Genetically Encoded Optical Probe for Detecting Release of Proteins

luciferase (Rluc) fragments by protein splicing with an Ssp. DnaE intein. A target mitochondrial protein connected with an N-terminal fragment of Rluc...
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Anal. Chem. 2006, 78, 8076-8081

Genetically Encoded Optical Probe for Detecting Release of Proteins from Mitochondria toward Cytosol in Living Cells and Mammals Akira Kanno,† Takeaki Ozawa,†,‡ and Yoshio Umezawa*,†

Department of Chemistry, School of Science, The University of Tokyo, 7-3-1 Hongo Bunkyo-ku, Tokyo 113-0033, Japan, and Japan Science and Technology Corporation, Tokyo, Japan, and Department of Molecular Structure, Institute for Molecular Science, 38 Nishigonaka, Myodaiji, Okazaki, Aichi 444-8585, and PREST, Japan Science and Technology Agency, 4-1-8 Honcho Kawaguchi, Saitama, Japan

We developed a genetically encoded bioluminescence indicator for monitoring the release of proteins from the mitochondria in living cells. The principle of this method is based on reconstitution of split Renilla reniformis luciferase (Rluc) fragments by protein splicing with an Ssp DnaE intein. A target mitochondrial protein connected with an N-terminal fragment of Rluc and an N-terminal fragment of DnaE is expressed in mammalian cells. If the target protein is released from the mitochondria toward the cytosol upon stimulation with a specific chemical, the N-terminal Rluc meets the C-terminal Rluc connected with C-terminal DnaE in the cytosol, and thereby, the fulllength Rluc is reconstituted by protein splicing. The extent of release of the target fusion protein is evaluated by measuring activities of the reconstituted Rluc. To test the feasibility of this method, here we monitored the release of Smac/DIABLO protein from mitochondria during apoptosis in living cells and mice. The present method allowed high-throughput screening of an apoptosis-inducing reagent, staurosporine, and imaging of the Smac/ DIABLO release in cells and in living mice. This rapid analysis can be used for screening and assaying chemicals that would increase or inhibit the release of mitochondrial proteins in living cells and animals. Eukaryotic cells are regulated by many processes on the proteome level such as protein expression, protein-protein interactions, additional posttranslational modifications, and dramatic redistribution of proteins. There is widespread agreement that the release of potentially harmful proteins from mitochondrial intermembrane spaces (IMSs) toward the cytoplasm plays key roles in programmed cell death (apoptosis). Apoptosis is an evolutionary conserved cellular process that is involved in development, tissue homeostasis, and the pathophysiology of proliferative and neurodegenerative disorders. Most cancer cells have the ability to escape apoptosis, which often correlates with tumor * To whom correspondence should be addressed: (phone) +81-3-5841-4351; (fax) +81-3-5841-8349; (e-mail) [email protected]. † The University of Tokyo. ‡ Institute for Molecular Science.

