Advertisement

Journal of Molecular Evolution

, Volume 79, Issue 5–6, pp 179–192 | Cite as

Spontaneous Encapsulation and Concentration of Biological Macromolecules in Liposomes: An Intriguing Phenomenon and Its Relevance in Origins of Life

  • Tereza Pereira de Souza
  • Alfred Fahr
  • Pier Luigi Luisi
  • Pasquale StanoEmail author
Review

Abstract

One of the main open questions in origin of life research focuses on the formation, by self-organization, of primitive cells composed by macromolecular compounds enclosed within a semi-permeable membrane. A successful experimental strategy for studying the emergence and the properties of primitive cells relies on a synthetic biology approach, consisting in the laboratory assembly of cell models of minimal complexity (semi-synthetic minimal cells). Despite the recent advancements in the construction and characterization of synthetic cells, an important physical aspect related to their formation is still not well known, namely, the mechanism of solute entrapment inside liposomes (in particular, the entrapment of macromolecules). In the past years, we have investigated this phenomenon and here we shortly review our experimental results. We show how the detailed cryo-transmission electron microscopy analyses of liposome populations created in the presence of ferritin (taken as model protein) or ribosomes have revealed that a small fraction of liposomes contains a high number of solutes, against statistical expectations. The local (intra-liposomal) macromolecule concentration in these liposomes largely exceeds the bulk concentration. A similar behaviour is observed when multi-molecular reaction mixtures are used, whereby the reactions occur effectively only inside those liposomes that have entrapped high number of molecules. If similar mechanisms operated in early times, these intriguing results support a scenario whereby the formation of lipid compartments plays an important role in concentrating the components of proto-metabolic systems—in addition to their well-known functions of confinement and protection.

Keywords

Concentration Crowding Encapsulation Ferritin Lipid vesicles (liposomes) Primitive cells 

Notes

Acknowledgments

This review summarizes the results obtained in the last years by the joint efforts of P. L. Luisi and A. Fahr groups, starting within the EU-FP6 SYNTHCELLS project (Approaches to the Bioengineering Synthetic Minimal Cells, Nr. 043359). We thank Fabio Mavelli (University of Bari, Italy) for discussion on stochastic simulations. T. Pereira de Souza was supported by the Alexander von Humboldt Foundation as a post-doctoral fellow in Jena. A more extensive version of this review can be found in Luisi (2012).

