Polysaccharide functionalization reduces lipid vesicle stiffness.

The biophysical properties of lipid vesicles are important for their stability and integrity, key parameters that control the performance when these vesicles are used for drug delivery. The vesicle properties are determined by the composition of lipids used to form the vesicle. However, for a given lipid composition, they can also be tailored by tethering polymers to the membrane. Typically, synthetic polymers like polyethyleneglycol are used to increase vesicle stability, but the use of polysaccharides in this context is much less explored. Here, we report a general method for functionalizing lipid vesicles with polysaccharides by binding them to cholesterol. We incorporate the polysaccharides on the outer membrane leaflet of giant unilamellar vesicles (GUVs) and investigate their effect on membrane mechanics using micropipette aspiration. We find that the presence of the glycolipid functionalization produces an unexpected softening of GUVs with fluid-like membranes. By contrast, the functionalization of GUVs with polyethylene glycol does not reduce their stretching modulus. This work provides the potential means to study membrane-bound meshworks of polysaccharides similar to the cellular glycocalyx; moreover, it can be used for tuning the mechanical properties of drug delivery vehicles.

Membranes on the move: The functional role of the extracellular vesicle membrane for contact-dependent cellular signalling.

Extracellular vesicles (EVs), lipid-enclosed structures released by virtually all life forms, have gained significant attention due to their role in intercellular and interorganismal communication. Despite their recognized importance in disease processes and therapeutic applications, fundamental questions about their primary function remain. Here, we propose a different perspective on the primary function of EVs, arguing that they serve as essential elements providing membrane area for long-distance, contact-dependent cellular communication based on protein-protein interaction. While EVs have been recognized as carriers of genetic information, additional unique advantages that they could provide for cellular communication remain unclear. Here, we introduce the concept that the substantial membrane area provided by EVs allows for membrane contact-dependent interactions that could be central to their function. This membrane area enables the lateral diffusion and sorting of membrane ligands like proteins, polysaccharides or lipids in two dimensions, promoting avidity-driven effects and assembly of co-stimulatory architectures at the EV-cell interface. The concept of vesicle-induced receptor sequestration (VIRS), for example, describes how EVs confine and focus receptors at the EV contact site, promoting a dense local concentration of receptors into signalosomes. This process can increase the signalling strength of EV-presented ligands by 10-1000-fold compared to their soluble counterparts. The speculations in this perspective advance our understanding of EV-biology and have critical implications for EV-based applications and therapeutics. We suggest a shift in perspective from viewing EVs merely as transporters of relevant nucleic acids and proteins to considering their unique biophysical properties as presentation platforms for long-distance, contact-dependent signalling. We therefore highlight the functional role of the EV membrane rather than their content. We further discuss how this signalling mechanism might be exploited by virus-transformed or cancer cells to enhance immune-evasive mechanisms.

Triggered contraction of self-assembled micron-scale DNA nanotube rings.

Contractile rings are formed from cytoskeletal filaments during cell division. Ring formation is induced by specific crosslinkers, while contraction is typically associated with motor protein activity. Here, we engineer DNA nanotubes and peptide-functionalized starPEG constructs as synthetic crosslinkers to mimic this process. The crosslinker induces bundling of ten to hundred DNA nanotubes into closed micron-scale rings in a one-pot self-assembly process yielding several thousand rings per microliter. Molecular dynamics simulations reproduce the detailed architectural properties of the DNA rings observed in electron microscopy. Theory and simulations predict DNA ring contraction - without motor proteins - providing mechanistic insights into the parameter space relevant for efficient nanotube sliding. In agreement between simulation and experiment, we obtain ring contraction to less than half of the initial ring diameter. DNA-based contractile rings hold promise for an artificial division machinery or contractile muscle-like materials.

Engineering DNA-based cytoskeletons for synthetic cells.

