Construction of an In Vitro Model of a Living Cellular System

  • K. YoshikawaEmail author
  • S. M. Nomura
  • K. Tsumoto
  • K. Takiguchi


The use of materials that are fully compatible with living cells may be the most reasonable plan for producing artificial models of cells. In this contribution, we present the results of some recent studies toward the construction of artificial cells, and explain methods for emulating several important specific functions of real cells in artificial liposomes. We discuss several aspects of the current experimental approaches, from the optimal construction of giant vesicles (GVs) to the realization of complex vesicle-based molecular system. The entrapment of DNA and enzymes inside GVs are discussed, as well as the synthesis of water soluble proteins inside vesicles. Emphasis is given on the new approaches to synthesize membrane-soluble proteins on the vesicle membrane. The case of connexin 43 (Cx43)-containing liposomes is described in details, as well as the extension of the proteoliposome technology to giant lipsomes, suitable for direct microscopy imaging. Application of the current in vitro synthetic approaches to the study of cell morphology is also discussed, by referring to the partial reconstitution of cytoskeleton inside GVs. In our recent study, G-actin and BBMI were simultaneously entrapped inside liposomes, through the method of natural swelling, resulting in efficient GV transformation. We conclude with some general consideration about the future work on cell models using vesicles.


Phosphatidic Acid Lipid Bilayer Membrane Liposome Membrane Actin Bundle Giant Vesicle 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


