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Cite this: Chem. Commun., 2014, 50, 2958 Received 27th October 2013, Accepted 14th January 2014
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Giant vesicles functionally expressing membrane receptors for an insect pheromone† Satoshi Hamada,‡a Masashi Tabuchi,‡b Taro Toyota,acd Takeshi Sakurai,e Tomohiro Hosoi,a Tomonori Nomoto,a Kei Nakatani,*f Masanori Fujinami*a and Ryohei Kanzaki*e
DOI: 10.1039/c3cc48216b www.rsc.org/chemcomm
To date, biochemical approaches to membrane receptors have been limited to the following methods: knockout or overexpression of membrane receptors by gene introduction and genome engineering or extraction of membrane receptor–surfactant complexes from innate cells and their introduction into model biomembranes. Here, we describe the development of a third method involving gene expression using cell-free in situ protein synthesis inside model biomembrane capsules. We verified this method by synthesizing olfactory receptors from the silkmoth Bombyx mori inside giant vesicles and found that they were excited in the presence of their ligand the Bombyx mori sex pheromone.
Membrane receptors are the outermost proteins of the cell that can detect the extracellular environment. The biochemical and molecular biological methods used to elucidate the mechanisms by which membrane receptors function primarily comprise the following two techniques: knockout or overexpression of membrane receptors by gene introduction and genome engineering or the extraction of membrane receptor–surfactant complexes from innate cells and their introduction into model biomembranes.1 However, in the former method, the potential
interference from the innate membrane proteins of host cells can be problematic. Furthermore, because the orientation and folding of receptors in model biomembranes are complex, the latter method is generally difficult to control. Here, we tackled these problems by establishing a system for the cell-free in situ synthesis of membrane receptors inside cell-sized lipid bilayer capsules called giant vesicles (GVs). The technique for cell-free protein synthesis inside of GVs was recently developed for the synthesis of ‘‘wet’’ artificial cells.2 Some of the membrane proteins were functionally expressed in the presence of lipid vesicles.3–9 However, to the best of our knowledge, there are no reports on the in situ synthesis of membrane receptors inside of GVs due to the difficultly of encapsulating all of the required reagents and organelles. Recently, a technique has been developed by the Libchaber3 and Weitz10 groups as well as our group11 to prepare GVs from water-in-oil emulsions, which play a key role in overcoming the difficulty of encapsulation. To verify that this technique could be used in the in situ synthesis of membrane receptors, we focused on olfactory receptors (ORs) and their co-receptors (Orco) in insects that form heteromeric complexes of ligand-gated ion channels12–16 because they comprise a huge protein family and have genomic variations.17 Among them, a sex
a
Department of Applied Chemistry and Biotechnology, Graduate School of Engineering, Chiba University, 1-33 Yayoi, Inage, Chiba, Chiba 263-8522, Japan. E-mail: [email protected]; Fax: +81-43-290-3503; Tel: +81-43-290-3503 b Department of Advanced Interdisciplinary Studies, Graduate School of Engineering, The University of Tokyo, 4-6-1 Komaba, Meguro, Tokyo 153-8904, Japan c Department of Basic Science, Graduate School of Arts and Sciences, The University of Tokyo, 3-8-1 Komaba, Meguro, Tokyo 153-8902, Japan d Research Center of Complex Systems Biology, Graduate School of Arts and Sciences, The University of Tokyo, 3-8-1 Komaba, Meguro, Tokyo 153-8902, Japan e Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Komaba, Meguro, Tokyo 153-8904, Japan. E-mail: [email protected]; Fax: +81-3-3469-2397; Tel: +81-3-5452-5195 f Faculty of Life and Environmental Sciences, University of Tsukuba, 1-1-1 Ten-noudai, Tsukuba, Ibaraki 305-8572, Japan. E-mail: [email protected]; Fax: +81-29-853-6614; Tel: +81-29-853-6672 † Electronic supplementary information (ESI) available: Fig. S1 and full experimental details. See DOI: 10.1039/c3cc48216b ‡ These authors contributed equally to this work.
2958 | Chem. Commun., 2014, 50, 2958--2961
Fig. 1 Schematic of GV encapsulating cell-free protein synthesizing reagents with canine pancreatic microsomal membranes and mRNA of BmOR1 and BmOrco, which form an olfactory receptor complex that could be excited by bombykol.
