Sequential bottom-up assembly of mechanically stabilized synthetic cells by microfluidics

Abstract

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.

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Figure 1: Formation and analysis of droplet-stabilized GUVs.
Figure 2: Sequential bottom-up assembly of dsGUV cell-like compartments.
Figure 3: Incorporation of transmembrane and cytoskeletal proteins into the dsGUV compartments by pico-injection technology.
Figure 4: Release of actin- and integrin-reconstituted GUVs.

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Acknowledgements

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.

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M.W. and J.P.F. realized experimentally the general concept of dsGUVs by microfluidic technology, established pico-injection technology and its application for the design of synthetic cells—this includes the formation of dsGUVs, release of GUVs from oil to water phase, FRAP measurements—and wrote parts of the manuscript. M.W. performed microtubule assembly and integration of functional ATP synthase and its analysis; J.P.F. reconstituted functional Integrin in liposomes and dsGUV and performed release of Integrin GUVs from oil to water phase; L.T.B. optimized the release of GUVs from oil to water phase, in particular the release of integrin functionalized GUVs, performed adhesion experiments of integrin functionalized GUVs to different matrices, and wrote parts of the manuscript; B.H. performed the experiments of dsGUVs with F-actin and its release from oil to water phase and performed Raman spectroscopy analysis; J.-W.J. performed the experiments of dsGUVs with F-actin and synthesized polymer-based surfactants; T.H. and M.B. prepared the labelled FoF1-ATP synthase, R.B.L., R.D. and R.L. developed and discussed lipid bilayer formation using droplets, E.B. and J.-C.B. helped install the pico-injection technology, T.V.-K. and K.S. supported the reconstitution of FoF1-ATP synthase, I.P. designed and supervised the experiments, and wrote the manuscript; J.P.S. invented the concept of synthetic cell formation by sequential bottom-up assembly in droplet-stabilized compartments, designed, supervised and managed the experiments, and wrote the manuscript.

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Correspondence to Ilia Platzman or Joachim P. Spatz.

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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

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