References

  1. Bangham AD, Standish MM, Watkins JC (1965) Diffusion of univalent ions across the lamellae of swollen phospholipids. J Mol Biol 13:238–252CrossRefPubMedGoogle Scholar
  2. Batzri S, Korn ED (1973) Single bilayer liposomes prepared without sonication. Biochim Biophys Acta 298:1015–1019. doi: 10.1016/0005-2736(73)90408-2 CrossRefPubMedGoogle Scholar
  3. Bell SH, Weir MP, Dickson DP et al (1984) Mössbauer spectroscopic studies of human haemosiderin and ferritin. Biochim Biophys Acta 787:227–236CrossRefPubMedGoogle Scholar
  4. Berclaz N, Blochliger E, Muller M, Luisi PL (2001a) Matrix effect of vesicle formation as investigated by cryotransmission electron microscopy. J Phys Chem B 105:1065–1071. doi: 10.1021/jp002151u CrossRefGoogle Scholar
  5. Berclaz N, Muller M, Walde P, Luisi PL (2001b) Growth and transformation of vesicles studied by ferritin labeling and cryotransmission electron microscopy. J Phys Chem B 105:1056–1064. doi: 10.1021/jp001298i CrossRefGoogle Scholar
  6. Blain JC, Szostak JW (2014) Progress toward synthetic cells. Annu Rev Biochem. doi: 10.1146/annurev-biochem-080411-124036 PubMedGoogle Scholar
  7. Boal et al., Steering Group for the Workshop on Size Limits of Very Small Microorganisms, National Research Council (1999) Size limits of very small microorganisms: proceedings of a workshop. The National Academies Press, Washington, D.CGoogle Scholar
  8. Calviello L, Stano P, Mavelli F et al (2013) Quasi-cellular systems: stochastic simulation analysis at nanoscale range. BMC Bioinformatics 14:S7. doi: 10.1186/1471-2105-14-S7-S7 PubMedCentralPubMedGoogle Scholar
  9. Caschera F, Sunami T, Matsuura T et al (2011) Programmed vesicle fusion triggers gene expression. Langmuir ACS J Surf Colloids 27:13082–13090. doi: 10.1021/la202648h CrossRefGoogle Scholar
  10. Dominak LM, Keating CD (2007) Polymer encapsulation within giant lipid vesicles. Langmuir 23:7148–7154. doi: 10.1021/la063687v CrossRefPubMedGoogle Scholar
  11. Dong H, Nilsson L, Kurland CG (1996) Co-variation of tRNA abundance and codon usage in Escherichia coli at different growth rates. J Mol Biol 260:649–663CrossRefPubMedGoogle Scholar
  12. Fischer A, Franco A, Oberholzer T (2002) Giant vesicles as microreactors for enzymatic mRNA synthesis. ChemBioChem 3:409–417. doi: 10.1002/1439-7633 CrossRefPubMedGoogle Scholar
  13. Hernández-Zapata E, Martínez-Balbuena L, Santamaría-Holek I (2009) Thermodynamics and dynamics of the formation of spherical lipid vesicles. J Biol Phys 35:297–308Google Scholar
  14. Hosoda K, Sunami T, Kazuta Y et al (2008) Quantitative study of the structure of multilamellar giant liposomes as a container of protein synthesis reaction. Langmuir ACS J Surf Colloids 24:13540–13548. doi: 10.1021/la802432f CrossRefGoogle Scholar
  15. Ichihashi N, Matsuura T, Kita H, Sunami T, Suzuki H, Yomo T (2012) Constructive approaches for the origin of life. In: Seckbach J (ed) Genesis-in the beginning: precursors of life, chemical models and early biological evolution. Cellular origin, life in extreme habitats and astrobiology (Springer) vol 22, pp 289–303. doi: 10.1007/978-94-007-2941-4_17
  16. Kato A, Yanagisawa M, Sato YT et al (2012) Cell-sized confinement in microspheres accelerates the reaction of gene expression. Sci Rep 2:283. doi: 10.1038/srep00283 PubMedCentralPubMedGoogle Scholar
  17. Kita H, Matsuura T, Sunami T et al (2008) Replication of genetic information with self-encoded replicase in liposomes. Chembiochem Eur J Chem Biol 9:2403–2410. doi: 10.1002/cbic.200800360 CrossRefGoogle Scholar