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Fig. 2 Western blot of synthesized EGFP-BmOR1 and EGFP-BmOrco. Asterisks indicate signals corresponding to the approximate molecular weight of synthesized proteins. The additional bands of low molecular weight are likely to be truncated synthesis products. Similar observations were previously reported in the cell-free synthesis of Drosophila melanogaster ORs.21
pheromone receptor BmOR1 and its co-receptor BmOrco from the silkmoth Bombyx mori18,19 were examined because their odorant– receptor interactions are highly specific. Thus, the purpose of this study was to construct GVs exhibiting OR functionalization (Fig. 1). Before preparing GVs, we synthesized N-terminally EGFP-tagged BmOR1 (EGFP-BmOR1) and EGFP-tagged BmOrco (EGFP-BmOrco) separately in a test tube by using insect cell-free protein synthesis reagents (see ESI†).20 Western blot analysis with anti-GFP revealed bands corresponding to the predicted molecular weight of each protein, confirming that full-length proteins were synthesized using this cell-free system (Fig. 2). GVs were subsequently produced using egg yolk lecithin, cholesterol, and polyethyleneglycol (PEG)-tagged phospholipids. We then encapsulated cell-free protein synthesis reagents of mRNA, cell-extract from insect cells, microsomal membranes from canine pancreatic cells, and sucrose in Tris-HCl buffer. To stabilize GV membranes, PEG-phospholipids and cholesterol were used as additives for lecithin GVs.22 Sucrose and canine pancreatic microsomal membranes were added to promote cellfree synthesis.23 Briefly, the cell-free synthesis reagent solution was emulsified in an oil mixture (liquid paraffin and squalene),10 which dissolved the lecithin, cholesterol, and PEG-phospholipids. The water-in-oil emulsion was subsequently layered onto a buffered solution containing glucose as an osmotic balancer. Centrifugation at 0 1C led to the formation of GVs via membrane bonding of the emulsion droplet monolayer and the water–emulsion interface monolayer. GV dispersions were obtained after incubation at 20–23 1C for 4 h. Using a differential interference contrast/fluorescence microscope equipped with a low magnitude objective lens, we observed that most GVs had a single-wall structure. Furthermore, the number density and average diameter of the GVs, which were stained by Texas Reds phospholipids, were 105–106 mL 1 and 11 5 mm (mean standard deviation) after the incubation, respectively (see Fig. S1 in ESI†). Although GVs were formed in the absence of PEG-phospholipids, almost all collapsed while
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dispersing during the 4 h incubation period. This finding suggests that PEG-chains grafted on GV membranes are necessary for prolonging the GV lifetime, which may be due to the steric effects of PEG-chains against the high ion-strength solution of the current cell-free system.22 When cholesterol was dissolved in the oil mixture, the number density of GVs did not change throughout the incubation period. When the concentration of cholesterol was greater than 4 mM, most GVs coagulated after centrifugation of the W/O emulsion layered on the buffered solution. Coagulation may have occurred due to cholesterol stabilizing the fused or semi-fused lipid bilayer membranes with its negative spontaneous curvature.24 As a result, we decided to include both PEGphospholipid and cholesterol (2 mM) in all oil mixtures. Images of GVs containing EGFP-BmOR1 and/or BmOrco mRNAs obtained by laser scanning confocal fluorescence microscopy showed that proteins of EGFP-BmOR1 and/or EGFP-BmOrco were transferred into GV membranes in approximately 50% of the GVs examined (Fig. 3a–d). In contrast, the localization of EGFP fluorescence on GV membranes was not observed in GVs encapsulating EGFP-BmOR1, which were synthesized in a plastic tube, even after 4 h of incubation (Fig. 3e). These data suggest that EGFP-BmOR1 is transferred into the vesicular membrane only when synthesized from mRNA inside GVs. In the absence of canine pancreatic microsomal membranes, both GVs expressing EGFP-BmOR1 and EGFP-BmOrco fluoresced after incubation for 4 h. However, the GVs expressing EGFP-BmOR1 exhibited only membrane fluorescence and those expressing EGFP-BmOrco did not show such an autonomous transfer of EGFP-BmOrco on GV membranes. These results may be due to microsomal membranes assisting in the maturation of EGFP-BmOrco. Lundin et al. reported that the fruit fly (Drosophila melanogaster) Orco synthesized by an E. coli cell-free system in the presence of rough Drosophila microsomes or rough canine pancreatic microsomes undergo N-linked glycosylation at the second extracellular loop.25 Because the same acceptor site for N-glycosylation is also present in BmOrco, we speculated that this site would be glycosylated in our system. Fig. 3b and d show the localization
Fig. 3 Laser scanning confocal fluorescence microscopic images of GVs synthesizing olfactory membrane receptors tagged with EGFP; (a) EGFP-BmOR1, (b) EGFP-BmOR1/BmOrco, (c) EGFP-BmOrco, and (d) EGFP-BmOrco/BmOR1. The white arrows show the localization of membrane receptors on the vesicular membranes. (e) A reference of a GV encapsulating EGFP-BmOR1 which was synthesized in a plastic tube.