  1. Akashi K, Miyata H, Itoh H, Kinosita K Jr (1996) Preparation of giant liposomes in physiological conditions and their characterization under an optical microscope. Biophys J 71:3242–3250CrossRefPubMedGoogle Scholar
  2. Akashi K, Miyata H, Itoh H, Kinosita K Jr (1998) Formation of giant liposomes promoted by divalent cations: critical role of electrostatic repulsion. Biophys J 74:2973–2982CrossRefPubMedGoogle Scholar
  3. Albrecht-Buehler G, Lancaster RM (1976) A quantitative description of the extension and retraction of surface protrusions in spreading 3T3 mouse fibroblasts. J Cell Biol 71:370–382CrossRefPubMedGoogle Scholar
  4. Angelova MI (2000) Liposome electroformation. In: Luisi PL, Walde P (eds) Giant vesicles. Wiley, New YorkGoogle Scholar
  5. Bangham AD (1995) Surrogate cells or Trojan horses. The discovery of liposomes. Bio Essays 17:1081–1088Google Scholar
  6. Bormann M, Kos J, Kurzmeier H et al (1992) In: Lipowsky R, Richter D, Kremer K (eds) The structure and conformation of amphiphilic membranes. Springer, HeidelbergGoogle Scholar
  7. Byers HR, Fujikawa K (1982) Stress fibers in cells in situ: immunofluorescence visualization with antiactin, antimyosin, and anti-a-actinin. J Cell Biol 93:804–811CrossRefPubMedGoogle Scholar
  8. Coluccio LM (1997) Myosin I. Am J Physiol 273:C347–C359PubMedGoogle Scholar
  9. Cortese JD, Schwab B III, Frieden C (1989) Actin polymerization induces a shape change in actin-containing vesicles. Proc Natl Acad Sci USA 86:5773–5777CrossRefPubMedGoogle Scholar
  10. Endo Y, Sawasaki T (2003) High-throughput, genome-scale protein production method based on the wheat germ cell-free expression system. Biotechnol Adv 21(8):695–713CrossRefPubMedGoogle Scholar
  11. Fischer A, Luisi PL, Oberholzer T, Walde P (2000) Formation of giant vesicles from different kinds of lipids using the electroformation method. In: Luisi PL, Walde P (eds) Giant vesicles. Wiley, New YorkGoogle Scholar
  12. Fukushima H, Mizutani M, Imamura K, Morino K, Kobayashi J, Okumura K, Tsumoto K, Yoshimura T (2008) Development of a novel preparation method of recombinant proteoliposomes using baculovirus gene expression systems. J Biochem 144:763–770CrossRefPubMedGoogle Scholar
  13. Fukushima H, Matsuo H, Imamura K, Morino K, Okumura K, Tsumoto K, Yoshimura T (2009) Diagnosis and discrimination of autoimmune Graves’ disease and Hashimoto’s disease using thyroid-stimulating hormone receptor-containing recombinant proteoliposomes. J Biosci Bioeng 108:551–556CrossRefPubMedGoogle Scholar
  14. Fygenson DK, Marko JF, Libchaber A (1997) Mechanics of microtubule-based membrane extension. Phys Rev Lett 79:4497–4500CrossRefGoogle Scholar
  15. Gibrat G, Pastoriza-Gallego M, Thiebot B et al (2008) Polyelectrolyte entry and transport through an asymmetric a-hemolysin channel. J Phys Chem B 112:14687–14691CrossRefPubMedGoogle Scholar
  16. Girard P, Pécréaux J, Lenoir G, Falson P, Rigaud JL, Bassereau P (2004) A new method for the reconstitution of membrane proteins into giant unilamellar vesicles. Biophys J 87:419–429CrossRefPubMedGoogle Scholar
  17. Häckl W, Bärmann M, Sackmann E (1998) Shape changes of self-assembled actin bilayer composite membranes. Phys Rev Lett 80:1786–1789CrossRefGoogle Scholar
  18. Hayashi I, Urano Y, Fukuda R, Isoo N, Kodama T, Hamakubo T, Tomita T, Iwatsubo T (2004) Selective reconstitution and recovery of functional γ-secretase complex on budded baculovirus particles. J Biol Chem 279:38040–38046CrossRefPubMedGoogle Scholar
  19. Hishida M, Seto H, Yoshikawa K (2005) Smooth/rough layering in liquid-crystalline/gel state of dry phospholipid film, in relation to its ability to generate giant vesicles. Chem Phys Lett 411:267–272CrossRefGoogle Scholar
  20. Hamada T, Miura Y, Komatsu Y et al (2008) Construction of asymmetric cell-sized lipid vesicles from lipid-coated water-in-oil microdroplets. J Phys Chem B 112:14678–14681CrossRefPubMedGoogle Scholar
