Exploring Giant Unilamellar Vesicle Production for Artificial Cells - Current Challenges and Future Directions.

Creating an artificial cell from the bottom up is a long-standing challenge and, while significant progress has been made, the full realization of this goal remains elusive. Arguably, one of the biggest hurdles that researchers are facing now is the assembly of different modules of cell function inside a single container. Giant unilamellar vesicles (GUVs) have emerged as a suitable container with many methods available for their production. Well-studied swelling-based methods offer a wide range of lipid compositions but at the expense of limited encapsulation efficiency. Emulsion-based methods, on the other hand, excel at encapsulation but are only effective with a limited set of membrane compositions and may entrap residual additives in the lipid bilayer. Since the ultimate artificial cell will need to comply with both specific membrane and encapsulation requirements, there is still no one-method-fits-all solution for GUV formation available today. This review discusses the state of the art in different GUV production methods and their compatibility with GUV requirements and operational requirements such as reproducibility and ease of use. It concludes by identifying the most pressing issues and proposes potential avenues for future research to bring us one step closer to turning artificial cells into a reality.

High-throughput mechanophenotyping of multicellular spheroids using a microfluidic micropipette aspiration chip.

Cell spheroids are in vitro multicellular model systems that mimic the crowded micro-environment of biological tissues. Their mechanical characterization can provide valuable insights in how single-cell mechanics and cell-cell interactions control tissue mechanics and self-organization. However, most measurement techniques are limited to probing one spheroid at a time, require specialized equipment and are difficult to handle. Here, we developed a microfluidic chip that follows the concept of glass capillary micropipette aspiration in order to quantify the viscoelastic behavior of spheroids in an easy-to-handle, more high-throughput manner. Spheroids are loaded in parallel pockets via a gentle flow, after which spheroid tongues are aspirated into adjacent aspiration channels using hydrostatic pressure. After each experiment, the spheroids are easily removed from the chip by reversing the pressure and new spheroids can be injected. The presence of multiple pockets with a uniform aspiration pressure, combined with the ease to conduct successive experiments, allows for a high throughput of tens of spheroids per day. We demonstrate that the chip provides accurate deformation data when working at different aspiration pressures. Lastly, we measure the viscoelastic properties of spheroids made of different cell lines and show how these are consistent with previous studies using established experimental techniques. In summary, our chip provides a high-throughput way to measure the viscoelastic deformation behavior of cell spheroids, in order to mechanophenotype different tissue types and examine the link between cell-intrinsic properties and overall tissue behavior.

DisGUVery: A Versatile Open-Source Software for High-Throughput Image Analysis of Giant Unilamellar Vesicles.

Giant unilamellar vesicles (GUVs) are cell-sized aqueous compartments enclosed by a phospholipid bilayer. Due to their cell-mimicking properties, GUVs have become a widespread experimental tool in synthetic biology to study membrane properties and cellular processes. In stark contrast to the experimental progress, quantitative analysis of GUV microscopy images has received much less attention. Currently, most analysis is performed either manually or with custom-made scripts, which makes analysis time-consuming and results difficult to compare across studies. To make quantitative GUV analysis accessible and fast, we present DisGUVery, an open-source, versatile software that encapsulates multiple algorithms for automated detection and analysis of GUVs in microscopy images. With a performance analysis, we demonstrate that DisGUVery's three vesicle detection modules successfully identify GUVs in images obtained with a wide range of imaging sources, in various typical GUV experiments. Multiple predefined analysis modules allow the user to extract properties such as membrane fluorescence, vesicle shape, and internal fluorescence from large populations. A new membrane segmentation algorithm facilitates spatial fluorescence analysis of nonspherical vesicles. Altogether, DisGUVery provides an accessible tool to enable high-throughput automated analysis of GUVs, and thereby to promote quantitative data analysis in synthetic cell research.

Actomyosin-Driven Division of a Synthetic Cell.

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.

Molecular Structure and Surface Accumulation Dynamics of Hyaluronan at the Water-Air Interface.

Hyaluronan is a biopolymer that is essential for many biological processes in the human body, like the regulation of tissue lubrication and inflammatory responses. Here, we study the behavior of hyaluronan at aqueous surfaces using heterodyne-detected vibrational sum-frequency generation spectroscopy (HD-VSFG). Low-molecular-weight hyaluronan (∼150 kDa) gradually covers the water-air interface within hours, leading to a negatively charged surface and a reorientation of interfacial water molecules. The rate of surface accumulation strongly increases when the bulk concentration of low-molecular-weight hyaluronan is increased. In contrast, high-molecular-weight hyaluronan (>1 MDa) cannot be detected at the surface, even hours after the addition of the polymer to the aqueous solution. The strong dependence on the polymer molecular weight can be explained by entanglements of the hyaluronan polymers. We also find that for low-molecular-weight hyaluronan the migration kinetics of hyaluronan in aqueous media shows an anomalous dependence on the pH of the solution, which can be explained from the interplay of hydrogen bonding and electrostatic interactions of hyaluronan polymers.

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 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.

Active particles induce large shape deformations in giant lipid vesicles.

Biological cells generate intricate structures by sculpting their membrane from within to actively sense and respond to external stimuli or to explore their environment1-4. Several pathogenic bacteria also provide examples of how localized forces strongly deform cell membranes from inside, leading to the invasion of neighbouring healthy mammalian cells5. Giant unilamellar vesicles have been successfully used as a minimal model system with which to mimic biological cells6-11, but the realization of a minimal system with localized active internal forces that can strongly deform lipid membranes from within and lead to dramatic shape changes remains challenging. Here we present a combined experimental and simulation study that demonstrates how self-propelled particles enclosed in giant unilamellar vesicles can induce a plethora of non-equilibrium shapes and active membrane fluctuations. Using confocal microscopy, in the experiments we explore the membrane response to local forces exerted by self-phoretic Janus microswimmers. To quantify dynamic membrane changes, we perform Langevin dynamics simulations of active Brownian particles enclosed in thin membrane shells modelled by dynamically triangulated surfaces. The most pronounced shape changes are observed at low and moderate particle loadings, with the formation of tether-like protrusions and highly branched, dendritic structures, whereas at high volume fractions globally deformed vesicle shapes are observed. The resulting state diagram predicts the conditions under which local internal forces generate various membrane shapes. A controlled realization of such distorted vesicle morphologies could improve the design of artificial systems such as small-scale soft robots and synthetic cells.

Charge-dependent interactions of monomeric and filamentous actin with lipid bilayers.

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.