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Fig. 4 (a) Current traces of BmOR1/BmOrco-expressing GVs before stimulation (left, no ligand) and during stimulation (right, 1 mM bombykol) recorded with a voltage clamp at 70 mV. The numbered traces show a time-expansion of 600 ms, corresponding to each numbered section in the top traces. (b) Current–voltage relationship of BmOR1/BmOrcoexpressing GVs before stimulation (green, no ligand) and during stimulation (magenta, 10 mM bombykol) over a voltage ramped from 80 to +40 mV. The traces are averages for each GV (n = 4). (c) Amplitude–frequency histograms of current before (top, dark green) and after (middle, magenta) addition of 1 mM bombykol shown in (a). Superimposed distributions are shown at the bottom.
of in situ synthesized proteins (indicated by white arrows) on GV membranes [EGFP-BmOR1/BmOrco, 38% (n = 31); BmOR1/ EGFP-BmOrco, 30% (n = 21)].
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Next, we examined the function of expressed BmOR1 and BmOrco in GV membranes by using the patch-clamp technique. At a holding potential of 70 mV, unitary-like inward currents were recorded while the silkmoth sex pheromone bombykol [(E,Z)-10,12-hexadecadien-1-ol]26 was simultaneously added to GVs at a concentration of 1 mM to demonstrate that GVs could be excited by an insect pheromone (Fig. 4a and b). In contrast, GVs containing no mRNAs of BmOR1 and BmOrco did not exhibit such characteristic current patterns under the same conditions (Fig. S2, ESI†). The corresponding all-point amplitude histogram in Fig. 4c shows that the amplitude of the current recorded in response to the addition of bombykol was within the range of the amplitude for single-channel conductance of insect ORs described previously.12 Moreover, the current–voltage relationship (Fig. 4b) was used to determine a reversal potential of 5.5 2.5 mV (mean SEM, n = 4), indicating that receptor complexes synthesized in situ in GVs react in a manner similar to those expressed in living cells.12,15 This successful functional reconstruction demonstrated that BmOR1 and BmOrco fold properly and form heteromeric complexes on GV membranes with the same odorant-gated cation channel functionality found in living cells. Although the precise mechanism of functionally expressing BmOR1 and BmOrco on artificial GV membranes remains to be fully elucidated, the current result is the first experimental proof of GVs simultaneously expressing a set of mRNA using a cell-free system. Because the entire process of each membrane receptor (i.e., translation from mRNA, transfer to membranes, folding and maturation, complex formation, and odorant-gated cation channel functionality) is stochastically variable inside GVs, we predicted that investigating functionalized GVs excited by bombykol would be difficult. However, we could approach them (less than 10% of all GVs formed) in this study because of the PEG-phospholipids anchored to GV membranes. The maturation and function of BmOR1 and/or BmOrco on GV membranes were probably assisted by the PEG chains anchoring to membrane surfaces as well as microsomal membranes and sucrose during cell-free protein synthesis. The insect OR family contains a large number of proteins with high odorant responses, selectivity, and sensitivity.27,28 These proteins can detect odorants without requiring cellular metabolism. Thus, the methodology developed in this study can potentially be applied to develop highly sensitive and selective odorant sensing artificial membranes that use insect ORs as sensor elements. The authors acknowledge financial support from KAKENHI [Grant-in Aid for Scientific Research, No. 21750073 (T.T.), 23658285 (R.K.), and 24658049 (T.S.) of the Ministry of Education, Culture, Sports, Science, and Technology of Japan/Japan Society for the Promotion of Science], the Kurata Memorial Hitachi Science and Technology Foundation (T.T.), and the SECOM Science and Technology Foundation (R.K.). The analysis of giant vesicles by confocal scanning laser fluorescence microscopy was performed with the help of the Association of Graduate Schools of Science and Technology, Chiba University.