  18. Kurihara K, Tamura M, Shohda K, Toyota T, Suzuki K, Sugawara T (2011) Self-reproduction of supramolecular giant vesicles combined with the amplification of encapsulated DNA. Nat Chem 3:775–781. doi: 10.1038/nchem.1127 CrossRefPubMedGoogle Scholar
  19. Lasic DD (1988) The mechanism of vesicle formation. Biochem J 256:1–11PubMedCentralCrossRefPubMedGoogle Scholar
  20. Lazzerini-Ospri L, Stano P, Luisi P, Marangoni R (2012) Characterization of the emergent properties of a synthetic quasi-cellular system. BMC Bioinformatics 13(Suppl 4):S9. doi: 10.1186/1471-2105-13-S4-S9 PubMedCentralCrossRefPubMedGoogle Scholar
  21. Lohse B, Bolinger P-Y, Stamou D (2008) Encapsulation efficiency measured on single small unilamellar vesicles. J Am Chem Soc 130:14372–14373. doi: 10.1021/ja805030w CrossRefPubMedGoogle Scholar
  22. Luisi PL (2006) The emergence of life: from origins of life to synthetic biology. Cambridge University Press, CambridgeCrossRefGoogle Scholar
  23. Luisi PL (2007) Question 3: the problem of macromolecular sequences: the forgotten stumbling block. Orig Life Evol Biosph 37:363–365CrossRefPubMedGoogle Scholar
  24. Luisi PL (2012) An open question on the origin of life: the first forms of metabolism. Chem Biodivers 9:2635–2647. doi: 10.1002/cbdv.201200281 CrossRefPubMedGoogle Scholar
  25. Luisi PL, Walde P, Oberholzer T (1999) Lipid vesicles as possible intermediates in the origin of life. Curr Opin Colloid Interface Sci 4:33–39. doi: 10.1016/S1359-0294(99)00012-6 CrossRefGoogle Scholar
  26. Luisi PL, Ferri F, Stano P (2006) Approaches to semi-synthetic minimal cells: a review. Naturwissenschaften 93:1–13. doi: 10.1007/s00114-005-0056-z CrossRefPubMedGoogle Scholar
  27. Luisi PL, Allegretti M, Souza TP et al (2010) Spontaneous protein crowding in liposomes: a new vista for the origin of cellular metabolism. ChemBioChem 11:1989–1992. doi: 10.1002/cbic.201000381 CrossRefPubMedGoogle Scholar
  28. Maeda YT, Nakadai T, Shin J et al (2012) Assembly of MreB filaments on liposome membranes: a synthetic biology approach. ACS Synth Biol 1:53–59. doi: 10.1021/sb200003v CrossRefPubMedGoogle Scholar
  29. Mansy SS, Schrum JP, Krishnamurthy M et al (2008) Template-directed synthesis of a genetic polymer in a model protocell. Nature 454:U10–U122. doi: 10.1038/nature07018 CrossRefGoogle Scholar
  30. Martini L, Mansy SS (2011) Cell-like systems with riboswitch controlled gene expression. Chem Commun 47:10734–10736. doi: 10.1039/c1cc13930d CrossRefGoogle Scholar
  31. Mayer LD, Hope MJ, Cullis PR, Janoff AS (1985) Solute distributions and trapping efficiencies observed in freeze-thawed multilamellar vesicles. Biochim Biophys Acta 817:193–196CrossRefPubMedGoogle Scholar
  32. Monnard P-A, Deamer DW (2002) Membrane self-assembly processes: steps toward the first cellular life. Anat Rec 268:196–207. doi: 10.1002/ar.10154 CrossRefPubMedGoogle Scholar
  33. Moritani Y, Nomura SM, Morita I, Akiyoshi K (2010) Direct integration of cell-free-synthesized connexin-43 into liposomes and hemichannel formation. FEBS J 277:3343–3352. doi: 10.1111/j.1742-4658.2010.07736.x CrossRefPubMedGoogle Scholar
  34. Murtas G, Kuruma Y, Bianchini P et al (2007) Protein synthesis in liposomes with a minimal set of enzymes. Biochem Biophys Res Commun 363:12–17. doi: 10.1016/j.bbrc.2007.07.201 CrossRefPubMedGoogle Scholar
  35. Nishimura K, Matsuura T, Nishimura K et al (2012) Cell-free protein synthesis inside giant unilamellar vesicles analyzed by flow cytometry. Langmuir 28:8426–8432. doi: 10.1021/la3001703 CrossRefPubMedGoogle Scholar
  36. Noireaux V, Libchaber A (2004) A vesicle bioreactor as a step toward an artificial cell assembly. Proc Natl Acad Sci USA 101:17669–17674. doi: 10.1073/pnas.0408236101 PubMedCentralCrossRefPubMedGoogle Scholar
