Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Opinion
  • Published:

Biology under construction: in vitro reconstitution of cellular function

Nature Reviews Molecular Cell Biology volume 10, pages 644–650 (2009)Cite this article

Abstract

We are much better at taking cells apart than putting them together. Reconstitution of biological processes from component molecules has been a powerful but difficult approach to studying functional organization in biology. Recently, the convergence of biochemical and cell biological advances with new experimental and computational tools is providing the opportunity to reconstitute increasingly complex processes. We predict that this bottom-up strategy will uncover basic processes that guide cellular assembly, advancing both basic and applied sciences.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: A cartoon of progress towards reconstitution of cellular processes.
Figure 2: Strategies for reconstituting cellular processes.

Similar content being viewed by others

References

  1. Groves, J. T. Bending mechanics and molecular organization in biological membranes. Annu. Rev. Phys. Chem. 58, 697–717 (2007).

    Article  CAS  Google Scholar 

  2. Garcia-Saez, A. J., Chiantia, S. & Schwille, P. Effect of line tension on the lateral organization of lipid membranes. J. Biol. Chem. 282, 33537–33544 (2007).

    Article  CAS  Google Scholar 

  3. Karsenti, E. Self-organization in cell biology: a brief history. Nature Rev. Mol. Cell Biol. 9, 255–262 (2008).

    Article  CAS  Google Scholar 

  4. Kron, S. J. & Spudich, J. A. Fluorescent actin filaments move on myosin fixed to a glass surface. Proc. Natl Acad. Sci. USA 83, 6272–6276 (1986).

    Article  CAS  Google Scholar 

  5. Hawking, S. W. The Universe in a Nutshell (Bantam, New York, 2001).

    Google Scholar 

  6. Kritikou, E. To see them contract for the first time. Nature Rev. Mol. Cell Biol. 9, S6 (2008).

    Google Scholar 

  7. Watson, J. D. & Crick, F. H. Molecular structure of nucleic acids; a structure for deoxyribose nucleic acid. Nature 171, 737–738 (1953).

    Article  CAS  Google Scholar 

  8. Kornberg, A. Biologic synthesis of deoxyribonucleic acid. Science 131, 1503–1508 (1960).

    Article  CAS  Google Scholar 

  9. Lehman, I. R., Bessman, M. J., Simms, E. S. & Kornberg, A. Enzymatic synthesis of deoxyribonucleic acid. I. Preparation of substrates and partial purification of an enzyme from Escherichia coli. J. Biol. Chem. 233, 163–170 (1958).

    CAS  PubMed  Google Scholar 

  10. Schekman, R. Protein localization and membrane traffic in yeast. Annu. Rev. Cell Biol. 1, 115–143 (1985).

    Article  CAS  Google Scholar 

  11. Orci, L., Glick, B. S. & Rothman, J. E. A new type of coated vesicular carrier that appears not to contain clathrin: its possible role in protein transport within the Golgi stack. Cell 46, 171–184 (1986).

    Article  CAS  Google Scholar 

  12. Barlowe, C. et al. COPII: a membrane coat formed by Sec proteins that drive vesicle budding from the endoplasmic reticulum. Cell 77, 895–907 (1994).

    Article  CAS  Google Scholar 

  13. Higgins, N. P., Peebles, C. L., Sugino, A. & Cozzarelli, N. R. Purification of subunits of Escherichia coli DNA gyrase and reconstitution of enzymatic activity. Proc. Natl Acad. Sci. USA 75, 1773–1777 (1978).

    Article  CAS  Google Scholar 

  14. Gorlich, D., Hartmann, E., Prehn, S. & Rapoport, T. A. A protein of the endoplasmic reticulum involved early in polypeptide translocation. Nature 357, 47–52 (1992).

    Article  CAS  Google Scholar 

  15. Nakajima, M. et al. Reconstitution of circadian oscillation of cyanobacterial KaiC phosphorylation in vitro. Science 308, 414–415 (2005).

    Article  CAS  Google Scholar 

  16. Surrey, T., Nedelec, F., Leibler, S. & Karsenti, E. Physical properties determining self-organization of motors and microtubules. Science 292, 1167–1171 (2001).

    Article  CAS  Google Scholar 

  17. Haviv, L. et al. Reconstitution of the transition from lamellipodium to filopodium in a membrane-free system. Proc. Natl Acad. Sci. USA 103, 4906–4911 (2006).