  21. Hase M, Yamada A, Hamada T et al (2007) Manipulation of cell-sized phospholipid-coated microdroplets and their use as biochemical microreactors. Langmuir 23:348–352CrossRefPubMedGoogle Scholar
  22. Hase M, Yoshikawa K (2006) Structural transition of actin filament in a cell-sized water droplet with a phospholipid membrane. J Chem Phys 124:Art. No. 104903Google Scholar
  23. Honda M, Takiguchi K, Ishikawa S et al (1999) Morphogenesis of liposomes encapsulating actin depends on the type of actin-crosslinking. J Mol Biol 287:293–300CrossRefPubMedGoogle Scholar
  24. Hotani H (1984) Transformation pathways of liposomes. J Mol Biol 178:113–120CrossRefPubMedGoogle Scholar
  25. Hotani H, Inaba T, Nomura F et al. (2003) Mechanical analyses of morphological and topological transformation of liposomes. Biosystems 71:93–100Google Scholar
  26. Janson LW, Kolega J, Taylor DL (1991) Modulation of contraction by gelation/solation in a reconstituted motile model. J Cell Biol 114:1005–1015CrossRefPubMedGoogle Scholar
  27. Kahya N, Pécheur EI, de Boeij WP, Wiersma DA, Hoekstra D (2001) Reconstitution of membrane proteins into giant unilamellar vesicles via peptide-induced fusion. Biophys J 81:1464–1474CrossRefPubMedGoogle Scholar
  28. Kalmbach R et al (2007) Functional cell-free synthesis of a seven helix membrane protein: in situ insertion of bacteriorhodopsin into liposomes. J Mol Biol 371:639–648CrossRefPubMedGoogle Scholar
  29. Kamps JAAM, Scherphof GL, Sullivan S, Gong Y, Hughes J (2003) In: Torchilin VP, Weissig V (eds) Liposomes, a practical approach, 2nd edn. Oxford University Press, New York, pp 267–301Google Scholar
  30. Kaneda M, Nomura SM, Ichinose S, Kondo S, Nakahama K, Akiyoshi K, Morita I (2009) Direct formation of proteo-liposomes by in vitro synthesis and cellular cytosolic delivery with connexin-expressing liposomes. Biomaterials 30:3971–3977CrossRefPubMedGoogle Scholar
  31. Katzen F, Peterson TC, KudlickiKatzen W (2009) Membrane protein expression: no cells required. Trends Biotechnol 27(8):455–460CrossRefPubMedGoogle Scholar
  32. Kost TA, Condreay JP, Jarvis DL (2005) Baculovirus as versatile vectors for protein expression in insect and mammalian cells. Nat Biotechnol 23:567–575CrossRefPubMedGoogle Scholar
  33. Kuruma Y, Stano P, Ueda T, Luisi PL (2009) A synthetic biology approach to the construction of membrane proteins in semi-synthetic minimal cells. Biochim Biophys Acta 1788:567–574CrossRefPubMedGoogle Scholar
  34. Lasic DD (1993) Liposomes: from Physics to applications. Elsevier, Amsterdam, pp 243–458Google Scholar
  35. Lasic DD (1995) Applications of liposomes. In: Lipowsky R, Sackmann E (eds) Structure and dynamics of membranes, vol 1a, pp 491–519. Elsevier, AmsterdamGoogle Scholar
  36. Limozin L, Bärmann M, Sackmann E (2003) On the organization of self-assembled actin networks in giant vesicles. Eur Phys J E 10:319–330CrossRefPubMedGoogle Scholar
  37. Limozin L, Roth A, Sackmann E (2005) Microviscoelastic moduli of biomimetic cell envelopes. Phys Rev Lett 95:Art. No. 178101Google Scholar
  38. Limozin L, Sackmann E (2002) Polymorphism of cross-linked actin networks in giant vesicles. Phys Rev Lett 89:Art. No. 168103Google Scholar
  39. Lipowsky R (1991) The conformation of membranes. Nature 349:475–481CrossRefPubMedGoogle Scholar
  40. Loisel TP, Ansanay H, St-Onge S, Gay B, Boulanger P, Strosberg AD, Marullo S, Bouvier M (1997) Recovery of homogeneous and functional β2-adrenergic receptors from extracellular baculovirus particles. Nat Biotechnol 15:1300–1304CrossRefPubMedGoogle Scholar
  41. Luisi PL (2000) Why gaiat vesicles? In: Luisi PL, Walde P (eds) Giant vesicles. Wiley, New YorkGoogle Scholar
  42. Maemichi H, Shikinaka K, Kakugo A et al (2008) Morphogenesis of liposomes caused by polycation-induced actin assembly formation. Langmuir 24:11975–11981CrossRefPubMedGoogle Scholar