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Notes and references 1 M. A. Cooper, J. Mol. Recognit., 2004, 17, 286; H.-H. Shen, T. Lithgow and L. Martin, Int. J. Mol. Sci., 2013, 14, 1589; R. Robelek, E. S. Lemker, B. Wiltschi, V. Kirste, R. Naumann, D. Oesterhelt and E. K. Sinner, Angew. Chem., Int. Ed., 2007, 46, 605. 2 P. Stano and P. L. Luisi, Chem. Commun., 2010, 46, 3639; The Minimal Cell, ed. P. L. Luisi and P. Stano, Springer, 2011. 3 V. Noireaux, Y. T. Maeda and A. Libchaber, Proc. Natl. Acad. Sci. U. S. A., 2011, 108, 3473. 4 S. M. Nomura, S. Kondo, W. Asayama, A. Asada, S. Nishikawa and K. Akiyoshi, J. Biotechnol., 2008, 133, 190. 5 M. Kaneda, S. M. Nomura, S. Ichinose, S. Kondo, K. Nakahama, K. Akiyoshi and I. Morita, Biomaterials, 2009, 30, 3971. 6 Y. Kuruma, P. Stano, T. Ueda and P. L. Luisi, Biochim. Biophys. Acta, Biomembr., 2009, 1788, 567. 7 Y. J. Liu, G. P. Hansen, A. Venancio-Marques and D. Baigl, ChemBioChem, 2013, 14, 2243. 8 Y. Moritani, S. M. Nomura, I. Morita and K. Akiyoshi, FEBS J., 2010, 277, 3343. 9 A. R. Long, C. C. O’Brien and N. N. Alder, PLoS One, 2012, 7, e46332. 10 S. Pautot, B. J. Frisken, J. X. Cheng, X. S. Xie and D. A. Weitz, Langmuir, 2003, 19, 10281. 11 T. Toyota, N. Ohguri, K. Maruyama, M. Fujinami, T. Saga and I. Aoki, Anal. Chem., 2012, 84, 3952. 12 K. Sato, M. Pellegrino, T. Nakagawa, T. Nakagawa, L. B. Vosshall and K. Touhara, Nature, 2008, 452, 1002. ¨fer, R. Bauernfeind, M. C. Stensmyr, R. Heller, 13 D. Wicher, R. Scha S. H. Heinemann and B. S. Hansson, Nature, 2008, 452, 1007.
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ChemComm 14 R. Smart, A. Kiely, M. Beale, E. Vargas, C. Carraher, A. V. Kralicek, D. L. Christie, C. Chen, R. D. Newcomb and C. G. Warr, Insect Biochem. Mol. Biol., 2008, 38, 770. 15 T. Nakagawa, M. Pellegrino, K. Sato, L. B. Vosshall and K. Touhara, PLoS One, 2012, 7, e32372. 16 K. Touhara and L. B. Vosshall, Annu. Rev. Physiol., 2009, 71, 307. 17 R. Glatz and K. Bailey-Hill, Prog. Neurobiol., 2011, 93, 270. 18 T. Sakurai, T. Nakagawa, H. Mitsuno, H. Mori, Y. Endo, S. Tanoue, Y. Yasukochi, K. Touhara and T. Nishioka, Proc. Natl. Acad. Sci. U. S. A., 2004, 101, 16653. 19 T. Nakagawa, T. Sakurai, T. Nishioka and K. Touhara, Science, 2005, 307, 1638. 20 T. Ezure, T. Suzuki, S. Higashide, E. Shintani, K. Endo, S. Kobayashi, M. Shikata, M. Ito, K. Tanimizu and O. Nishimura, Biotechnol. Prog., 2006, 22, 1570. 21 C. Carraher, A. R. Nazmi, R. D. Newcomb and A. Kralicek, Protein Expression Purif., 2013, 90, 160. 22 S. M. Moghimi, A. C. Hunter and J. C. Murray, Pharmacol. Rev., 2001, 53, 283. 23 Promega Corp., Canine Pancreatic Microsomal Membranes: Technical. Manual, Madison, WI, 2000. 24 M. A. Churchward, T. Rogasevskaia, D. M. Brandman, H. Khosravani, P. Nava, J. K. Atkinson and J. R. Coorssen, Biophys. J., 2008, 94, 3976. ¨ll, S. A. Kreher, K. Kapp, E. L. Sonnhammer, 25 C. Lundin, L. Ka J. R. Carlson, G. von Hijne and I. Nilsson, FEBS Lett., 2007, 581, 5601. 26 A. Butenandt, R. Beckmann, D. Stamm and E. Hecker, Z. Naturforsch., 1959, 14b, 283. 27 E. Hallem and J. R. Carlson, Cell, 2006, 125, 143. 28 A. F. Carey, G. Wang, C.-Y. Su, L. J. Zwiebel and J. R. Carlson, Nature, 2010, 464, 66.