    Article  CAS  Google Scholar 

  18. Karsenti, E., Nedelec, F. & Surrey, T. Modelling microtubule patterns. Nature Cell Biol. 8, 1204–1211 (2006).

    Article  CAS  Google Scholar 

  19. Pollard, T. D. The cytoskeleton, cellular motility and the reductionist agenda. Nature 422, 741–745 (2003).

    Article  CAS  Google Scholar 

  20. Heald, R. et al. Self-organization of microtubules into bipolar spindles around artificial chromosomes in Xenopus egg extracts. Nature 382, 420–425 (1996).

    Article  CAS  Google Scholar 

  21. Pistor, S., Chakraborty, T., Niebuhr, K., Domann, E. & Wehland, J. The ActA protein of Listeria monocytogenes acts as a nucleator inducing reorganization of the actin cytoskeleton. EMBO J. 13, 758–763 (1994).

    Article  CAS  Google Scholar 

  22. Loisel, T. P., Boujemaa, R., Pantaloni, D. & Carlier, M. F. Reconstitution of actin-based motility of Listeria and Shigella using pure proteins. Nature 401, 613–616 (1999).

    Article  CAS  Google Scholar 

  23. Akin, O. & Mullins, R. D. Capping protein increases the rate of actin-based motility by promoting filament nucleation by the Arp2/3 complex. Cell 133, 841–851 (2008).

    Article  CAS  Google Scholar 

  24. Vignjevic, D. et al. Formation of filopodia-like bundles in vitro from a dendritic network. J. Cell Biol. 160, 951–962 (2003).

    Article  CAS  Google Scholar 

  25. Brieher, W. M., Coughlin, M. & Mitchison, T. J. Fascin-mediated propulsion of Listeria monocytogenes independent of frequent nucleation by the Arp2/3 complex. J. Cell Biol. 165, 233–242 (2004).

    Article  CAS  Google Scholar 

  26. Shih, Y. L. & Rothfield, L. The bacterial cytoskeleton. Microbiol. Mol. Biol. Rev. 70, 729–754 (2006).

    Article  CAS  Google Scholar 

  27. Garner, E. C., Campbell, C. S., Weibel, D. B. & Mullins, R. D. Reconstitution of DNA segregation driven by assembly of a prokaryotic actin homolog. Science 315, 1270–1274 (2007).

    Article  CAS  Google Scholar 

  28. Garner, E. C., Campbell, C. S. & Mullins, R. D. Dynamic instability in a DNA-segregating prokaryotic actin homolog. Science 306, 1021–1025 (2004).

    Article  CAS  Google Scholar 

  29. Sackmann, E. & Tanaka, M. Supported membranes on soft polymer cushions: fabrication, characterization and applications. Trends Biotechnol. 18, 58–64 (2000).

    Article  CAS  Google Scholar 

  30. Groves, J. T., Wulfing, C. & Boxer, S. G. Electrical manipulation of glycan-phosphatidyl inositol-tethered proteins in planar supported bilayers. Biophys. J. 71, 2716–2723 (1996).

    Article  CAS  Google Scholar 

  31. Grakoui, A. et al. The immunological synapse: a molecular machine controlling T cell activation. Science 285, 221–227 (1999).

    Article  CAS  Google Scholar 

  32. Pautot, S., Lee, H., Isacoff, E. Y. & Groves, J. T. Neuronal synapse interaction reconstituted between live cells and supported lipid bilayers. Nature Chem. Biol. 1, 283–289 (2005).

    Article  CAS  Google Scholar 

  33. Loose, M., Fischer-Friedrich, E., Ries, J., Kruse, K. & Schwille, P. Spatial regulators for bacterial cell division self-organize into surface waves in vitro. Science 320, 789–792 (2008).

    Article  CAS  Google Scholar 

  34. Co, C., Wong, D. T., Gierke, S., Chang, V. & Taunton, J. Mechanism of actin network attachment to moving membranes: barbed end capture by N-WASP WH2 domains. Cell 128, 901–913 (2007).

    Article  CAS  Google Scholar 

  35. Pucadyil, T. J. & Schmid, S. L. Real-time visualization of dynamin-catalyzed membrane fission and vesicle release. Cell 135, 1263–1275 (2008).

