High-throughput sensing and noninvasive imaging of protein nuclear transport by using reconstitution of split Renilla luciferase

Abstract

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.

Fig. 1.

Basic strategy for the detection of AR translocation. (A) Principle for monitoring translocation of a particular protein (X) into the nucleus using protein splicing of split-Rluc. RLuc-N (1∼229 aa) is connected with DnaE-N (1∼123 aa) and NLS [(DPKKKRKV)₃], which is predominantly localized in the nucleus. DnaE-C (1∼36 aa) is connected with RLuc-C (230∼311 aa) and a protein X, which is localized in the cytosol. When the tandem fusion protein consisting of DnaE-C, Rluc-C, and protein X translocates into the nucleus, the DnaE-C interacts with DnaE-N, and protein splicing results. Rluc-N and -C are linked by a peptide bond, and the reconstituted Rluc recovers its bioluminescence activity. FLAG means epitope (DYKDDDDK). (B) Schematic structures of cDNA constructs. All italics mean the genes of their corresponding proteins. Additional sequences at the boundaries between rLuc and dnaE are shown under the bars. GC linker, cDNA sequence of amino acids GGGGSG.

Fig. 2.

Characterization of the indicators in vitro.(A) The immunocytochemical images of COS-7 cells transiently transfected with pcRDn-NLS or pcDRc-AR. The COS-7 cells were cultured for 36 h, and then the cells were incubated for 2 h in the absence or presence of 1 μM DHT. (Top) The two expressed proteins were recognized by anti-AR and anti-FLAG antibodies, respectively, and stained with Cy-5-labeled secondary antibody. (Middle) The nuclei stained with YO-PRO-1; (Bottom) their merged images are shown with the transmission. (B) Western blot of protein extracts from COS-7 cells (lane 1) and from the cells cotransfected with pcRDn-NLS and pcDRc-AR in the absence (lane 2) or presence (lane 3) of 1 μM DHT. As a reference for the amounts of the electrophoresed proteins, β-actin was stained with its specific antibody. (C) Quantitative analysis of the Rluc activity for nuclear and cytoplasmic fractions. The cellular fractions were obtained from cotransfected cells with pcRDn-NLS and pcDRc-AR in the absence or presence of 1 μM DHT (n = 3).

Fig. 3.

Quantitative analysis of AR translocation for various chemical compounds. (A) Dose–response curves for DHT based on the luminescence intensity of the reconstituted Rluc. The COS-7 cells were transiently transfected with pcDRc-AR or cotransfected with pcRDn-NLS and pcDRc-AR, and Rluc activities were tested. The mean luminescence intensities (n = 3) were determined at each DHT concentration. (B) Dose–response curves for steroid hormones based on the luminescence intensity of Rluc. The COS-7 cells were cotransfected with pcRDn-NLS and pcDRc-AR, and Rluc activities were tested upon addition of each hormone (n = 3). (C) Dose–response curves for synthetic chemical compounds based on the luminescence intensity of Rluc. (D) An inhibitory effect of procymidone or PCB on the bioluminescence developed by 0.1 μM DHT. Red dotted curves were obtained from the cells cotransfected with pcRDn-NLS and pcDRc-AR, whereas blue dotted curves were from the cells with pcRDn-NLS and pcDRc-NLS (positive control, n = 3). The circles and squares are the means of luminescence intensities obtained from the cotransfected cells stimulated with procymidone and PCB, respectively. Left and right y axes are for red and blue dotted lines, respectively.

Fig. 4.

DHT-dependent translocation of AR in living mice. (A) In vivo optical CCD imaging of mice carrying transiently transfected COS-7 cells in the presence or absence of DHT (100 μg/kg of body weight). The mice were imaged after s.c. implantation of COS-7 cells transiently cotransfected with pcRDn-NLS and pcDRc-AR. One group of mice was injected with DHT and the other group was left uninjected. (B) The average of photon counts from each implanted site in A (n = 4). The graph shows the observed photon counts for uninjected and DHT-injected group of mice with mean values over four mice, respectively. The average values of photon counts for 4 mice were (1.23 ± 0.15) × 10⁵ (–DHT), and (3.70 ± 0.87) × 10⁵ (+DHT) (photons per sec per cm²).

Fig. 5.

An inhibitory effect of procymidone or PCB on the bioluminescence developed by DHT (10 μg/kg of body weight) in living mice. (A) The inhibitory effect of chemicals on AR translocation into the nucleus in the mouse brain. The COS-7 cells transiently cotransfected with pcRDn-NLS and pcDRc-AR were implanted in the forebrain of the nude mice at a depth of 3 mm through a 1-mm burrhole. Of mouse groups 1–4, groups 1 and 2 were stimulated with 1% DMSO, whereas groups 3 and 4 were stimulated with procymidone (10 mg/kg body weight) and PCB (10 mg/kg of body weight), respectively. Two hours after the stimulation, mouse groups 2–4 were then stimulated with DHT (10μg/kg of body weight). Two hours after DHT stimulation, the mice were imaged in 2-min intervals until reaching the maximum photon counts after intercerebral injection of coelenterazine (1.4 mg/kg of body weight). (B) The average of photon counts from each implanted site in A (n = 3). The averages of three mice were (5.53 ± 0.53) × 10⁴ (group 1), (7.68 ± 0.91) × 10⁴ (group 2), (5.07 ± 0.23) × 10⁴ (group 3), and (4.18 ± 0.55) × 10⁴ (group 4) (photons per sec per cm²).