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Cite this: Chem. Commun., 2014, 50, 2958 Received 27th October 2013, Accepted 14th January 2014
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Giant vesicles functionally expressing membrane receptors for an insect pheromone† Satoshi Hamada,‡a Masashi Tabuchi,‡b Taro Toyota,acd Takeshi Sakurai,e Tomohiro Hosoi,a Tomonori Nomoto,a Kei Nakatani,*f Masanori Fujinami*a and Ryohei Kanzaki*e
DOI: 10.1039/c3cc48216b www.rsc.org/chemcomm
To date, biochemical approaches to membrane receptors have been limited to the following methods: knockout or overexpression of membrane receptors by gene introduction and genome engineering or extraction of membrane receptor–surfactant complexes from innate cells and their introduction into model biomembranes. Here, we describe the development of a third method involving gene expression using cell-free in situ protein synthesis inside model biomembrane capsules. We verified this method by synthesizing olfactory receptors from the silkmoth Bombyx mori inside giant vesicles and found that they were excited in the presence of their ligand the Bombyx mori sex pheromone.
Membrane receptors are the outermost proteins of the cell that can detect the extracellular environment. The biochemical and molecular biological methods used to elucidate the mechanisms by which membrane receptors function primarily comprise the following two techniques: knockout or overexpression of membrane receptors by gene introduction and genome engineering or the extraction of membrane receptor–surfactant complexes from innate cells and their introduction into model biomembranes.1 However, in the former method, the potential
interference from the innate membrane proteins of host cells can be problematic. Furthermore, because the orientation and folding of receptors in model biomembranes are complex, the latter method is generally difficult to control. Here, we tackled these problems by establishing a system for the cell-free in situ synthesis of membrane receptors inside cell-sized lipid bilayer capsules called giant vesicles (GVs). The technique for cell-free protein synthesis inside of GVs was recently developed for the synthesis of ‘‘wet’’ artificial cells.2 Some of the membrane proteins were functionally expressed in the presence of lipid vesicles.3–9 However, to the best of our knowledge, there are no reports on the in situ synthesis of membrane receptors inside of GVs due to the difficultly of encapsulating all of the required reagents and organelles. Recently, a technique has been developed by the Libchaber3 and Weitz10 groups as well as our group11 to prepare GVs from water-in-oil emulsions, which play a key role in overcoming the difficulty of encapsulation. To verify that this technique could be used in the in situ synthesis of membrane receptors, we focused on olfactory receptors (ORs) and their co-receptors (Orco) in insects that form heteromeric complexes of ligand-gated ion channels12–16 because they comprise a huge protein family and have genomic variations.17 Among them, a sex
a
Department of Applied Chemistry and Biotechnology, Graduate School of Engineering, Chiba University, 1-33 Yayoi, Inage, Chiba, Chiba 263-8522, Japan. E-mail: [email protected]; Fax: +81-43-290-3503; Tel: +81-43-290-3503 b Department of Advanced Interdisciplinary Studies, Graduate School of Engineering, The University of Tokyo, 4-6-1 Komaba, Meguro, Tokyo 153-8904, Japan c Department of Basic Science, Graduate School of Arts and Sciences, The University of Tokyo, 3-8-1 Komaba, Meguro, Tokyo 153-8902, Japan d Research Center of Complex Systems Biology, Graduate School of Arts and Sciences, The University of Tokyo, 3-8-1 Komaba, Meguro, Tokyo 153-8902, Japan e Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Komaba, Meguro, Tokyo 153-8904, Japan. E-mail: [email protected]; Fax: +81-3-3469-2397; Tel: +81-3-5452-5195 f Faculty of Life and Environmental Sciences, University of Tsukuba, 1-1-1 Ten-noudai, Tsukuba, Ibaraki 305-8572, Japan. E-mail: [email protected]; Fax: +81-29-853-6614; Tel: +81-29-853-6672 † Electronic supplementary information (ESI) available: Fig. S1 and full experimental details. See DOI: 10.1039/c3cc48216b ‡ These authors contributed equally to this work.
2958 | Chem. Commun., 2014, 50, 2958--2961
Fig. 1 Schematic of GV encapsulating cell-free protein synthesizing reagents with canine pancreatic microsomal membranes and mRNA of BmOR1 and BmOrco, which form an olfactory receptor complex that could be excited by bombykol.
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Fig. 2 Western blot of synthesized EGFP-BmOR1 and EGFP-BmOrco. Asterisks indicate signals corresponding to the approximate molecular weight of synthesized proteins. The additional bands of low molecular weight are likely to be truncated synthesis products. Similar observations were previously reported in the cell-free synthesis of Drosophila melanogaster ORs.21
pheromone receptor BmOR1 and its co-receptor BmOrco from the silkmoth Bombyx mori18,19 were examined because their odorant– receptor interactions are highly specific. Thus, the purpose of this study was to construct GVs exhibiting OR functionalization (Fig. 1). Before preparing GVs, we synthesized N-terminally EGFP-tagged BmOR1 (EGFP-BmOR1) and EGFP-tagged BmOrco (EGFP-BmOrco) separately in a test tube by using insect cell-free protein synthesis reagents (see ESI†).20 Western blot analysis with anti-GFP revealed bands corresponding to the predicted molecular weight of each protein, confirming that full-length proteins were synthesized using this cell-free system (Fig. 2). GVs were subsequently produced using egg yolk lecithin, cholesterol, and polyethyleneglycol (PEG)-tagged phospholipids. We then encapsulated cell-free protein synthesis reagents of mRNA, cell-extract from insect cells, microsomal membranes from canine pancreatic cells, and sucrose in Tris-HCl buffer. To stabilize GV membranes, PEG-phospholipids and cholesterol were used as additives for lecithin GVs.22 Sucrose and canine pancreatic microsomal membranes were added to promote cellfree synthesis.23 Briefly, the cell-free synthesis reagent solution was emulsified in an oil mixture (liquid paraffin and squalene),10 which dissolved the lecithin, cholesterol, and PEG-phospholipids. The water-in-oil emulsion was subsequently layered onto a buffered solution containing glucose as an osmotic balancer. Centrifugation at 0 1C led to the formation of GVs via membrane bonding of the emulsion droplet monolayer and the water–emulsion interface monolayer. GV dispersions were obtained after incubation at 20–23 1C for 4 h. Using a differential interference contrast/fluorescence microscope equipped with a low magnitude objective lens, we observed that most GVs had a single-wall structure. Furthermore, the number density and average diameter of the GVs, which were stained by Texas Reds phospholipids, were 105–106 mL 1 and 11 5 mm (mean standard deviation) after the incubation, respectively (see Fig. S1 in ESI†). Although GVs were formed in the absence of PEG-phospholipids, almost all collapsed while
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ChemComm
dispersing during the 4 h incubation period. This finding suggests that PEG-chains grafted on GV membranes are necessary for prolonging the GV lifetime, which may be due to the steric effects of PEG-chains against the high ion-strength solution of the current cell-free system.22 When cholesterol was dissolved in the oil mixture, the number density of GVs did not change throughout the incubation period. When the concentration of cholesterol was greater than 4 mM, most GVs coagulated after centrifugation of the W/O emulsion layered on the buffered solution. Coagulation may have occurred due to cholesterol stabilizing the fused or semi-fused lipid bilayer membranes with its negative spontaneous curvature.24 As a result, we decided to include both PEGphospholipid and cholesterol (2 mM) in all oil mixtures. Images of GVs containing EGFP-BmOR1 and/or BmOrco mRNAs obtained by laser scanning confocal fluorescence microscopy showed that proteins of EGFP-BmOR1 and/or EGFP-BmOrco were transferred into GV membranes in approximately 50% of the GVs examined (Fig. 3a–d). In contrast, the localization of EGFP fluorescence on GV membranes was not observed in GVs encapsulating EGFP-BmOR1, which were synthesized in a plastic tube, even after 4 h of incubation (Fig. 3e). These data suggest that EGFP-BmOR1 is transferred into the vesicular membrane only when synthesized from mRNA inside GVs. In the absence of canine pancreatic microsomal membranes, both GVs expressing EGFP-BmOR1 and EGFP-BmOrco fluoresced after incubation for 4 h. However, the GVs expressing EGFP-BmOR1 exhibited only membrane fluorescence and those expressing EGFP-BmOrco did not show such an autonomous transfer of EGFP-BmOrco on GV membranes. These results may be due to microsomal membranes assisting in the maturation of EGFP-BmOrco. Lundin et al. reported that the fruit fly (Drosophila melanogaster) Orco synthesized by an E. coli cell-free system in the presence of rough Drosophila microsomes or rough canine pancreatic microsomes undergo N-linked glycosylation at the second extracellular loop.25 Because the same acceptor site for N-glycosylation is also present in BmOrco, we speculated that this site would be glycosylated in our system. Fig. 3b and d show the localization
Fig. 3 Laser scanning confocal fluorescence microscopic images of GVs synthesizing olfactory membrane receptors tagged with EGFP; (a) EGFP-BmOR1, (b) EGFP-BmOR1/BmOrco, (c) EGFP-BmOrco, and (d) EGFP-BmOrco/BmOR1. The white arrows show the localization of membrane receptors on the vesicular membranes. (e) A reference of a GV encapsulating EGFP-BmOR1 which was synthesized in a plastic tube.