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metastasis and resistance to traditional anticancer drug treatments. Researchers make much effort to develop new chemical compounds or peptides that accelerate the apoptotic program in tumor cells.1-3 Development of a rapid screening system to detect the release of mitochondrial proteins, therefore, is essential for the discovery of novel anticancer drugs or for screening the toxicity of chemicals. Optical imaging to monitor the dynamics of protein movement inside individual living cells relies on immunochemically labeling the protein with fluorescent dyes or genetically tagging the protein with green fluorescent protein (GFP).4,5 Immunocytochemical analysis requires a complicated procedure for sample preparations, which prevents high-throughput drug screening or risk assessment. Although these systems are powerful for revealing the spatiotemporal dynamics of proteins within individual cells, the time required for taking images is so long. The obtained results are qualitative rather than quantitative, because the number of analyzed cells is limited. Automated fluorescence microscopy has been developed for high-throughput screening of the protein movement.4 However, the algorithm to determine regions of interest left room for improvement in accuracy and precision. To address such limitations, we have developed a high-throughput screening method for detecting a particular protein transported from the cytosol into the nucleus.6-9 Taking advantage of the bioluminescence of Renilla luciferase (Rluc) with background-fluorescence-free and highly sensitive bioluminescence detection, we developed a new indicator for detecting release of a mitochondrial protein toward the cytosol in living cells and animals. The indicator is composed of two fragments of an Ssp DnaE intein10,11 connected with split Rluc.12 The enzymatic activity of split Rluc to generate bioluminescence (1) Walensky, L. D.; Kung, A. L.; Escher, I.; Malia, T. J.; Barbuto, S.; Wright, R. D.; Wagner, G.; Verdine, G. L.; Korsmeyer, S. J. Science 2004, 305, 14661470. (2) Li, L.; Thomas, R. M.; Suzuki, H.; De Brabander, J. K.; Wang, X. D.; Harran, P. G. Science 2004, 305, 1471-1474. (3) Bockbrader, K. M.; Tan, M. J.; Sun, Y. Oncogene 2005, 24, 7381-7388. (4) Kau, T. R.; Way, J. C.; Silver, P. A. Nat. Rev. Cancer 2004, 4, 106-117. (5) Gross, S.; Piwnica-Worms, D. Cancer Cell 2005, 7, 5-15. (6) Kim, S. B.; Takao, R.; Ozawa, T.; Umezawa, Y. Anal. Chem. 2005, 77, 69286934. (7) Kim, S. B.; Ozawa, T.; Watanabe, S.; Umezawa, Y. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 11542-11547. (8) Kim, S. B.; Ozawa, T.; Umezawa, Y. Anal. Chem. 2005, 77, 6588-6593. (9) Kim, S. B.; Ozawa, T.; Umezawa, Y. Anal. Biochem. 2005, 347, 213-220. 10.1021/ac061488a CCC: $33.50

© 2006 American Chemical Society Published on Web 10/19/2006

DIABLO release toward the cytosol with an apoptosis-inducing agent in living cells and living mice. This rapid analyzing system overcomes the limitations of the method with fluorescent proteintagged Smac/DIABLO:16 (i) the number of cells monitored in a batch with a fluorescence microscope is small; (ii) determination of regions of interest still remains imprecise and inaccurate. Moreover, the system opens the door to the discovery of novel anticancer drugs and screening cytotoxicity of chemicals.

Figure 1. (a) Principle of the presented split Rluc system. DnaEn fused to RlucN (N-Rluc) is connected with a targeted mitochondrial protein (Target protein). RlucC (C-Rluc) is fused to DnaEc. Upon a particular stimulation, the target protein is released from the mitochondria toward the cytosol, and DnaEn and DnaEc are brought in proximity and undergo correct folding, which induces protein splicing. RlucN and RlucC are linked together by a peptide bond to obtain a full-length Rluc. The extent of the release of the target protein is evaluated by measuring the activity of the reconstituted full-length Rluc. (b) Schematic representation of plasmids used in this work. “V5” is V5 epitope tag, GKPIPNPLLGLDST. “FLAG” is FLAG epitope tag, DYKDDDDK. For efficient protein splicing reaction, 5 and 6 amino acid residues are inserted into the splicing junctions of DnaEn and DnaEc, respectively. The fusion proteins expressed from pSmacRDn-V5 and pDRc-FLAG localize in the mitochondria and in the cytosol, respectively.

