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 biological 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. @en

One of the major challenges of bottom-up synthetic biology is rebuilding a minimal cell division machinery. From a reconstitution perspective, the animal cell division apparatus is mechanically the simplest and therefore attractive to rebuild. An actin-based ring produces contractile force to constrict the membrane. By contrast, microbes and plant cells have a cell wall, so division requires concerted membrane constriction and cell wall synthesis. Furthermore, reconstitution of the actin division machinery helps in understanding the physical and molecular mechanisms of cytokinesis in animal cells and thus our own cells. In this review, we describe the state-of-the-art research on reconstitution of minimal actin-mediated cytokinetic machineries. Based on the conceptual requirements that we obtained from the physics of the shape changes involved in cell division, we propose two major routes for building a minimal actin apparatus capable of division. Importantly, we acknowledge both the passive and active roles that the confining lipid membrane can play in synthetic cytokinesis. We conclude this review by identifying the most pressing challenges for future reconstitution work, thereby laying out a roadmap for building a synthetic cell equipped with a minimal actin division machinery. @en

Cell replication is a fascinating biological process that ensures the proliferation of a species via division of a mother cell into two daughter ones. This process in bacteria is performed by a complex protein machinery named the Z-ring, which assembles at the cell mid-plane and promotes progressive membrane constriction down to division. A central element of this machinery is FtsZ, a protein that polymerizes into filaments that constitute the main scaffold of the ring. Even though this protein was found to be essential, many aspects concerning the Z-ring assembly, the role of FtsZ-associated proteins, and the contribution of FtsZ to the constricting force in the division process are not yet clearly determined. To address these questions, in this thesis we aimed to reconstitute a minimal divisome in vitro, in order to isolate single components from the complex cellular environment, and study their functionality in a bottom-up way. Following this approach, we aimed to better understand the dynamics and the parameters involved in FtsZ filament association into bundles, and to verify whether such structures are capable to generate a force on the lipid membrane. @en

Recently, both the cellular and synthetic biology communities have expressed a strong interest in coacervates, membrane‐less liquid droplets composed of densely packed multivalent molecules that form as a result of spontaneous phase separation. Here, it is studied how FtsZ, a protein that plays a key role in the bacterial division process, remodels coacervates made of polylysine (pLL) and guanosine triphosphate (GTP). It is shown that FtsZ strongly partitions at the surface of the coacervates and induces their disassembly due to the hydrolysis of GTP by FtsZ. Surprisingly, the coacervates are found to promote lateral interactions between FtsZ filaments, inducing the formation of an emanating network of FtsZ bundles that interconnect neighboring coacervates. Under mechanical stress, coacervates are shown to fracture, resulting in profound invaginations along their circumference. The results bring out the potential of coacervates for their use as membrane‐free scaffolds for building synthetic cells as well as are possibly relevant for coacervation in prokaryotic cells.@en

Bottom-up biology is an expanding research field that aims to understand the mechanisms underlying biological processes via in vitro assembly of their essential components in synthetic cells. As encapsulation and controlled manipulation of these elements is a crucial step in the recreation of such cell-like objects, microfluidics is increasingly used for the production of minimal artificial containers such as single-emulsion droplets, double-emulsion droplets, and liposomes. Despite the importance of cell morphology on cellular dynamics, current synthetic-cell studies mainly use spherical containers, and methods to actively shape manipulate these have been lacking. In this paper, we describe a microfluidic platform to deform the shape of artificial cells into a variety of shapes (rods and discs) with adjustable cell-like dimensions below 5 μm, thereby mimicking realistic cell morphologies. To illustrate the potential of our method, we reconstitute three biologically relevant protein systems (FtsZ, microtubules, collagen) inside rod-shaped containers and study the arrangement of the protein networks inside these synthetic containers with physiologically relevant morphologies resembling those found in living cells.@en