  37. Nomura S, Tsumoto K, Hamada T et al (2003) Gene expression within cell-sized lipid vesicles. ChemBioChem 4:1172–1175. doi: 10.1002/cbic.200300630 CrossRefPubMedGoogle Scholar
  38. Nourian Z, Danelon C (2013) Linking genotype and phenotype in protein synthesizing liposomes with external supply of resources. ACS Synth Biol 2:186–193. doi: 10.1021/sb300125z CrossRefPubMedGoogle Scholar
  39. Oberholzer T, Luisi PL (2002) The use of liposomes for constructing cell models. J Biol Phys 28:733–744. doi: 10.1023/A:1021267512805 PubMedCentralCrossRefPubMedGoogle Scholar
  40. Oberholzer T, Wick R, Luisi PL, Biebricher CK (1995) Enzymatic RNA replication in self-reproducing vesicles: an approach to a minimal cell. Biochem Biophys Res Commun 207:250–257. doi: 10.1006/bbrc.1995.1180 CrossRefPubMedGoogle Scholar
  41. Oberholzer T, Nierhaus KH, Luisi PL (1999) Protein expression in liposomes. Biochem Biophys Res Commun 261:238–241. doi: 10.1006/bbrc.1999.0404 CrossRefPubMedGoogle Scholar
  42. Pohorille A, Deamer D (2002) Artificial cells: prospects for biotechnology. Trends Biotechnol 20:123–128. doi: 10.1016/S0167-7799(02)01909-1 CrossRefPubMedGoogle Scholar
  43. Rasmussen S, Chen LH, Nilsson M, Abe S (2003) Bridging nonliving and living matter. Artif Life 9:269–316. doi: 10.1162/106454603322392479 CrossRefPubMedGoogle Scholar
  44. Rasmussen S, Bedau MA, Chen L, Deamer D, Krakauer DC, Packard NH, Stadler PF (eds) (2009) Protocells. bridging nonliving and living matter. MIT Press, Cambridge MAGoogle Scholar
  45. Saito H, Kato Y, Le Berre M et al (2009) Time-resolved tracking of a minimum gene expression system reconstituted in giant liposomes. ChemBioChem 10:1640–1643. doi: 10.1002/cbic.200900205 CrossRefPubMedGoogle Scholar
  46. Sakakura T, Nishimura K, Suzuki H, Yomo T (2012) Statistical analysis of discrete encapsulation of nanomaterials in colloidal capsules. Anal Methods 4:1648–1655. doi: 10.1039/C2AY25105A CrossRefGoogle Scholar
  47. Shimizu Y, Inoue A, Tomari Y et al (2001) Cell-free translation reconstituted with purified components. Nat Biotechnol 19:751–755. doi: 10.1038/90802 CrossRefPubMedGoogle Scholar
  48. Shimizu Y, Kanamori T, Ueda T (2005) Protein synthesis by pure translation systems. Methods 36:299–304. doi: 10.1016/j.ymeth.2005.04.006 CrossRefPubMedGoogle Scholar
  49. Souza TP, Stano P, Luisi PL (2009) The minimal size of liposome-based model cells brings about a remarkably enhanced entrapment and protein synthesis. ChemBioChem 10:1056–1063. doi: 10.1002/cbic.200800810 CrossRefGoogle Scholar
  50. Souza TP, Steiniger F, Stano P et al (2011) Spontaneous crowding of ribosomes and proteins inside vesicles: a possible mechanism for the origin of cell metabolism. ChemBioChem 12:2325–2330. doi: 10.1002/cbic.201100306 CrossRefGoogle Scholar
  51. Souza TP, Stano P, Steiniger F, D’Aguanno E, Altamura E, Fahr A, Luisi PL (2012) Encapsulation of ferritin, ribosomes, and ribo-peptidic complexes inside liposomes: insights into the origin of metabolism. Orig Life Evol Biosph 42:421–428. doi: 10.1007/s11084-012-9303-4 CrossRefPubMedGoogle Scholar
  52. Stano P, Kuruma Y, Souza TP, Luisi PL (2010) Biosynthesis of proteins inside liposomes. Methods Mol Biol 606:127–145. doi: 10.1007/978-1-60761-447-0_11
  53. Stano P, Carrara P, Kuruma Y et al (2011) Compartmentalized reactions as a case of soft-matter biotechnology: synthesis of proteins and nucleic acids inside lipid vesicles. J Mater Chem 21:18887–18902. doi: 10.1039/c1jm12298c CrossRefGoogle Scholar
  54. Stano P, Rampioni G, Carrara P et al (2012) Semi-synthetic minimal cells as a tool for biochemical ICT. Biosystems 109:24–34. doi: 10.1016/j.biosystems.2012.01.002 CrossRefPubMedGoogle Scholar
