Van de Cauter, L. | bioRxiv Science

bioRxiv2023

Uncovering the hidden mechanisms of giant unilamellar vesicle formation in cDICE

Giant unilamellar vesicles (GUVs) are widely used as in vitro model membranes in biophysics and as cell-sized containers in synthetic biology. Despite their ubiquitous use, there is no one-size-fits-all method for their production. Numerous methods have been developed to meet the demanding requirements of reproducibility, reliability, and high yield, while simultaneously achieving robust encapsulation. Emulsion-based methods are often praised for their apparent simplicity and good yields; hence, methods like continuous droplet interface crossing encapsulation (cDICE) that make use of this principle, have gained popularity. However, the underlying physical principles governing the formation of GUVs in cDICE and related methods remain poorly understood. To this end, we have developed a high-speed microscopy setup that allows us to visualize GUV formation in real-time. Our experiments reveal a complex droplet formation process occurring at the capillary orifice, generating both larger droplets and, likely, GUV-sized satellite droplets. According to existing theoretical models, the oil-water interface should allow for crossing of all droplets, but based on our observations and theoretical modelling of the fluid dynamics within the system, we find a size-selective crossing of GUV-sized droplets only. Finally, we demonstrate that proteins in the inner solution affect GUV formation by increasing the viscosity and altering lipid adsorption kinetics. These results will not only contribute to a better understanding of GUV formation processes in cDICE, but ultimately also aid the development of more reliable and efficient methods for GUV production. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=98 SRC="FIGDIR/small/562183v2_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@9764cforg.highwire.dtl.DTLVardef@15b63c7org.highwire.dtl.DTLVardef@f39e83org.highwire.dtl.DTLVardef@2bb63a_HPS_FORMAT_FIGEXP M_FIG C_FIG We developed a high-speed microscopy setup to study giant unilamellar vesicle formation in cDICE, revealing a complex droplet formation process occurring at the capillary orifice and size-selectivity at the oil-water interface.

synthetic biology↗

bioRxiv2021

Optimized cDICE for efficient reconstitution of biological systems in giant unilamellar vesicles

Giant unilamellar vesicles (GUVs) are often used to mimic biological membranes in reconstitution experiments. They are also widely used in research on synthetic cells as they provide a mechanically responsive reaction compartment that allows for controlled exchange of reactants with the environment. However, while many methods exist to encapsulate functional biomolecules in GUVs, there is no one-size-fits-all solution and reliable GUV fabrication still remains a major experimental hurdle in the field. Here, we show that defect-free GUVs containing complex biochemical systems can be generated by optimizing a double-emulsion method for GUV formation called continuous droplet interface crossing encapsulation (cDICE). By tightly controlling environmental conditions and tuning the lipid-in-oil dispersion, we show that it is possible to significantly improve the reproducibility of high-quality GUV formation as well as the encapsulation efficiency. We demonstrate efficient encapsulation for a range of minimal systems including a minimal actin cytoskeleton, membrane-anchored DNA nanostructures, and a functional PURE (Protein synthesis Using Recombinant Elements) system. Our optimized cDICE method displays promising potential to become a standard method in biophysics and bottom-up synthetic biology.

synthetic biology↗