from coelenterate luciferin (coelenterazine) is completely lost (Figure 1a), because the C-terminal fragment stays in the cytosol, whereas the N-terminal fragment, which is fused with a particular mitochondrial protein, is localized in the mitochondrial IMS. Release of the protein toward the cytosol triggers protein splicing with the DnaE intein, which produces full-length Rluc. For the proof of the principle, we chose an IMS protein, Smac/DIABLO. It is well known that increase in mitochondrial membrane permeability during apoptosis results in release of several proteins,13 including Smac/DIABLO,14-16 AIF,17,18 Omi/HtrA2,19,20 endo G,21 and cytochrome c,22-24 which normally reside in the IMS. Here we describe a method for analyzing the extent of Smac/ (10) Wu, H.; Hu, Z. M.; Liu, X. Q. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 92269231. (11) Evans, T. C.; Martin, D.; Kolly, R.; Panne, D.; Sun, L.; Ghosh, I.; Chen, L. X.; Benner, J.; Liu, X. Q.; Xu, M. Q. J. Biol. Chem. 2000, 275, 9091-9094. (12) Paulmurugan, R.; Gambhir, S. S. Anal. Chem. 2003, 75, 1584-1589. (13) Martinou, J. C.; Green, D. R. Nat. Rev. Mol. Cell Biol. 2001, 2, 63-67. (14) Du, C. Y.; Fang, M.; Li, Y. C.; Li, L.; Wang, X. D. Cell 2000, 102, 33-42. (15) Verhagen, A. M.; Ekert, P. G.; Pakusch, M.; Silke, J.; Connolly, L. M.; Reid, G. E.; Moritz, R. L.; Simpson, R. J.; Vaux, D. L. Cell 2000, 102, 43-53. (16) Rehm, M.; Dussmann, H.; Prehn, J. H. M. J. Cell Biol. 2003, 162, 10311043. (17) Ye, H.; Cande, C.; Stephanou, N. C.; Jiang, S. L.; Gurbuxani, S.; Larochette, N.; Daugas, E.; Garrido, C.; Kroemer, G.; Wu, H. Nat. Struct. Biol. 2002, 9, 680-684. (18) Punj, V.; Chakrabarty, A. M. Cell. Microbiol. 2003, 5, 225-231.

EXPERIMENTAL SECTION Materials. DNA-modifying enzymes were from Takara Bio Inc. (Tokyo, Japan). A mammalian expression vector, pcDNA3.1 (+) and mouse anti-V5 antibody were obtained from Invitrogen Co. (Carlsbad, CA). Renilla luciferase assay kit, pRL-TK, and pGL4.74[hRluc/TK] encoding Rluc cDNA were purchased from Promega Co. (Madison, WI). An apoptosis inducer, staurosporine (STS), digitonin, gelatin from cold water fish skin (FSG), minimal essential medium (MEM), and phosphate-buffered saline (PBS) tablets were from Sigma (St. Louis, MO). Fetal bovine serum (FBS), 0.05% trypsin-EDTA, Hank’s balanced buffered saline (HBSS), penicillin and streptomycin solution, sodium pyruvate solution, and nonessential amino acids solution were obtained from Gibco BRL (Rockville, MD). A transfection reagent, TransIT-LT1, was purchased from Mirus Bio Co. (Madison, WI). Mouse antiHSP60 monoclonal antibody and mouse anti-cytochrome c monoclonal antibody were from BD Biosciences (Franklin Lakes, NJ). An ECL western blotting detection kit, an ECL advance western blotting detection kit, horseradish peroxidase (HRP)-linked sheep anti-mouse Ig antibody, and a nitrocellulose membrane (a HybondECL) were purchased from GE Healthcare (Buckinghamshire, England). A protein assay kit was from Bio-Rad (Hercules, CA). Alexa Fluor 488-conjugate anti-mouse IgG and MitoTracker Orange were from Molecular Probes (Eugene, OR). Construction of Plasmids for Mammalian Cell Expression. An Escherichia coli strain, DH5R, was used as a bacterial host for construction of all plasmids. To express proteins encoded by the vectors in mammalian cells, we used pcDNA3.1 (+), which has a human cytomegalovirus immediate-early promoter. The constructed plasmids were named pSmac-RDn-V5 and pDRcFLAG (Figure 1b). The sequences of all plasmids were verified by sequencing with a genetic analyzer ABI prism 310 (PE Biosystems, Tokyo, Japan). Cell Culture and Transfection. Human metastatic mammary carcinoma-derived MCF-7 cells were cultured in MEM supplemented with 10% heat-inactivated FBS, 100 unit/mL penicillin, 100 µg/mL streptomycin, 1 mM sodium pyruvate, and 0.1 mM (19) Suzuki, Y.; Imai, Y.; Nakayama, H.; Takahashi, K.; Takio, K.; Takahashi, R. Mol. Cell 2001, 8, 613-621. (20) van Loo, G.; van Gurp, M.; Depuydt, B.; Srinivasula, S. M.; Rodriguez, I.; Alnemri, E. S.; Gevaert, K.; Vandekerckhove, J.; Declercq, W.; Vandenabeele, P. Cell Death Differ. 2002, 9, 20-26. (21) van Loo, G.; Schotte, P.; van Gurp, M.; Demol, H.; Hoorelbeke, B.; Gevaert, K.; Rodriguez, I.; Ruiz-Carrillo, A.; Vandekerckhove, J.; Declercq, W.; Beyaert, R.; Vandenabeele, P. Cell Death Differ. 2001, 8, 1136-1142. (22) Liu, X. S.; Kim, C. N.; Yang, J.; Jemmerson, R.; Wang, X. D. Cell 1996, 86, 147-157. (23) Goldstein, J. C.; Waterhouse, N. J.; Juin, P.; Evan, G. I.; Green, D. R. Nat. Cell Biol. 2000, 2, 156-162. (24) Luetjens, C. M.; Kogel, D.; Reimertz, C.; Dussmann, H.; Renz, A.; SchulzeOsthoff, K.; Nieminen, A. L.; Poppe, M.; Prehn, J. H. M. Mol. Pharmacol. 2001, 60, 1008-1019.

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nonessential amino acids at 37 °C in an atmosphere of 5% CO2. Cells were seeded onto multiwell culture plates, transfected with cDNA plasmids by using TransIT-LT1 reagent, and left at 37 °C in an atmosphere of 5% CO2 for 48 h. Evaluation of Renilla Luciferase Activities. MCF-7 cells were cotransfected with pSmac-RDn-V5 and pDRc-FLAG and were incubated for 48 h. After the cells were stimulated with an apoptosis-inducing reagent for 2-6 h, the cells were scraped out with HBSS. Luciferase activities of 20 µL of the cell suspensions were measured by using a Renilla luciferase assay kit with a luminometer (MiniLumat LB9506; Berthold, Nashua, NH) for 20 s. After the measurements, the cells were lysed with a lysis buffer of the Renilla luciferase assay kit, and protein concentrations of the cell lysates in the supernatants were assessed with the BioRad protein assay kit. Cell Fractionation. Approximately 107 cells were transfected with pSmac-RDn-V5 for 48 h, harvested, and suspended in a 2 mL of fractionation buffer (0.25 M sucrose, 5 mM EDTA, 20 mM Tris-HCl, pH 7.4).25 The cells were broken with a Teflon-glass homogenizer, and the homogenates were centrifuged at 600g for 15 min to remove nuclei and cell debris. The supernatants were centrifuged at 9000g for 15 min to yield the mitochondrial fractions as previously described.25 The pellets were denatured in an SDSloading buffer and subjected to western blotting analysis. Selective Membrane Permeabilization. Selective permeabilization of MCF-7 cells with digitonin was performed to analyze the release of the fusion protein encoded by pSmac-RDn-V5 (Smac-RDn). This method eliminates a possible artifact due to mechanical breakage of the outer mitochondrial membrane by homogenization.24 Six-well culture dishes with 106 cells/well were placed on ice, and the culture medium was removed. The cells were washed once with ice-cold PBS and subsequently incubated in a 100 µL of a permeabilization buffer (210 mM D-mannitol, 70 mM sucrose, 10 mM HEPES, 5 mM succinic acid, 0.2 mM EGTA, and 250 µg/mL digitonin, pH 7.2). To determine an optimal incubation time for acquisition of cytosolic fractions without contamination of mitochondrial proteins, we treated the cells with the permeabilization buffer containing 250 µg/mL digitonin at 4 °C for differing times. At the indicated time points (Figure S-1, Supporting Information), the permeabilization buffer was transferred to a reaction tube and centrifuged for 10 min at 13000g. Subsequently, the supernatants were transferred to new reaction tubes and denatured in an SDS-loading buffer. Pellets were removed from the remainings on six-well plates with an SDSloading buffer. Equal amounts of proteins were analyzed by western blotting using polyacrylamide gels. Figure S-1 in Supporting Information shows that only the sample permeabilized for 1 min presents the cytosolic fraction without a mitochondrial component, cytochrome c. Hereafter, selective permeabilizations with digitonin were performed at 4 °C for 1 min. Western Blotting Analysis. Lysates of cells transfected with the pSmac-RDn-V5, pDRc-FLAG, or both were separated with 10 or 15% SDS-PAGE gels and electrophoretically transferred onto a nitrocellulose membrane. The membrane was probed with a particular antibody and then with HRP-labeled anti-mouse IgG antibody. This secondary antibody was visualized by using the (25) Ozawa, T.; Sako, Y.; Sato, M.; Kitamura, T.; Umezawa, Y. Nat. Biotechnol. 2003, 21, 287-293.