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Fig. 4 (a) Current traces of BmOR1/BmOrco-expressing GVs before stimulation (left, no ligand) and during stimulation (right, 1 mM bombykol) recorded with a voltage clamp at 70 mV. The numbered traces show a time-expansion of 600 ms, corresponding to each numbered section in the top traces. (b) Current–voltage relationship of BmOR1/BmOrcoexpressing GVs before stimulation (green, no ligand) and during stimulation (magenta, 10 mM bombykol) over a voltage ramped from 80 to +40 mV. The traces are averages for each GV (n = 4). (c) Amplitude–frequency histograms of current before (top, dark green) and after (middle, magenta) addition of 1 mM bombykol shown in (a). Superimposed distributions are shown at the bottom.
of in situ synthesized proteins (indicated by white arrows) on GV membranes [EGFP-BmOR1/BmOrco, 38% (n = 31); BmOR1/ EGFP-BmOrco, 30% (n = 21)].
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Next, we examined the function of expressed BmOR1 and BmOrco in GV membranes by using the patch-clamp technique. At a holding potential of 70 mV, unitary-like inward currents were recorded while the silkmoth sex pheromone bombykol [(E,Z)-10,12-hexadecadien-1-ol]26 was simultaneously added to GVs at a concentration of 1 mM to demonstrate that GVs could be excited by an insect pheromone (Fig. 4a and b). In contrast, GVs containing no mRNAs of BmOR1 and BmOrco did not exhibit such characteristic current patterns under the same conditions (Fig. S2, ESI†). The corresponding all-point amplitude histogram in Fig. 4c shows that the amplitude of the current recorded in response to the addition of bombykol was within the range of the amplitude for single-channel conductance of insect ORs described previously.12 Moreover, the current–voltage relationship (Fig. 4b) was used to determine a reversal potential of 5.5 2.5 mV (mean SEM, n = 4), indicating that receptor complexes synthesized in situ in GVs react in a manner similar to those expressed in living cells.12,15 This successful functional reconstruction demonstrated that BmOR1 and BmOrco fold properly and form heteromeric complexes on GV membranes with the same odorant-gated cation channel functionality found in living cells. Although the precise mechanism of functionally expressing BmOR1 and BmOrco on artificial GV membranes remains to be fully elucidated, the current result is the first experimental proof of GVs simultaneously expressing a set of mRNA using a cell-free system. Because the entire process of each membrane receptor (i.e., translation from mRNA, transfer to membranes, folding and maturation, complex formation, and odorant-gated cation channel functionality) is stochastically variable inside GVs, we predicted that investigating functionalized GVs excited by bombykol would be difficult. However, we could approach them (less than 10% of all GVs formed) in this study because of the PEG-phospholipids anchored to GV membranes. The maturation and function of BmOR1 and/or BmOrco on GV membranes were probably assisted by the PEG chains anchoring to membrane surfaces as well as microsomal membranes and sucrose during cell-free protein synthesis. The insect OR family contains a large number of proteins with high odorant responses, selectivity, and sensitivity.27,28 These proteins can detect odorants without requiring cellular metabolism. Thus, the methodology developed in this study can potentially be applied to develop highly sensitive and selective odorant sensing artificial membranes that use insect ORs as sensor elements. The authors acknowledge financial support from KAKENHI [Grant-in Aid for Scientific Research, No. 21750073 (T.T.), 23658285 (R.K.), and 24658049 (T.S.) of the Ministry of Education, Culture, Sports, Science, and Technology of Japan/Japan Society for the Promotion of Science], the Kurata Memorial Hitachi Science and Technology Foundation (T.T.), and the SECOM Science and Technology Foundation (R.K.). The analysis of giant vesicles by confocal scanning laser fluorescence microscopy was performed with the help of the Association of Graduate Schools of Science and Technology, Chiba University.