The development and bottom-up assembly of synthetic cells with a functional cytoskeleton sets a major milestone to understand cell mechanics and to develop man-made machines on the nano- and microscale. However, natural cytoskeletal components can be difficult to purify, deliberately engineer and reconstitute within synthetic cells which therefore limits the realization of multifaceted functions of modern cytoskeletons in synthetic cells. Here, we review recent progress in the development of synthetic cytoskeletons made from deoxyribonucleic acid (DNA) as a complementary strategy. In particular, we explore the capabilities and limitations of DNA cytoskeletons to mimic functions of natural cystoskeletons like reversible assembly, cargo transport, force generation, mechanical support and guided polymerization. With recent examples, we showcase the power of rationally designed DNA cytoskeletons for bottom-up assembled synthetic cells as fully engineerable entities. Nevertheless, the realization of dynamic instability, self-replication and genetic encoding as well as contractile force generating motors remains a fruitful challenge for the complete integration of multifunctional DNA-based cytoskeletons into synthetic cells.

Modulating Lipid Membrane Morphology by Dynamic DNA Origami Networks.

Membrane morphology and its dynamic adaptation regulate many cellular functions, which are often mediated by membrane proteins. Advances in DNA nanotechnology have enabled the realization of various protein-inspired structures and functions with precise control at the nanometer level, suggesting a viable tool to artificially engineer membrane morphology. In this work, we demonstrate a DNA origami cross (DOC) structure that can be anchored onto giant unilamellar vesicles (GUVs) and subsequently polymerized into micrometer-scale reconfigurable one-dimensional (1D) chains or two-dimensional (2D) lattices. Such DNA origami-based networks can be switched between left-handed (LH) and right-handed (RH) conformations by DNA fuels and exhibit potent efficacy in remodeling the membrane curvatures of GUVs. This work sheds light on designing hierarchically assembled dynamic DNA systems for the programmable modulation of synthetic cells for useful applications.

A DNA Segregation Module for Synthetic Cells.

The bottom-up construction of an artificial cell requires the realization of synthetic cell division. Significant progress has been made toward reliable compartment division, yet mechanisms to segregate the DNA-encoded informational content are still in their infancy. Herein, droplets of DNA Y-motifs are formed by liquid-liquid phase separation. DNA droplet segregation is obtained by cleaving the linking component between two populations of DNA Y-motifs. In addition to enzymatic cleavage, photolabile sites are introduced for spatio-temporally controlled DNA segregation in bulk as well as in cell-sized water-in-oil droplets and giant unilamellar lipid vesicles (GUVs). Notably, the segregation process is slower in confinement than in bulk. The ionic strength of the solution and the nucleobase sequences are employed to regulate the segregation dynamics. The experimental results are corroborated in a lattice-based theoretical model which mimics the interactions between the DNA Y-motif populations. Altogether, engineered DNA droplets, reconstituted in GUVs, can represent a strategy toward a DNA segregation module within bottom-up assembled synthetic cells.

Genotype-phenotype mapping with polyominos made from DNA origami tiles.

The correlation between genetic information and characteristics of a living cell-its genotype and its phenotype-constitutes the basis of genetics. Here, we experimentally realize a primitive form of genotype-phenotype mapping with DNA origami. The DNA origami can polymerize into two-dimensional lattices (phenotype) via blunt-end stacking facilitated by edge staples at the seam of the planar DNA origami. There are 80 binding positions for edge staples, which allow us to translate an 80-bit long binary code (genotype) onto the DNA origami. The presence of an edge staple thus corresponds to a "1" and its absence to a "0." The interactions of our DNA-based system can be reproduced by a polyomino model. Polyomino growth simulations qualitatively reproduce our experimental results. We show that not only the absolute number of base stacks but also their sequence position determine the cluster size and correlation length of the orientation of single DNA origami within the cluster. Importantly, the mutation of a few bits can result in major morphology changes of the DNA origami cluster, while more often, major sequence changes have no impact. Our experimental realization of a correlation between binary information ("genotype") and cluster morphology ("phenotype") thus reproduces key properties of genotype-phenotype maps known from living systems.

Evolution and Single-Droplet Analysis of Fuel-Driven Compartments by Droplet-Based Microfluidics.