    Article  CAS  Google Scholar 

  36. Takei, K. et al. Generation of coated intermediates of clathrin-mediated endocytosis on protein-free liposomes. Cell 94, 131–141 (1998).

    Article  CAS  Google Scholar 

  37. Matsuoka, K. et al. COPII-coated vesicle formation reconstituted with purified coat proteins and chemically defined liposomes. Cell 93, 263–275 (1998).

    Article  CAS  Google Scholar 

  38. Palade, G. E. The organization of living matter. Proc. Natl Acad. Sci. USA 52, 613–634 (1964).

    Article  CAS  Google Scholar 

  39. Roux., A., Uyhazi, K., Frost, A. & De Camilli, P. GTP-dependent twisting of dynamin implicates constriction and tension in membrane fission. Nature 441, 528–531 (2006).

    Article  CAS  Google Scholar 

  40. Koster, G., VanDuijn, M., Hofs, B. & Dogterom, M. Membrane tube formation from giant vesicles by dynamic association of motor proteins. Proc. Natl Acad. Sci. USA 100, 15583–15588 (2003).

    Article  CAS  Google Scholar 

  41. Giardini, P. A., Fletcher, D. A. & Theriot, J. A. Compression forces generated by actin comet tails on lipid vesicles. Proc. Natl Acad. Sci. USA 100, 6493–6498 (2003).

    Article  CAS  Google Scholar 

  42. Upadhyaya, A. & van Oudenaarden, A. Biomimetic systems for studying actin-based motility. Curr. Biol. 13, R734–R744 (2003).

    Article  CAS  Google Scholar 

  43. Cortese, J. D., Schwab, B., Frieden, C. & Elson, E. L. Actin polymerization induces a shape change in actin-containing vesicles. Proc. Natl Acad. Sci. USA 86, 5773–5777 (1989).

    Article  CAS  Google Scholar 

  44. Fygenson, D. K., Marko, J. F. & Libchaber, A. Mechanics of microtubule-based membrane extension. Phys. Rev. Lett. 79, 4497 (1997).

    Article  CAS  Google Scholar 

  45. Pontani, L. L. et al. Reconstitution of an actin cortex inside a liposome. Biophys. J. 96, 192–198 (2009).

    Article  CAS  Google Scholar 

  46. Liu, A. P. & Fletcher, D. A. Actin polymerization serves as a membrane domain switch in model lipid bilayers. Biophys. J. 91, 4064–4070 (2006).

    Article  CAS  Google Scholar 

  47. Holowka, D., Sheets, E. D. & Baird, B. Interactions between FcɛRI and lipid raft components are regulated by the actin cytoskeleton. J. Cell Sci. 113, 1009–1019 (2000).

    CAS  PubMed  Google Scholar 

  48. Liu, A. P. et al. Membrane-induced bundling of actin filaments. Nature Phys. 4, 789–793 (2008).

    Article  CAS  Google Scholar 

  49. Pronk, S., Geissler, P. L. & Fletcher, D. A. Limits of filopodium stability. Phys. Rev. Lett. 100, 258102 (2008).

    Article  Google Scholar 

  50. Osawa, M., Anderson, D. E. & Erickson, H. P. Reconstitution of contractile FtsZ rings in liposomes. Science 320, 792–794 (2008).

    Article  CAS  Google Scholar 

  51. Doh, J. & Irvine, D. J. Immunological synapse arrays: patterned protein surfaces that modulate immunological synapse structure formation in T cells. Proc. Natl Acad. Sci. USA 103, 5700–5705 (2006).

    Article  CAS  Google Scholar 

  52. Cosentino Lagomarsino, M. et al. Microtubule organization in three-dimensional confined geometries: evaluating the role of elasticity through a combined in vitro and modeling approach. Biophys. J. 92, 1046–1057 (2007).

    Article  Google Scholar 

  53. Fernandez-Suarez, M. & Ting, A. Y. Fluorescent probes for super-resolution imaging in living cells. Nature Rev. Mol. Cell Biol. 9, 929–943 (2008).

    Article  CAS  Google Scholar 

  54. Alberts, J. B. & Odell, G. M. In silico reconstitution of Listeria propulsion exhibits nano-saltation. PLoS Biol. 2, e412 (2004).

    Article  Google Scholar 

  55. Pautot, S., Frisken, B. J. & Weitz, D. A. Engineering asymmetric vesicles. Proc. Natl Acad. Sci. USA 100, 10718–10721 (2003).

