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 base...

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Published inProceedings of the National Academy of Sciences - PNAS Vol. 101; no. 32; pp. 11542 - 11547
Main Authors Kim, Sung Bae, Ozawa, Takeaki, Watanabe, Shigeaki, Umezawa, Yoshio, Hastings, J. Woodland
Format Journal Article
LanguageEnglish
Published United States National Academy of Sciences 10.08.2004
National Acad Sciences
Subjects
Active Transport, Cell Nucleus
Animals
Antibodies
Biological Sciences
Biology
Body weight
Brain - metabolism
Cell Line
Cell nucleus
Complementary DNA
COS cells
Cytosol
Diagnostic Imaging - methods
DNA Polymerase III - genetics
Gene expression
Genes, Reporter
Imaging
Luminescence
Medical imaging
Mice
Protein Engineering
Protein Splicing
Protein Transport
Proteins
Receptors, Androgen - metabolism
Renilla reniformis
Splicing
Sulfotransferases - genetics
Transfection
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Summary: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.
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S.B.K. and T.O. contributed equally to this work.
This paper was submitted directly (Track II) to the PNAS office.
To whom correspondence should be addressed. E-mail: umezawa@chem.s.u-tokyo.ac.jp.
Abbreviations: AR, androgen receptor; DHT, 5α-dihydrotestosterone; NLS, nuclear localization signal; CCD, charge-coupled device; Rluc, Renilla luciferase; Rluc-N, N-terminal domain of Rluc; Rluc-C, C-terminal domain of Rluc; PCB, polychlorinated biphenyls; DnaE-N, N-terminal splicing domain of DnaE; DnaE-C, C-terminal splicing domain of DnaE.
Edited by J. Woodland Hastings, Harvard University, Cambridge, MA, and approved June 30, 2004
ISSN:0027-8424
1091-6490
DOI:10.1073/pnas.0401722101