Active droplets are a great model for membraneless organelles. However, the analysis of these systems remains challenging and is often limited due to the short timescales of their kinetics. We used droplet-based microfluidics to encapsulate a fuel-driven cycle that drives phase separation into coacervate-based droplets to overcome this challenge. This approach enables the analysis of every coacervate-based droplet in the reaction container throughout its lifetime. We discovered that the fuel concentration dictates the formation of the coacervate-based droplets and their properties. We observed that coacervate-based droplets grow through fusion, decay simultaneously independent of their volume, and shrinkage rate scales with their initial volume. This method helps to further understand the regulation of membraneless organelles, and we believe the analysis of individual coacervate-based droplets enables future selection- or evolution-based studies.

Functional DNA-based cytoskeletons for synthetic cells.

The cytoskeleton is an essential component of a cell. It controls the cell shape, establishes the internal organization, and performs vital biological functions. Building synthetic cytoskeletons that mimic key features of their natural counterparts delineates a crucial step towards synthetic cells assembled from the bottom up. To this end, DNA nanotechnology represents one of the most promising routes, given the inherent sequence specificity, addressability and programmability of DNA. Here we demonstrate functional DNA-based cytoskeletons operating in microfluidic cell-sized compartments. The synthetic cytoskeletons consist of DNA tiles self-assembled into filament networks. These filaments can be rationally designed and controlled to imitate features of natural cytoskeletons, including reversible assembly and ATP-triggered polymerization, and we also explore their potential for guided vesicle transport in cell-sized confinement. Also, they possess engineerable characteristics, including assembly and disassembly powered by DNA hybridization or aptamer-target interactions and autonomous transport of gold nanoparticles. This work underpins DNA nanotechnology as a key player in building synthetic cells.

Bottom-Up Assembly of Synthetic Cells with a DNA Cytoskeleton.

Cytoskeletal elements, like actin and myosin, have been reconstituted inside lipid vesicles toward the vision to reconstruct cells from the bottom up. Here, we realize the de novo assembly of entirely artificial DNA-based cytoskeletons with programmed multifunctionality inside synthetic cells. Giant unilamellar lipid vesicles (GUVs) serve as cell-like compartments, in which the DNA cytoskeletons are repeatedly and reversibly assembled and disassembled with light using the cis-trans isomerization of an azobenzene moiety positioned in the DNA tiles. Importantly, we induced ordered bundling of hundreds of DNA filaments into more rigid structures with molecular crowders. We quantify and tune the persistence length of the bundled filaments to achieve the formation of ring-like cortical structures inside GUVs, resembling actin rings that form during cell division. Additionally, we show that DNA filaments can be programmably linked to the compartment periphery using cholesterol-tagged DNA as a linker. The linker concentration determines the degree of the cortex-like network formation, and we demonstrate that the DNA cortex-like network can deform GUVs from within. All in all, this showcases the potential of DNA nanotechnology to mimic the diverse functions of a cytoskeleton in synthetic cells.

Actomyosin-Assisted Pulling of Lipid Nanotubes from Lipid Vesicles and Cells.

Molecular motors are pivotal for intracellular transport as well as cell motility and have great potential to be put to use outside cells. Here, we exploit engineered motor proteins in combination with self-assembly of actin filaments to actively pull lipid nanotubes from giant unilamellar vesicles (GUVs). In particular, actin filaments are bound to the outer GUV membrane and the GUVs are seeded on a heavy meromyosin-coated substrate. Upon addition of ATP, hollow lipid nanotubes with a length of tens of micrometer are pulled from single GUVs due to the motor activity. We employ the same mechanism to pull lipid nanotubes from different types of cells. We find that the length and number of nanotubes critically depends on the cell type, whereby suspension cells form bigger networks than adherent cells. This suggests that molecular machines can be used to exert forces on living cells to probe membrane-to-cortex attachment.

Two-Photon 3D Laser Printing Inside Synthetic Cells.

Toward the ambitious goal of manufacturing synthetic cells from the bottom up, various cellular components have already been reconstituted inside lipid vesicles. However, the deterministic positioning of these components inside the compartment has remained elusive. Here, by using two-photon 3D laser printing, 2D and 3D hydrogel architectures are manufactured with high precision and nearly arbitrary shape inside preformed giant unilamellar lipid vesicles (GUVs). The required water-soluble photoresist is brought into the GUVs by diffusion in a single mixing step. Crucially, femtosecond two-photon printing inside the compartment does not destroy the GUVs. Beyond this proof-of-principle demonstration, early functional architectures are realized. In particular, a transmembrane structure acting as a pore is 3D printed, thereby allowing for the transport of biological cargo, including DNA, into the synthetic compartment. These experiments show that two-photon 3D laser microprinting can be an important addition to the existing toolbox of synthetic biology.

pH-Triggered Assembly of Endomembrane Multicompartments in Synthetic Cells.