    Article  CAS  Google Scholar 

  56. Noireaux, V. & Libchaber, A. A vesicle bioreactor as a step toward an artificial cell assembly. Proc. Natl Acad. Sci. USA 101, 17669–17674 (2004).

    Article  CAS  Google Scholar 

  57. Okushima, S., Nisisako, T., Torii, T. & Higuchi, T. Controlled production of monodisperse double emulsions by two-step droplet breakup in microfluidic devices. Langmuir 20, 9905–9908 (2004).

    Article  CAS  Google Scholar 

  58. Utada, A. S. et al. Monodisperse double emulsions generated from a microcapillary device. Science 308, 537–541 (2005).

    Article  CAS  Google Scholar 

  59. Shum, H. C., Kim, J. W. & Weitz, D. A. Microfluidic fabrication of monodisperse biocompatible and biodegradable polymersomes with controlled permeability. J. Am. Chem. Soc. 130, 9543–9549 (2008).

    Article  CAS  Google Scholar 

  60. Funakoshi, K., Suzuki, H. & Takeuchi, S. Formation of giant lipid vesicle-like compartments from a planar lipid membrane by a pulsed jet flow. J. Am. Chem. Soc. 129, 12608–12609 (2007).

    Article  CAS  Google Scholar 

  61. Stachowiak, J. C. et al. Unilamellar vesicle formation and encapsulation by microfluidic jetting. Proc. Natl Acad. Sci. USA 105, 4697–4702 (2008).

    Article  CAS  Google Scholar 

  62. Mossman, K. D., Campi, G., Groves, J. T. & Dustin, M. L. Altered TCR signaling from geometrically repatterned immunological synapses. Science 310, 1191–1193 (2005).

    Article  CAS  Google Scholar 

  63. Theriot, J. A., Mitchison, T. J., Tilney, L. G. & Portnoy, D. A. The rate of actin-based motility of intracellular Listeria monocytogenes equals the rate of actin polymerization. Nature 357, 257–260 (1992).

    Article  CAS  Google Scholar 

  64. Cameron, L. A., Footer, M. J., van Oudenaarden, A. & Theriot, J. A. Motility of ActA protein-coated microspheres driven by actin polymerization. Proc. Natl Acad. Sci. USA 96, 4908–4913 (1999).

    Article  CAS  Google Scholar 

  65. Gaetz, J., Gueroui, Z., Libchaber, A. & Kapoor, T. M. Examining how the spatial organization of chromatin signals influences metaphase spindle assembly. Nature Cell Biol. 8, 924–932 (2006).

    Article  CAS  Google Scholar 

  66. Roux., A. et al. A minimal system allowing tubulation with molecular motors pulling on giant liposomes. Proc. Natl Acad. Sci. USA 99, 5394–5399 (2002).

    Article  CAS  Google Scholar 

  67. Wollert, T., Wunder, C., Lippincott-Schwartz, J. & Hurley, J. H. Membrane scission by the ESCRT-III complex. Nature 458, 172–177 (2009).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We apologize to those colleagues whose original and important work could not be cited owing to space limitations. We thank D. Richmond, J. Stachowiak and the rest of the Fletcher laboratory, as well as T. Pucadyil, for helpful discussion. A.P.L. is supported by the Natural Sciences and Engineering Research Council of Canada. D.A.F. is funded by National Institutes of Health R01 grants and a Nanomedicine Development Centre grant.

Author information

Authors and Affiliations

  1. Allen P. Liu is at the Department of Cell Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, MB-6, La Jolla, California 92037, USA.,

    Allen P. Liu

  2. Daniel A. Fletcher is at the Biophysics Group and Bioengineering Department, USA and the Physical Biosciences Division, University of California, Berkeley, California 94702, Lawrence Berkeley National Laboratory, Berkeley, California 94720, USA.,

    Daniel A. Fletcher

Authors
  1. Allen P. Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Daniel A. Fletcher
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Daniel A. Fletcher.

Related links

Related links

FURTHER INFORMATION

Daniel A. Fletcher's homepage

Rights and permissions

Reprints and permissions

About this article

Cite this article

Liu, A., Fletcher, D. Biology under construction: in vitro reconstitution of cellular function. Nat Rev Mol Cell Biol 10, 644–650 (2009). https://doi.org/10.1038/nrm2746

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nrm2746

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing