Compartments for the spatially and temporally controlled assembly of biological processes are essential towards cellular life. Synthetic mimics of cellular compartments based on lipid-based protocells lack the mechanical and chemical stability to allow their manipulation into a complex and fully functional synthetic cell. Here, we present a high-throughput microfluidic method to generate stable, defined sized liposomes termed ‘droplet-stabilized giant unilamellar vesicles (dsGUVs)’. The enhanced stability of dsGUVs enables the sequential loading of these compartments with biomolecules, namely purified transmembrane and cytoskeleton proteins by microfluidic pico-injection technology. This constitutes an experimental demonstration of a successful bottom-up assembly of a compartment with contents that would not self-assemble to full functionality when simply mixed together. Following assembly, the stabilizing oil phase and droplet shells are removed to release functional self-supporting protocells to an aqueous phase, enabling them to interact with physiologically relevant matrices.
This is a preview of subscription content, access via your institution
Open Access articles citing this article.
Nature Communications Open Access 02 November 2022
Nature Communications Open Access 02 September 2022
Nature Communications Open Access 23 August 2022
Subscribe to Nature+
Get immediate online access to Nature and 55 other Nature journal
Subscribe to Journal
Get full journal access for 1 year
only $9.92 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Tax calculation will be finalised during checkout.
Get time limited or full article access on ReadCube.
All prices are NET prices.
Szostak, J. W., Bartel, D. P. & Luisi, P. L. Synthesizing life. Nature 409, 387–390 (2001).
Tawfik, D. S. & Griffiths, A. D. Man-made cell-like compartments for molecular evolution. Nat. Biotechnol. 16, 652–656 (1998).
Diekmann, Y. & Pereira-Leal, J. B. Evolution of intracellular compartmentalization. Biochem. J. 449, 319–331 (2013).
Agapakis, C. M., Boyle, P. M. & Silver, P. A. Natural strategies for the spatial optimization of metabolism in synthetic biology. Nat. Chem. Biol. 8, 527–535 (2012).
Yoshida, M., Muneyuki, E. & Hisabori, T. ATP synthase—A marvellous rotary engine of the cell. Nat. Rev. Mol. Cell Biol. 2, 669–677 (2001).
Geiger, B., Spatz, J. P. & Bershadsky, A. D. Environmental sensing through focal adhesions. Nat. Rev. Mol. Cell Biol. 10, 21–33 (2009).
Nomura, S. M. et al. Gene expression within cell-sized lipid vesicles. Chembiochem 4, 1172–1175 (2003).
Merkle, D., Kahya, N. & Schwille, P. Reconstitution and anchoring of cytoskeleton inside giant unilamellar vesicles. Chembiochem 9, 2673–2681 (2008).
Hardy, G. J., Nayak, R. & Zauscher, S. Model cell membranes: techniques to form complex biomimetic supported lipid bilayers via vesicle fusion. Curr. Opin. Colloid Interface Sci. 18, 448–458 (2013).
Seantier, B. & Kasemo, B. Influence of mono- and divalent ions on the formation of supported phospholipid bilayers via vesicle adsorption. Langmuir 25, 5767–5772 (2009).
Shigematsu, T., Koshiyama, K. & Wada, S. Effects of stretching speed on mechanical rupture of phospholipid/cholesterol bilayers: molecular dynamics simulation. Sci. Rep. 5, 15369 (2015).
Jorgensen, I. L., Kemmer, G. C. & Pomorski, T. G. Membrane protein reconstitution into giant unilamellar vesicles: a review on current techniques. Eur. Biophys. J. 46, 103–119 (2016).
Discher, B. M. et al. Polymersomes: tough vesicles made from diblock copolymers. Science 284, 1143–1146 (1999).
Palivan, C. G. et al. Bioinspired polymer vesicles and membranes for biological and medical applications. Chem. Soc. Rev. 45, 377–411 (2016).
Onaca, O., Nallani, M., Ihle, S., Schenk, A. & Schwaneberg, U. Functionalized nanocompartments (Synthosomes): limitations and prospective applications in industrial biotechnology. Biotechnol. J. 1, 795–805 (2006).
Picker, A., Nuss, H., Guenoun, P. & Chevallard, C. Polymer vesicles as microreactors for bioinspired calcium carbonate precipitation. Langmuir 27, 3213–3218 (2011).
Lee, J. C. M., Santore, M., Bates, F. S. & Discher, D. E. From membranes to melts, rouse to reptation: diffusion in polymersome versus lipid bilayers. Macromolecules 35, 323–326 (2002).
Duncombe, T. A., Tentori, A. M. & Herr, A. E. Microfluidics: reframing biological enquiry. Nat. Rev. Mol. Cell Biol. 16, 554–567 (2015).
Martino, C. & deMello, A. J. Droplet-based microfluidics for artificial cell generation: a brief review. Interface Focus 6, 20160011 (2016).
Schaerli, Y. et al. Continuous-flow polymerase chain reaction of single-copy DNA in microfluidic microdroplets. Anal. Chem. 81, 302–306 (2009).
Platzman, I., Janiesch, J.-W. & Spatz, J. P. Synthesis of nanostructured and biofunctionalized water-in-oil droplets as tools for homing T cells. J. Am. Chem. Soc. 135, 3339–3342 (2013).
Huebner, A. et al. Quantitative detection of protein expression in single cells using droplet microfluidics. Chem. Commun. 12, 1218–1220 (2007).
Janiesch, J. W. et al. Key factors for stable retention of fluorophores and labeled biomolecules in droplet-based microfluidics. Anal. Chem. 87, 2063–2067 (2015).
Abate, A. R., Hung, T., Mary, P., Agresti, J. J. & Weitz, D. A. High-throughput injection with microfluidics using picoinjectors. Proc. Natl Acad. Sci. USA 107, 19163–19166 (2010).
Itel, F. et al. Molecular organization and dynamics in polymersome membranes: a lateral diffusion study. Macromolecules 47, 7588–7596 (2014).
Bhatia, T., Husen, P., Ipsen, J. H., Bagatolli, L. A. & Simonsen, A. C. Fluid domain patterns in free-standing membranes captured on a solid support. Biochim. Biophys. Acta Biomembr. 1838, 2503–2510 (2014).
Machan, R. & Hof, M. Lipid diffusion in planar membranes investigated by fluorescence correlation spectroscopy. Biochim. Biophys. Acta 1798, 1377–1391 (2010).
Przybylo, M. et al. Lipid diffusion in giant unilamellar vesicles is more than 2 times faster than in supported phospholipid bilayers under identical conditions. Langmuir 22, 9096–9099 (2006).
Goennenwein, S., Tanaka, M., Hu, B., Moroder, L. & Sackmann, E. Functional incorporation of integrins into solid supported membranes on ultrathin films of cellulose: impact on adhesion. Biophys. J. 85, 646–655 (2003).
Erb, E. M., Tangemann, K., Bohrmann, B., Muller, B. & Engel, J. Integrin alpha IIb beta 3 reconstituted into lipid bilayers is nonclustered in its activated state but clusters after fibrinogen binding. Biochemistry 36, 7395–7402 (1997).
Edel, J. B., Wu, M., Baird, B. & Craighead, H. G. High spatial resolution observation of single-molecule dynamics in living cell membranes. Biophys. J. 88, L43–L45 (2005).
Frohnmayer, J. P. et al. Minimal synthetic cells to study integrin-mediated adhesion. Angew. Chem. Int. Ed. 54, 12472–12478 (2015).
Fischer, S. & Graber, P. Comparison of Delta pH- and Delta phi-driven ATP synthesis catalyzed by the H + -ATPases from Escherichia coli or chloroplasts reconstituted into liposomes. FEBS Lett. 457, 327–332 (1999).
Wolff, J. Plasma membrane tubulin. Biochim. Biophys. Acta Biomembr. 1788, 1415–1433 (2009).
Dimova, R. Recent developments in the field of bending rigidity measurements on membranes. Adv. Colloid Interface Sci. 208, 225–234 (2014).
Streicher, P. et al. Integrin reconstituted in GUVs: a biomimetic system to study initial steps of cell spreading. Biochim. Biophys. Acta Biomembr. 1788, 2291–2300 (2009).
Karamdad, K., Law, R. V., Seddon, J. M., Brooks, N. J. & Ces, O. Preparation and mechanical characterisation of giant unilamellar vesicles by a microfluidic method. Lab Chip 15, 557–562 (2015).
Matosevic, S. & Paegel, B. M. Stepwise synthesis of giant unilamellar vesicles on a microfluidic assembly line. J. Am. Chem. Soc. 133, 2798–2800 (2011).
Deng, N.-N., Yelleswarapu, M. & Huck, W. T. S. Monodisperse uni- and multicompartment liposomes. J. Am. Chem. Soc. 138, 7584–7591 (2016).
Matosevic, S. & Paegel, B. M. Layer-by-layer cell membrane assembly. Nat. Chem. 5, 958–963 (2013).
Kamiya, K., Kawano, R., Osaki, T., Akiyoshi, K. & Takeuchi, S. Cell-sized asymmetric lipid vesicles facilitate the investigation of asymmetric membranes. Nat. Chem. 8, 881–889 (2016).
Morita, M. et al. Droplet-shooting and size-filtration (DSSF) method for synthesis of cell-sized liposomes with controlled lipid compositions. ChemBioChem 16, 2029–2035 (2015).
Niu, X., Gulati, S., Edel, J. B. & deMello, A. J. Pillar-induced droplet merging in microfluidic circuits. Lab Chip 8, 1837–1841 (2008).
Eberhard, C. Online-Ressource (Heidelberg Univ., 2012).
Zimmermann, B., Diez, M., Zarrabi, N., Graber, P. & Borsch, M. Movements of the epsilon-subunit during catalysis and activation in single membrane-bound H + -ATP synthase. EMBO J. 24, 2053–2063 (2005).
Heitkamp, T., Deckers-Hebestreit, G. & Borsch, M. in Single Molecule Spectroscopy and Superresolution Imaging IX Vol. 9714 (eds Enderlein, J., Gregor, I., Gryczynski, Z. K., Erdmann, R. & Koberling, F.) (Spie-Int Soc Optical Engineering, 2016).
Mashaghi, S. & van Oijen, A. M. External control of reactions in microdroplets. Sci. Rep. 5, 11837 (2015).
Gan, B. S., Krump, E., Shrode, L. D. & Grinstein, S. Loading pyranine via purinergic receptors or hypotonic stress for measurement of cytosolic pH by imaging. Am. J. Physiol. 275, C1158–C1166 (1998).
Castoldi, M. & Popov, A. V. Purification of brain tubulin through two cycles of polymerization-depolymerization in a high-molarity buffer. Protein Expr. Purif. 32, 83–88 (2003).
Hyman, A. et al. Preparation of modified tubulins. Methods Enzymol. 196, 478–485 (1991).
Parts of the research leading to these results have received funding from the European Research Council/ERC Grant Agreement no. 294852, SynAd. This work is also part of the MaxSynBio consortium, which is jointly funded by the Federal Ministry of Education and Research of Germany and the Max Planck Society. The work was also partly supported by the SFB 1129 of the German Science Foundation and the VolkswagenStiftung (priority call ‘Life?’). J.P.S. is the Weston Visiting Professor at the Weizmann Institute of Science and part of the excellence cluster CellNetworks at the University of Heidelberg. J.-C.B. acknowledges financial support by the ERC (FP7/2007-2013/ERC Grant agreement 306385-SofI). I.P. acknowledges the support of the Alexander von Humboldt Foundation. The authors acknowledge the help of P. Gruner and B. Riechers for their technical assistance with preliminary microfluidic experiments and A. Richter (WITec GmbH, Germany) for her technical assistance with Raman microscopy. The support of N. Grunze for editing the manuscript as well as of J. Ricken and Ch. Mollenhauer for their general support in protein purification and chemical synthesis is highly acknowledged. The Max Planck Society is appreciated for its general support in all aspects of our research.
The authors declare no competing financial interests.
About this article
Cite this article
Weiss, M., Frohnmayer, J., Benk, L. et al. Sequential bottom-up assembly of mechanically stabilized synthetic cells by microfluidics. Nature Mater 17, 89–96 (2018). https://doi.org/10.1038/nmat5005
This article is cited by
Nature Communications (2022)
Nature Chemistry (2022)
Nature Communications (2022)
Nature Communications (2022)