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ECL western blotting detection kit or the ECL advance western blotting detection kit with a LAS-1000 plus image analyzer (Fuji Film Co., Tokyo, Japan). Immunocytochemistry. MCF-7 cells were cultured on microscope glass slides and were transfected with pSmac-RDnV5. The transfected cells were stained with growth medium containing 1 µM MitoTracker Orange at 37 °C in an atmosphere of 5% CO2 for 30 min. After the staining, the cells were fixed with MEM for their growth containing 3.7% formaldehyde at 37 °C in an atmosphere of 5% CO2 for 15 min and were permeabilized in PBS containing 0.2% Triton X-100 at room temperature for 5 min. The cells were blocked with 0.2% fish skin gelatin and then incubated with mouse anti-V5 antibody. The antibody was reacted with Alexa Fluor 488-conjugate secondary antibody. The fluorescence was recorded by using a confocal laser-scanning microscope (LSM510 META; Carl Zeiss, Jena, Germany) provided with a Plan-Neofluar 100× /1.3 oil objective (Carl Zeiss). The confocal laser scanning unit was equipped with a 488-nm argon laser (Alexa Fluor 488 excitation) and a 543-nm helium/neon laser (MitoTracker Orange excitation). Fluorescence was detected after transmission of a 505-530-nm band-pass filter (Alexa Fluor 488 emission) or a 567-nm long-pass filter (MitoTracker Orange emission). Imaging of Living Mice. MCF-7 cells were transfected with pRL-TK or cotransfected with pSmac-RDn-V5 and pDRc-FLAG. The cells were harvested with rubber scrapers after incubation in MEM with 10% FBS for 60 h. The cells were suspended in PBS, and an aliquot of 1 × 106 cells was implanted in two different sites on the back of an anesthetized BALB/c-nu/nu mouse (female, 5week-old, ∼17 g of body weight; Sankyo Labo Services, Tokyo, Japan). After the implantation of the cells, 10% DMSO solution of STS (10 µg/kg of body weight) was intraperitoneally (ip) injected. Immediately, DMSO containing coelenterazine (2.0 mg/kg of body weight) was injected ip, and the mouse was imaged. All mice were imaged with a CCD camera (IVIS100 system, Xenogen, Alameda, CA). Photons emitted from the implanted cells were collected and integrated for 1 min. Image processing was performed by a LIVING IMAGE software (Xenogen). To quantify the measured luminescence, regions of interest were drawn over the cellimplanted areas, and the luminescence intensities (photons s-1 cm-2) were evaluated. RESULTS AND DISCUSSION Design of Fusion Proteins for Monitoring Release of Organellar Proteins. Using Saccharomyces cerevisiae VDE or Ssp DnaE inteins, we previously reported methods for monitoring protein-protein interactions,26-29 identifying organellar proteins,25,30 and determining protein nuclear transportation.6-9 The basic concept of the above is based on the reconstitution of split reporter proteins by protein splicing with the inteins.31-33 The (26) Ozawa, T.; Nogami, S.; Sato, M.; Ohya, Y.; Umezawa, Y. Anal. Chem. 2000, 72, 5151-5157. (27) Ozawa, T.; Kaihara, A.; Sato, M.; Tachihara, K.; Umezawa, Y. Anal. Chem. 2001, 73, 2516-2521. (28) Kanno, A.; Ozawa, T.; Umezawa, Y. Anal. Chem. 2006, 78, 556-560. (29) Ozawa, T.; Takeuchi, M.; Kaihara, A.; Sato, M.; Umezawa, Y. Anal. Chem. 2001, 73, 5866-5874. (30) Ozawa, T.; Nishitani, K.; Sako, Y.; Umezawa, Y. Nucleic Acids Res. 2005, 33, e34. (31) Ozawa, T.; Umezawa, Y. Curr. Opin. Chem. Biol. 2001, 5, 578-583. (32) Ozawa, T. Bull. Chem. Soc. Jpn. 2005, 78, 739-751.

Figure 3. Renilla luciferase activities upon release of the Smac/ DIABLO probe in MCF-7 cells. The cells were cultured in 6-well plates and cotransfected with 1 µg of pSmac-RDn-V5 and pDRc-FLAG. Observed luminescence intensities were normalized against amounts of total protein. After the cells were incubated for 72 h, the cells were treated with 50 nM STS (solid square) or 0.1% DMSO (open square) for 2-6 h.

Figure 2. Characterization of the recombinant protein indicators within cell lines. (a) Western blotting analysis of mitochondrial fractions. Mitochondrial fractions were obtained from intact MCF-7 cells (ctrl) and MCF-7 cells transfected with pSmac-RDn-V5 (pSmac-RDn-V5) at 37 °C in an atmosphere of 5% CO2 for 48 h and were separated by SDS-PAGE. Proteins were transferred to nitrocellulose membranes and stained with the indicated monoclonal antibodies. HSP60 is a mitochondrial marker protein. (b) The immunocytochemistry images of MCF-7 cells transiently transfected with pSmac-RDn-V5. MCF-7 cells were cultured for 48 h after the transfection, and then the cells (pSmac-RDn-V5) and intact MCF-7 cells (ctrl) were subjected to immunocytochemical procedure. SmacRDn was recognized by anti-V5 antibody and stained with Alexa Fluor 488-labeled secondary antibody (Anti-V5). The mitochondria were stained with MitoTracker Orange (MitoTracker). Images of the cells (transmission) were also obtained. After the observations, the images of fluorescence were superimposed (merge). Bar, 20 µm. (c) Monitoring the release of the Smac/DIABLO fusion protein from the mitochondria toward the cytosol with western blotting. MCF-7 cells were transfected with pSmac-RDn-V5 and incubated at 37 °C in an atmosphere of 5% CO2 for 48 h. In order to obtain cytosolic fractions, the cells were subjected to a selective membrane permeabilization procedure at the indicated time points after stimulation with 50 nM STS. The control was not treated with STS. The release of SmacRDn from the mitochondria toward the cytosol was analyzed by western blotting. β-Actin is as a cytoplasm-specific marker.38 Experiments were performed three times with similar results.

principle of the presented method is a further extension of an earlier method to identify organellar proteins.25,30 We used Rluc as a reporter protein for detecting release of proteins from mitochondria toward the cytosol, because Rluc is a desirable monomeric reporter protein. The features of Rluc and its substrate, coelenterazine, are as follows: (i) the molecular weight of Rluc is small; (ii) the substrate of Rluc, coelenterazine, rapidly penetrates membranes of living cells; (iii) ATP is not required for the enzymatic activity of Rluc.34,35 These are suitable for nondestructive (33) Ozawa, T. Anal. Chim. Acta 2006, 556, 58-68.

analysis monitoring the release of proteins. Rluc was divided into two fragments, Rluc(1-229) (RlucN) and Rluc(230-311) (RlucC), so that the luminescence was completely lost. We constructed a cDNA encoding a fusion protein consisting of RlucN, the Nterminus of DnaE intein (DnaEn), and a targeted mitochondrial protein (Figure 1b). In order to express a cytosolic bait protein that captures the target protein, we constructed a cDNA encoding RlucC fused to the C-terminus of DnaE intein (DnaEc). For efficient protein splicing reaction to occur, five and six amino acid residues (KFAEY and CFNLSH) were inserted at the end of the splicing junctions in DnaEn and DnaEc, respectively. As a model of proteins released from mitochondria, we chose an IMS protein, Smac/DIABLO, which diffuses toward the cytosol at an early stage of apoptosis process. The scheme of the present system is shown in Figure 1a. When the Smac/DIABLO fusion protein is released from mitochondria due to the apoptosis-inducing reagent staurosporine, it spreads over the cytosol. Consequently, the DnaE fragments are brought in proximity and undergo correct folding, which induces protein splicing. As a result, RlucN and RlucC directly link to each other by a peptide bond. The extent of the release of Smac/DIABLO is, therefore, evaluated by measuring the bioluminescence intensity originating from the reconstituted the full-length Rluc. Monitoring Release of Smac/DIABLO from Mitochondria toward Cytosol. We analyzed whether the fusion protein expressed with pSmac-RDn-V5 localizes in the mitochondria by western blotting analysis. After MCF-7 cells were transiently transfected with pSmac-RDn-V5 and incubated for 48 h, the cells were harvested and the assay was carried out. The results indicate that Smac-RDn localized in the mitochondria (Figure 2a). To confirm this, we optically determined the localization of SmacRDn by immunofluorescence microscopy. MCF-7 cells were (34) Matthews, J. C.; Hori, K.; Cormier, M. J. Biochemistry 1977, 16, 85-91. (35) Lorenz, W. W.; McCann, R. O.; Longiaru, M.; Cormier, M. J. Proc. Natl. Acad. Sci. U.S.A. 1991, 88, 4438-4442. (36) Umezawa, Y. Trends Anal. Chem. 2005, 24, 138-146. (37) Umezawa, Y. Biosens. Bioelectron. 2005, 20, 2504-2511. (38) Ohtsuka, T.; Ryu, H.; Minamishima, Y. A.; Macip, S.; Sagara, J.; Nakayama, K. I.; Aaronson, S. A.; Lee, S. W. Nat. Cell Biol. 2004, 6, 121-128.

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Figure 4. STS-dependent release of the Smac/DIABLO probe in living mice. (a) In vivo imaging of mice implanted with transiently transfected MCF-7 cells. The mice were subcutaneously implanted with MCF-7 cells carrying pSmac-RDn-V5 and pDRc-FLAG in the right side and MCF-7 cells carrying a full-length Rluc in the left side. The images were obtained after the ip injection of STS (10 µg/kg of body weight). Acquisition of images was performed at the indicated times. A representative of three mice with similar results is displayed. (b) Analysis of RLU change in (a). The graph shows observed RLUs normalized against the RLU at 0 h. Indicated time corresponds with the time indicated (a). The normalized RLU values are averages among three mice with standard deviation.

transfected with pSmac-RDn-V5 and were subjected to immunostaining. The results shown in Figure 2b demonstrate that Smac-RDn predominantly localized in the mitochondria. Next, we monitored release of Smac/DIABLO toward the cytosol with western blotting analysis. MCF-7 cells transfected with pSmacRDn-V5 were stimulated with 50 nM STS. STS is a protein kinase C inhibitor that triggers the release of IMS proteins including Smac/DIABLO16 and cytochrome c.24 To obtain the cytosolic fractions of the cells, we incubated the cells with permeabilization buffer containing digitonin for 1 min. The amount of Smac-RDn increased in the cytosol after the treatment with STS (Figure 2c). These results suggest that STS induces the release of SmacRDn fusion protein predominantly localizing in the mitochondria toward the cytosol within 2 h. Increase in Renilla Luciferase Activity upon Staurosporine-Induced Smac/DIABLO Release. To demonstrate the nondestructive assay for detecting the Smac/DIABLO release from the mitochondria toward the cytosol, bioluminescence was 8080