  55. Stano P, D’Aguanno E, Bolz J et al (2013) A remarkable self-organization process as the origin of primitive functional cells. Angew Chem Int Ed Engl 52:13397–13400. doi: 10.1002/anie.201306613 CrossRefPubMedGoogle Scholar
  56. Stano P, Souza TP, Carrara P, Altamura E, D’Aguanno E, Caputo M, Luisi PL, Mavelli F (2014) Recent biophysical issues about the preparation of solute-filled lipid vesicles. Mech Adv Mater Struct. doi: 10.1080/15376494.2013.857743 Google Scholar
  57. Sun BY, Chiu DT (2005) Determination of the encapsulation efficiency of individual vesicles using single-vesicle photolysis and confocal single-molecule detection. Anal Chem 77:2770–2776. doi: 10.1021/ac048439n CrossRefPubMedGoogle Scholar
  58. Sunami T, Sato K, Matsuura T et al (2006) Femtoliter compartment in liposomes for in vitro selection of proteins. Anal Biochem 357:128–136. doi: 10.1016/j.ab.2006.06.040 CrossRefPubMedGoogle Scholar
  59. Szostak JW, Bartel DP, Luisi PL (2001) Synthesizing life. Nature 409:387–390. doi: 10.1038/35053176 CrossRefPubMedGoogle Scholar
  60. Tanford Charles (1973) The hydrophobic effect: formation of micelles and biological membranes. Wiley, New York, NYGoogle Scholar
  61. Tokuriki N, Kinjo M, Negi S et al (2004) Protein folding by the effects of macromolecular crowding. Protein Sci 13:125–133. doi: 10.1110/ps.03288104 PubMedCentralCrossRefPubMedGoogle Scholar
  62. Torino D, Martini L, Mansy SS (2013) Piecing together cell-like systems. Curr Org Chem 17:1751–1757. doi: 10.2174/13852728113179990082 PubMedCentralCrossRefPubMedGoogle Scholar
  63. Van Hoof B, Markvoort AJ, van Santen RA, Hilbers PAJ (2012) On protein crowding and bilayer bulging in spontaneous vesicle formation. J Phys Chem B 116:12677–12683. doi: 10.1021/jp3062306 CrossRefPubMedGoogle Scholar
  64. Van Hoof B, Markvoort AJ, van Santen RA, Hilbers PAJ (2014) Molecular simulation of protein encapsulation in vesicle formation. J Phys Chem B 118:3346–3354. doi: 10.1021/jp410612k CrossRefPubMedGoogle Scholar
  65. Van Nies P, Nourian Z, Kok M et al (2013) Unbiased tracking of the progression of mRNA and protein synthesis in bulk and in liposome-confined reactions. ChemBioChem 14:1963–1966. doi: 10.1002/cbic.201300449 CrossRefPubMedGoogle Scholar
  66. Walde P (2006) Surfactant assemblies and their various possible roles for the origin(s) of life. Orig Life Evol Biosph 36:109–150. doi: 10.1007/s11084-005-9004-3 CrossRefPubMedGoogle Scholar
  67. Walde P (2010) Building artificial cells and protocell models: experimental approaches with lipid vesicles. BioEssays 32:296–303. doi: 10.1002/bies.200900141 CrossRefPubMedGoogle Scholar
  68. Yamaji K, Kanai T, Nomura SM, Akiyoshi K, Negishi M, Chen Y, Atomi H, Yoshikawa K, Imanaka T (2009) Protein synthesis in giant liposomes using the in vitro translation system of Thermococcus kodakaraensis. IEEE Trans Nanobiosci 8:325–331CrossRefGoogle Scholar
  69. Yu W, Sato K, Wakabayashi M et al (2001) Synthesis of functional protein in liposome. J Biosci Bioeng 92:590–593CrossRefPubMedGoogle Scholar
  70. Zhou H-X, Rivas G, Minton AP (2008) Macromolecular crowding and confinement: biochemical, biophysical, and potential physiological consequences. Annu Rev Biophys 37:375–397. doi: 10.1146/annurev.biophys.37.032807.125817 PubMedCentralCrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Tereza Pereira de Souza
    • 1
  • Alfred Fahr
    • 1
  • Pier Luigi Luisi
    • 2
  • Pasquale Stano
    • 2
    Email author
  1. 1.Institut für PharmazieFriedrich Schiller Universität JenaJenaGermany
  2. 2.Science DepartmentRoma Tre UniversityRomeItaly

Personalised recommendations