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), which 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 >30 μm-sized droplets and only in some cases GUV-sized (∼15 μm) satellite droplets. According to existing theoretical models, the oil-water interface should allow for the crossing of all droplets, but based on our observations and scaling arguments on the fluid dynamics within the system, we find a size-selective crossing of GUV-sized droplets only. The origin of these droplets remains partly unclear; we hypothesize that some small GUVs might be formed from large droplets sitting at the second interface. Finally, we demonstrate that proteins in the inner solution affect GUV formation by increasing the viscosity and altering the lipid adsorption kinetics. These results will not only contribute to a better understanding of GUV formation processes in cDICE but ultimately also aid in the development of more reliable and efficient methods for GUV production.

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The actin cortex is a complex cytoskeletal machinery that drives and responds to changes in cell shape. It must generate or adapt to plasma membrane curvature to facilitate diverse functions such as cell division, migration, and phagocytosis. Due to the complex molecular makeup of the actin cortex, it remains unclear whether actin networks are inherently able to sense and generate membrane curvature, or whether they rely on their diverse binding partners to accomplish this. Here, we show that curvature sensing is an inherent capability of branched actin networks nucleated by Arp2/3 and VCA. We develop a robust method to encapsulate actin inside giant unilamellar vesicles (GUVs) and assemble an actin cortex at the inner surface of the GUV membrane. We show that actin forms a uniform and thin cortical layer when present at high concentration and distinct patches associated with negative membrane curvature at low concentration. Serendipitously, we find that the GUV production method also produces dumbbell-shaped GUVs, which we explain using mathematical modeling in terms of membrane hemifusion of nested GUVs. We find that branched actin networks preferentially assemble at the neck of the dumbbells, which possess a micrometer-range convex curvature comparable with the curvature of the actin patches found in spherical GUVs. Minimal branched actin networks can thus sense membrane curvature, which may help mammalian cells to robustly recruit actin to curved membranes to facilitate diverse cellular functions such as cytokinesis and migration.

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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.

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Cells are the fundamental units of life. They make up all living things, from bacteria that live in the soil, to archea that give thermal springs their bright colors, to trees and humans. All of these cells share some common functions: they build themselves from basic building blocks, following the instructions of their genetic blueprint, and procreate by growing and dividing. The building blocks must be taken up from the environment and metabolized, and cell division requires the cell to be able to control its own shape. While these basic tasks are shared across the tree of life, different types of organisms have evolved distinct molecular machineries to complete them. In this thesis, we take a close look at animal cells, and ask how they control their shape as they must do in order to move, eat, sense, and divide. In animal cells, shape is controlled by the cytoskeleton, and in particular by the actin cortex. This cortex is a thin layer of actin _laments that sit underneath the plasma membrane, supporting the cell surface. The _laments are highly dynamic: they are constantly growing, shrinking and being remodeled, with well over 100 different proteins regulating their length and architecture. This molecular complexity, combined with the small size and high density of _laments and their rapid remodeling, makes it extremely difficult to disentangle different functions performed by the actin cortex in living cells. To better understand what fundamental principles govern cortex-based shape control of animal cells, we thus pursue a different approach: instead of studying living cells directly, we build minimal versions, so-called ‘synthetic cells’, from the bottom up. In such a bottom-up reconstitution approach, we isolate proteins (for instance actin) from their native environment, purify them, and bring them back together in vitro, following rational design principles. Consequently, we drastically reduce the complexity of the system, giving us a chance to actually understand what is going on. This allows us to test our assumptions about how cellular processes work in vivo, and discover new functions that are normally hidden in the complexity of the living cell. In this thesis, we use bottom-up reconstitution to ask how animal cells control their shape, with the ultimate aim to build a minimal actin-based cell division machinery…@en

Creating materials with structure that is independently controllable at a range of scales requires breaking naturally occurring hierarchies. Breaking these hierarchies can be achieved via the decoupling of building block attributes from structure during assembly. Here, we demonstrate, through computer simulations and experiments, that shape and interaction decoupling occur in colloidal cuboids suspended in evaporating emulsion droplets. The resulting colloidal clusters serve as “preassembled” mesoscale building blocks for larger-scale structures. We show that clusters of up to nine particles form mesoscale building blocks with geometries that are independent of the particles’ degree of faceting and dipolar magnetic interactions. To highlight the potential of these superball clusters for hierarchical assembly, we demonstrate, using computer simulations, that clusters of six to nine particles can assemble into high-order structures that differ from bulk self-assembly of individual particles. Our results suggest that preassembled building blocks present a viable route to hierarchical materials design.

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Molecular catch bonds are ubiquitous in biology and essential for processes like leucocyte extravasion¹ and cellular mechanosensing². Unlike normal (slip) bonds, catch bonds strengthen under tension. The current paradigm is that this feature provides ‘strength on demand³’, thus enabling cells to increase rigidity under stress^1,4–6. However, catch bonds are often weaker than slip bonds because they have cryptic binding sites that are usually buried^7,8. Here we show that catch bonds render reconstituted cytoskeletal actin networks stronger than slip bonds, even though the individual bonds are weaker. Simulations show that slip bonds remain trapped in stress-free areas, whereas weak binding allows catch bonds to mitigate crack initiation by moving to high-tension areas. This ‘dissociation on demand’ explains how cells combine mechanical strength with the adaptability required for shape change, and is relevant to diseases where catch bonding is compromised^7,9, including focal segmental glomerulosclerosis¹⁰ caused by the α-actinin-4 mutant studied here. We surmise that catch bonds are the key to create life-like materials.

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Understanding the relationship between colloidal building block shape and self-assembled material structure is important for the development of novel materials by self-assembly. In this regard, colloidal superballs are unique building blocks because their shape can smoothly transition between spherical and cubic. Assembly of colloidal superballs under spherical confinement results in macroscopic clusters with ordered internal structure. By utilizing Small Angle X-Ray Scattering (SAXS), we probe the internal structure of colloidal superball dispersion droplets during confinement. We observe and identify four distinct drying regimes that arise during compression via evaporating droplets, and we track the development of the assembled macrostructure. As the superballs assemble, we found that they arrange into the predicted paracrystalline, rhombohedral C₁-lattice that varies by the constituent superballs’ shape. This provides insights in the behavior between confinement and particle shape that can be applied in the development of new functional materials.

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The cytoskeletal protein actin polymerizes into filaments that are essential for the mechanical stability of mammalian cells. In vitro experiments showed that direct interactions between actin filaments and lipid bilayers are possible and that the net charge of the bilayer as well as the presence of divalent ions in the buffer play an important role. In vivo, colocalization of actin filaments and divalent ions are suppressed, and cells rely on linker proteins to connect the plasma membrane to the actin network. Little is known, however, about why this is the case and what microscopic interactions are important. A deeper understanding is highly beneficial, first, to obtain understanding in the biological design of cells and, second, as a possible basis for the building of artificial cortices for the stabilization of synthetic cells. Here, we report the results of coarse-grained molecular dynamics simulations of monomeric and filamentous actin in the vicinity of differently charged lipid bilayers. We observe that charges on the lipid head groups strongly determine the ability of actin to adsorb to the bilayer. The inclusion of divalent ions leads to a reversal of the binding affinity. Our in silico results are validated experimentally by reconstitution assays with actin on lipid bilayer membranes and provide a molecular-level understanding of the actin-membrane interaction.

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