By using electrostatic interactions as driving force to assemble vesicles, the droplet-stabilized method was recently applied to reconstitute and encapsulate proteins, or compartments, inside giant unilamellar vesicles (GUVs) to act as minimal synthetic cells. However, the droplet-stabilized approach exhibits low production efficiency associated with the troublesome release of the GUVs from the stabilized droplets, corresponding to a major hurdle for the droplet-stabilized approach. Herein, we report the use of pH as a potential trigger to self-assemble droplet-stabilized GUVs (dsGUVs) by either bulk or droplet-based microfluidics. Moreover, pH enables the generation of compartmentalized GUVs with flexibility and robustness. By co-encapsulating pH-sensitive small unilamellar vesicles (SUVs), negatively charged SUVs, and/or proteins, we show that acidification of the droplets efficiently produces dsGUVs while sequestrating the co-encapsulated material. Most importantly, the pH-mediated assembly of dsGUVs significantly improves the production efficiency of free-standing GUVs (i.e., released from the stabilizing-droplets) compared to its previous implementation.

Tuning Epithelial Cell-Cell Adhesion and Collective Dynamics with Functional DNA-E-Cadherin Hybrid Linkers.

The binding strength between epithelial cells is crucial for tissue integrity, signal transduction and collective cell dynamics. However, there is no experimental approach to precisely modulate cell-cell adhesion strength at the cellular and molecular level. Here, we establish DNA nanotechnology as a tool to control cell-cell adhesion of epithelial cells. We designed a DNA-E-cadherin hybrid system consisting of complementary DNA strands covalently bound to a truncated E-cadherin with a modified extracellular domain. DNA sequence design allows to tune the DNA-E-cadherin hybrid molecular binding strength, while retaining its cytosolic interactions and downstream signaling capabilities. The DNA-E-cadherin hybrid facilitates strong and reversible cell-cell adhesion in E-cadherin deficient cells by forming mechanotransducive adherens junctions. We assess the direct influence of cell-cell adhesion strength on intracellular signaling and collective cell dynamics. This highlights the scope of DNA nanotechnology as a precision technology to study and engineer cell collectives.

Building a community to engineer synthetic cells and organelles from the bottom-up.

Employing concepts from physics, chemistry and bioengineering, 'learning-by-building' approaches are becoming increasingly popular in the life sciences, especially with researchers who are attempting to engineer cellular life from scratch. The SynCell2020/21 conference brought together researchers from different disciplines to highlight progress in this field, including areas where synthetic cells are having socioeconomic and technological impact. Conference participants also identified the challenges involved in designing, manipulating and creating synthetic cells with hierarchical organization and function. A key conclusion is the need to build an international and interdisciplinary research community through enhanced communication, resource-sharing, and educational initiatives.

Light-Triggered Cargo Loading and Division of DNA-Containing Giant Unilamellar Lipid Vesicles.

A minimal synthetic cell should contain a substrate for information storage and have the capability to divide. Notable efforts were made to assemble functional synthetic cells from the bottom up, however often lacking the capability to reproduce. Here, we develop a mechanism to fully control reversible cargo loading and division of DNA-containing giant unilamellar vesicles (GUVs) with light. We make use of the photosensitizer Chlorin e6 (Ce6) which self-assembles into lipid bilayers and leads to local lipid peroxidation upon illumination. On the time scale of minutes, illumination induces the formation of transient pores, which we exploit for cargo encapsulation or controlled release. In combination with osmosis, complete division of two daughter GUVs can be triggered within seconds of illumination due to a spontaneous curvature increase. We ultimately demonstrate the division of a selected DNA-containing GUV with full spatiotemporal control-proving the relevance of the division mechanism for bottom-up synthetic biology.

Proton gradients from light-harvesting E. coli control DNA assemblies for synthetic cells.