  43. Magome N, Takemura T, Yoshikawa K (1997) Spontaneous formation of giant liposomes from neutral phospholipids. Chem Lett 26:205–206CrossRefGoogle Scholar
  44. Masuda K, Itoh H, Sakihama T, Akiyama C, Takahashi K, Fukuda R, Yokomizo T, Shimizu T, Kodama T, Hamakubo T (2003) A combinatorial G protein-coupled receptor reconstitution system on budded baculovirus. Evidence for Gαi and Gαo coupling to a human leukotriene B4 receptor. J Biol Chem 278:24552–24562CrossRefPubMedGoogle Scholar
  45. Mitchison TJ, Cramer LP (1996) Actin-based cell motility and cell locomotion. Cell 84:371–379CrossRefPubMedGoogle Scholar
  46. Mitchison TJ, Kirschner M (1988) Cytoskeletal dynamics and nerve growth. Neuron 1:761–772CrossRefPubMedGoogle Scholar
  47. Miyata H, Hotani H (1992) Morphological changes in liposomes caused by polymerization of encapsulated actin and spontaneous formation of actin bundles. Proc Natl Acad Sci USA 89:11547–11551CrossRefPubMedGoogle Scholar
  48. Miyata H, Nishiyama S, Akashi K et al (1999) Protrusive growth from giant liposomes driven by actin polymerization. Proc Natl Acad Sci USA 96:2048–2053CrossRefPubMedGoogle Scholar
  49. Nakano H, Kawarasaki Y, Yamane T (2004) Cell-free protein synthesis systems: increasing their performance and applications. Adv Biochem Eng Biotechnol 90:135–149PubMedGoogle Scholar
  50. Noireaux V, Libchaber A (2004) A vesicle bioreactor as a step toward an artificial cell assembly. Proc Natl Acad Sci USA 101:17669–17674CrossRefPubMedGoogle Scholar
  51. Nomura S, Yoshikawa K (2000) Giant phospholipid vesicles entrapping giant DNA. In: Luisi PL, Walde P (eds) Giant vesicles. Wiley, New YorkGoogle Scholar
  52. Nomura SM, Yoshikawa Y, Yoshikawa K, Dannenmuller O, Chasserot-Golez S, Ourisson G, Nakatani Y (2001) Towards proto-cells:”primitive” lipid vesicles encapsulating giant DNA and its histone complex. Chembiochem 2(6):457–459CrossRefPubMedGoogle Scholar
  53. Nomura SM, Kondoh S, Asayama W, Asada A, Nishikawa S, Akiyoshi K (2008) Direct preparation of giant proteo-liposomes by in vitro membrane protein synthesis. J Biotechnol 133(2):190–195CrossRefPubMedGoogle Scholar
  54. Nomura SM, Tsumoto K, Hamada T, Akiyoshi K, Nakatani Y, Yoshikawa K (2003) Gene expression within cell-sized lipid vesicles. Chembiochem 4(11):1172–1175CrossRefPubMedGoogle Scholar
  55. Pautot S, Frisken BJ, Weitz DA (2003a) Engineering asymmetric vesicles. Proc Natl Acad Sci USA 100:10718–10721CrossRefPubMedGoogle Scholar
  56. Pautot S, Frisken BJ, Weitz DA (2003b) Production of unilamellar vesicles using an inverted emulsion. Langmuir 19:2870–2879CrossRefGoogle Scholar
  57. Pollard TD, Blanchoin L, Mullins RD (2000) Molecular mechanisms controlling actin filament dynamics in nonmuscle cells. Annu Rev Biophys Biomol Struct 29:545–576CrossRefPubMedGoogle Scholar
  58. Pontani L-L, van der Gucht J, Salbreux G et al (2009) Reconstitution of an actin cortex inside a liposome. Biophys J 96:192–198CrossRefPubMedGoogle Scholar
  59. Rigaud JL, Lévy D (2003) Reconstitution of membrane proteins into liposomes. Methods Enzymol 372:65–86Google Scholar
  60. Robelek R, Lemker ES, Wiltschi B, Kirste V, Naumann R, Oesterhelt D, Sinner E-K (2007) Incorporation of in vitro synthesized gpcr into a tethered artificial lipid membrane system. Angew Chem Int Ed Eng 46:605–608CrossRefGoogle Scholar
  61. Rodriguez N, Pincet F, Cribier S (2005) Giant vesicles formed by gentle hydration and electroformation: a comparison by fluorescence microscopy. Colloids Surf B Biointerfaces 42:125–130CrossRefPubMedGoogle Scholar
  62. Rodriguez OC, Schaefer AW, Mandato CA et al (2003) Conserved microtubule-actin interactions in cell movement and morphogenesis. Nat Cell Biol 5:599–609CrossRefPubMedGoogle Scholar
  63. Saitoh A, Takiguchi K, Tanaka Y et al (1998) Opening-up of liposomal membranes by talin. Proc Natl Acad Sci USA 95:1026–1031CrossRefPubMedGoogle Scholar
