase III; D015238:Sulfotransferases; C022568:Renilla-luciferin sulfotransferase