Bottom-up and top-down approaches to synthetic biology each employ distinct methodologies with the common aim to harness living systems. Here, we realize a strategic merger of both approaches to convert light into proton gradients for the actuation of synthetic cellular systems. We genetically engineer E. coli to overexpress the light-driven inward-directed proton pump xenorhodopsin and encapsulate them in artificial cell-sized compartments. Exposing the compartments to light-dark cycles, we reversibly switch the pH by almost one pH unit and employ these pH gradients to trigger the attachment of DNA structures to the compartment periphery. For this purpose, a DNA triplex motif serves as a nanomechanical switch responding to the pH-trigger of the E. coli. When DNA origami plates are modified with the pH-sensitive triplex motif, the proton-pumping E. coli can trigger their attachment to giant unilamellar lipid vesicles (GUVs) upon illumination. A DNA cortex is formed upon DNA origami polymerization, which sculpts and deforms the GUVs. We foresee that the combination of bottom-up and top down approaches is an efficient way to engineer synthetic cells.

Choice of fluorophore affects dynamic DNA nanostructures.

The ability to dynamically remodel DNA origami structures or functional nanodevices is highly desired in the field of DNA nanotechnology. Concomitantly, the use of fluorophores to track and validate the dynamics of such DNA-based architectures is commonplace and often unavoidable. It is therefore crucial to be aware of the side effects of popular fluorophores, which are often exchanged without considering the potential impact on the system. Here, we show that the choice of fluorophore can strongly affect the reconfiguration of DNA nanostructures. To this end, we encapsulate a triple-stranded DNA (tsDNA) into water-in-oil compartments and functionalize their periphery with a single-stranded DNA handle (ssDNA). Thus, the tsDNA can bind and unbind from the periphery by reversible opening of the triplex and subsequent strand displacement. Using a combination of experiments, molecular dynamics (MD) simulations, and reaction-diffusion modelling, we demonstrate for 12 different fluorophore combinations that it is possible to alter or even inhibit the DNA nanostructure formation-without changing the DNA sequence. Besides its immediate importance for the design of pH-responsive switches and fluorophore labelling, our work presents a strategy to precisely tune the energy landscape of dynamic DNA nanodevices.

Division and Regrowth of Phase-Separated Giant Unilamellar Vesicles*.

Success in the bottom-up assembly of synthetic cells will depend on strategies for the division of protocellular compartments. Here, we describe the controlled division of phase-separated giant unilamellar lipid vesicles (GUVs). We derive an analytical model based on the vesicle geometry, which makes four quantitative predictions that we verify experimentally. We find that the osmolarity ratio required for division is 2 , independent of the GUV size, while asymmetric division happens at lower osmolarity ratios. Remarkably, we show that a suitable osmolarity change can be triggered by water evaporation, enzymatic decomposition of sucrose or light-triggered uncaging of CMNB-fluorescein. The latter provides full spatiotemporal control, such that a target GUV undergoes division whereas the surrounding GUVs remain unaffected. Finally, we grow phase-separated vesicles from single-phased vesicles by targeted fusion of the opposite lipid type with programmable DNA tags to enable subsequent division cycles.

Engineering Light-Responsive Contractile Actomyosin Networks with DNA Nanotechnology.

External control and precise manipulation is key for the bottom-up engineering of complex synthetic cells. Minimal actomyosin networks have been reconstituted into synthetic cells; however, their light-triggered symmetry breaking contraction has not yet been demonstrated. Here, light-activated directional contractility of a minimal synthetic actomyosin network inside microfluidic cell-sized compartments is engineered. Actin filaments, heavy-meromyosin-coated beads, and caged ATP are co-encapsulated into water-in-oil droplets. ATP is released upon illumination, leading to a myosin-generated force which results in a motion of the beads along the filaments and hence a contraction of the network. Symmetry breaking is achieved using DNA nanotechnology to establish a link between the network and the compartment periphery. It is demonstrated that the DNA-linked actin filaments contract to one side of the compartment forming actin asters and quantify the dynamics of this process. This work exemplifies that an engineering approach to bottom-up synthetic biology, combining biological and artificial elements, can circumvent challenges related to active multi-component systems and thereby greatly enrich the complexity of synthetic cellular systems.