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Notes and references 1 M. A. Cooper, J. Mol. Recognit., 2004, 17, 286; H.-H. Shen, T. Lithgow and L. Martin, Int. J. Mol. Sci., 2013, 14, 1589; R. Robelek, E. S. Lemker, B. Wiltschi, V. Kirste, R. Naumann, D. Oesterhelt and E. K. Sinner, Angew. Chem., Int. Ed., 2007, 46, 605. 2 P. Stano and P. L. Luisi, Chem. Commun., 2010, 46, 3639; The Minimal Cell, ed. P. L. Luisi and P. Stano, Springer, 2011. 3 V. Noireaux, Y. T. Maeda and A. Libchaber, Proc. Natl. Acad. Sci. U. S. A., 2011, 108, 3473. 4 S. M. Nomura, S. Kondo, W. Asayama, A. Asada, S. Nishikawa and K. Akiyoshi, J. Biotechnol., 2008, 133, 190. 5 M. Kaneda, S. M. Nomura, S. Ichinose, S. Kondo, K. Nakahama, K. Akiyoshi and I. Morita, Biomaterials, 2009, 30, 3971. 6 Y. Kuruma, P. Stano, T. Ueda and P. L. Luisi, Biochim. Biophys. Acta, Biomembr., 2009, 1788, 567. 7 Y. J. Liu, G. P. Hansen, A. Venancio-Marques and D. Baigl, ChemBioChem, 2013, 14, 2243. 8 Y. Moritani, S. M. Nomura, I. Morita and K. Akiyoshi, FEBS J., 2010, 277, 3343. 9 A. R. Long, C. C. O’Brien and N. N. Alder, PLoS One, 2012, 7, e46332. 10 S. Pautot, B. J. Frisken, J. X. Cheng, X. S. Xie and D. A. Weitz, Langmuir, 2003, 19, 10281. 11 T. Toyota, N. Ohguri, K. Maruyama, M. Fujinami, T. Saga and I. Aoki, Anal. Chem., 2012, 84, 3952. 12 K. Sato, M. Pellegrino, T. Nakagawa, T. Nakagawa, L. B. Vosshall and K. Touhara, Nature, 2008, 452, 1002. ¨fer, R. Bauernfeind, M. C. Stensmyr, R. Heller, 13 D. Wicher, R. Scha S. H. Heinemann and B. S. Hansson, Nature, 2008, 452, 1007.
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ChemComm 14 R. Smart, A. Kiely, M. Beale, E. Vargas, C. Carraher, A. V. Kralicek, D. L. Christie, C. Chen, R. D. Newcomb and C. G. Warr, Insect Biochem. Mol. Biol., 2008, 38, 770. 15 T. Nakagawa, M. Pellegrino, K. Sato, L. B. Vosshall and K. Touhara, PLoS One, 2012, 7, e32372. 16 K. Touhara and L. B. Vosshall, Annu. Rev. Physiol., 2009, 71, 307. 17 R. Glatz and K. Bailey-Hill, Prog. Neurobiol., 2011, 93, 270. 18 T. Sakurai, T. Nakagawa, H. Mitsuno, H. Mori, Y. Endo, S. Tanoue, Y. Yasukochi, K. Touhara and T. Nishioka, Proc. Natl. Acad. Sci. U. S. A., 2004, 101, 16653. 19 T. Nakagawa, T. Sakurai, T. Nishioka and K. Touhara, Science, 2005, 307, 1638. 20 T. Ezure, T. Suzuki, S. Higashide, E. Shintani, K. Endo, S. Kobayashi, M. Shikata, M. Ito, K. Tanimizu and O. Nishimura, Biotechnol. Prog., 2006, 22, 1570. 21 C. Carraher, A. R. Nazmi, R. D. Newcomb and A. Kralicek, Protein Expression Purif., 2013, 90, 160. 22 S. M. Moghimi, A. C. Hunter and J. C. Murray, Pharmacol. Rev., 2001, 53, 283. 23 Promega Corp., Canine Pancreatic Microsomal Membranes: Technical. Manual, Madison, WI, 2000. 24 M. A. Churchward, T. Rogasevskaia, D. M. Brandman, H. Khosravani, P. Nava, J. K. Atkinson and J. R. Coorssen, Biophys. J., 2008, 94, 3976. ¨ll, S. A. Kreher, K. Kapp, E. L. Sonnhammer, 25 C. Lundin, L. Ka J. R. Carlson, G. von Hijne and I. Nilsson, FEBS Lett., 2007, 581, 5601. 26 A. Butenandt, R. Beckmann, D. Stamm and E. Hecker, Z. Naturforsch., 1959, 14b, 283. 27 E. Hallem and J. R. Carlson, Cell, 2006, 125, 143. 28 A. F. Carey, G. Wang, C.-Y. Su, L. J. Zwiebel and J. R. Carlson, Nature, 2010, 464, 66.
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