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measured upon adding STS. After MCF-7 cells were transfected with pSmac-RDn-V5 and pDRc-FLAG and incubated for 72 h, the cells were stimulated with 50 nM STS for 2-6 h. To eliminate errors due to variation of the number of cells in each culture plate, the luminescence was assessed as a relative light unit (RLU), which is calculated by normalizing the luminescence intensity against amount of total proteins. Figure 3 indicates that the increase in RLU is in parallel with the Smac-RDn release (see also Figure 2c). We conclude that the present method is applicable to nondestructive analysis for monitoring release of organellar proteins in living mammalian cells. The cells without STS treatment showed a subtle RLU. This signal may be because SmacRDn was mislocalized in the cytosol (Figure 2b and c). We reasoned that overexpression of Smac-RDn in several cells caused the mislocalization. In Vivo Imaging of Smac/DIABLO Release in Living Mice. For a further application of the present method, we imaged Smac/ DIABLO release triggered by STS in living mice. We implanted

MCF-7 cells expressing full-length Rluc from wild-type codons under the control of a herpes simplex virus thymidine kinase promoter in the left side on the back of mice in a depth of 1 mm. Also, we implanted MCF-7 cells expressing the recombinant protein indicators from preferred mammalian codons under the control of a human cytomegalovirus immediate-early promoter/ enhancer in the right side on the back of mice in a depth of 1 mm. We observed the luminescence intensity (photons s-1 cm-2) from the right side (LumR) and that from the left side (LumL). The extent of Smac-RDn release was evaluated as RLU; RLU ) LumR/ LumL. This method of calculation eliminates variations in some experimental conditions such as transfection efficiency, the numbers of the implanted cells, and the amount of injected coelenterazine circulating within mice. Figure 4a shows the luminescence intensities from a mouse stimulated with STS for 0-4 h, while the percentage of the RLU during this time is shown in Figure 4b. Two hours after ip injections of STS (10 µg/kg of body weight), the ratio of RLU reached ∼150% of RLU at 0 h (Figure 4a and b). Agreement between this result and the result of an in vitro assay (Figure 3) indicates that the release of organellar proteins toward the cytosol can be detected in living mammals with our system. CONCLUSION Several techniques have been reported to image the movement of proteins within single living cells with fluorescent tags. Although automated fluorescence microscopy,4 for example, is a representative high-throughput analysis tool for monitoring the movement of fluorescent dye- or GFP-tagged proteins, it is difficult in each cell to accurately sort out and distinguish the fluorescence of tagged proteins localized only in an organelle from that left in the cytosol.36,37 To overcome such limitation, we used split Rluc fragments and its reconstitution-dependent bioluminescence signal by protein splicing as a reporter protein for monitoring movement of an IMS protein, Smac/DIABLO, in this study. We described herein a new system, with which we evaluated the extent of the Smac/DIABLO release from the mitochondria toward the cytosol

in living cells and mice upon stimulation with STS. This system relies on a set of two genetically encoded indicators consisting of an IMS protein, inteins, and split Rluc fragments. Using this system, we determined the subcellular distribution of Smac/ DIABLO by the luminescence signals. The luminescence is generated only when the Smac/DIABLO fusion proteins were released from the mitochondria toward the cytosol. The Smac/ DIABLO fusion proteins remaining in the mitochondria did not induce reconstitution of split Rluc fragments, and therefore, no background luminescence was observed. In addition, the number of the analyzed cells in a batch was ∼105, which was enough to precisely evaluate the extent of Smac/DIABLO release toward the cytosol. Thus, this indicator enabled fluorescent-backgroundfree, accurate, precise, and sensitive detection, which is of great advantage for monitoring the release of IMS proteins in a highthroughput manner. The presented method may allow a versatile application to pharmacological or toxicological analyses including assessment of novel anticancer drugs and evaluation of risk factors of toxic chemical compounds on living cells or mammals. Also, the generation of transgenic mammals carrying the genetically encoded bioluminescent indicator is a possible application to monitoring apoptotic events in development or in tissue homeostasis. ACKNOWLEDGMENT This work was supported by grants from Japan Science and Technology Agency (JST) and Japan Society for the Promotion of Science (JSPS). SUPPORTING INFORMATION AVAILABLE Determination of an optimal permeabilization with digitonin. This material is available free of charge via the Internet at http:// pubs.acs.org. Received for review August 10, 2006. Accepted September 14, 2006. AC061488A

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