  64. Sato Y, Nomura SM, Yoshikawa K (2003) Enhanced uptake of giant DNA in cell-sized liposomes. Chem Phys Lett 380:279–285CrossRefGoogle Scholar
  65. Schroeder TE (1973) Actin in dividing cells: contractile ring filaments bind heavy meromyosin. Proc Natl Acad Sci USA 70:1688–1692CrossRefPubMedGoogle Scholar
  66. Shimanouchi T, Umakoshi H, Kuboi R (2009) Kinetic study on giant vesicle formation with electroformation method. Langmuir 25:4835–4840CrossRefPubMedGoogle Scholar
  67. Shimizu Y, Kuruma Y, Ying BW, Umekage S, Ueda T (2006) Cell-free translation systems for protein engineering. FEBS J 273:4133–4140CrossRefPubMedGoogle Scholar
  68. Takeda S, Saitoh A, Furuta M et al (2006) Opening of holes in liposomal membranes is induced by proteins possessing the FERM domain. J Mol Biol 362:403–413CrossRefPubMedGoogle Scholar
  69. Takiguchi K (1991) Heavy meromyosin induces sliding movements between antiparallel actin filaments. J Biochem 109:520–527PubMedGoogle Scholar
  70. Takiguchi K, Yamada A, Negishi M et al (2008) Entrapping desired amounts of actin filaments and molecular motor proteins in giant liposomes. Langmuir 24:11323–11326CrossRefPubMedGoogle Scholar
  71. Tanaka-Takiguchi Y, Kakei T, Tanimura A et al (2004) The elongation and contraction of actin bundles are induced by double-headed myosins in a motor concentration-dependent manner. J Mol Biol 341:467–476CrossRefPubMedGoogle Scholar
  72. Taylor DL, Condeelis JS (1979) Cytoplasmic structure and contractility in amoeboid cells. Int Rev Cytol 56:57–144CrossRefPubMedGoogle Scholar
  73. Tsumoto K, Matsuo H, Tomita M, Yoshimura T (2009) Efficient formation of giant liposomes through the gentle hydration of phosphatidylcholine films doped with sugar. Colloids Surf B Biointerfaces 68:98–105CrossRefPubMedGoogle Scholar
  74. Tsumoto K, Nomura SM, Nakatani Y, Yoshikawa K (2001) Giant liposome as a biochemical reactor: transcription of DNA and transportation by laser tweezers. Langmuir 17(23):7225–7228CrossRefGoogle Scholar
  75. Tsumoto K, Yoshimura T (2009) Recombinant proteoliposomes prepared using baculovirus expression systems. Methods Enzymol 465:95–109CrossRefPubMedGoogle Scholar
  76. Vinarov DA, Loushin Newman CL, Markley JL (2006) Wheat germ cell-free platform for eukaryotic protein production. FEBS J 273(18):4160–4169CrossRefPubMedGoogle Scholar
  77. Yamada A, Yamanaka Y, Hamada T et al (2006) Spontaneous transfer of phospholipid-coated oil-in-oil and water-in-oil micro-droplets through an oil/water interface. Langmuir 22:9824–9828CrossRefPubMedGoogle Scholar
  78. Yamada NL, Hishida M, Seto H, Tsumoto K, Yoshimura T (2007) Unbinding of lipid bilayers induced by osmotic pressure in relation to unilamellar vesicle formation. EPL 80:48002CrossRefGoogle Scholar
  79. Yamaji K, Kanai T, Nomura SM, Akiyoshi K, Negishi M, Atomi H, Yoshikawa K, Imanaka T (2009) Protein synthesis in giant liposomes using the in vitro translation system of Thermococcus kodakaraensis. IEEE trans Nanobiosci 8:325–331Google Scholar
  80. Yamashita Y, Oka M, Tanaka T, Yamazaki M (2002) A new method for the preparation of giant liposomes in high salt concentrations and growth of protein microcrystals in them. Biochim Biophys Acta 1561:129–134CrossRefPubMedGoogle Scholar
  81. Yu W, Sato K, Wakabayashi M, Nakaishi T, Ko-Mitamura EP, Shima Y, Urabe I, Yomo T (2001) Synthesis of functional protein in liposome. J Biosci Bioeng 92(6):590–593CrossRefPubMedGoogle Scholar

Copyright information

© Springer Netherlands 2011

Authors and Affiliations

  • K. Yoshikawa
    • 1
    Email author
  • S. M. Nomura
    • 2
  • K. Tsumoto
    • 3
  • K. Takiguchi
    • 4
  1. 1.Graduate School of ScienceKyoto UniversityKyotoJapan
  2. 2.Institute for Integrated Cell-Material Sciences, JST PRESTOKyoto UniversityKyotoJapan
  3. 3.Graduate School of EngineeringMie UniversityTsu-shiJapan
  4. 4.Graduate School of ScienceNagoya UniversityNagoyaJapan

Personalised recommendations