Increased efficiency of charge-mediated fusion in polymer/lipid hybrid membranes.

Due to their augmented properties, biomimetic polymer/lipid hybrid compartments are a promising substitute for natural liposomes in multiple applications, but the protein-free fusion of those semisynthetic membranes is unexplored to date. Here, we study the charge-mediated fusion of hybrid vesicles composed of poly(dimethylsiloxane)-graft-poly(ethylene oxide) and different lipids and analyze the process by size distribution and the mixing of membrane species at µm and nano scales. Remarkably, the membrane mixing of oppositely charged hybrids surpasses by far the degree in liposomes, which we correlate with properties like membrane disorder, rigidity, and ability of amphiphiles for flip-flop. Furthermore, we employ the integration of two respiratory proteins as a functional content mixing assay for different membrane compositions. This reveals that fusion is also attainable with neutral and cationic hybrids and that the charge is not the sole determinant of the final adenosine triphosphate synthesis rate, substantiating the importance of reconstitution environment. Finally, we employ this fusion strategy for the delivery of membrane proteins to giant unilamellar vesicles as a way to automate the assembly of synthetic cells.

Protein-Rich Rafts in Hybrid Polymer/Lipid Giant Unilamellar Vesicles.

Considerable attention has been dedicated to lipid rafts due to their importance in numerous cell functions such as membrane trafficking, polarization, and signaling. Next to studies in living cells, artificial micrometer-sized vesicles with a minimal set of components are established as a major tool to understand the phase separation dynamics and their intimate interplay with membrane proteins. In parallel, mixtures of phospholipids and certain amphiphilic polymers simultaneously offer an interface for proteins and mimic this segregation behavior, presenting a tangible synthetic alternative for fundamental studies and bottom-up design of cellular mimics. However, the simultaneous insertion of complex and sensitive membrane proteins is experimentally challenging and thus far has been largely limited to natural lipids. Here, we present the co-reconstitution of the proton pump bo3 oxidase and the proton consumer ATP synthase in hybrid polymer/lipid giant unilamellar vesicles (GUVs) via fusion/electroformation. Variations of the current method allow for tailored reconstitution protocols and control of the vesicle size. In particular, mixing of protein-free and protein-functionalized nanosized vesicles in the electroformation film results in larger GUVs, while separate reconstitution of the respiratory enzymes enables higher ATP synthesis rates. Furthermore, protein labeling provides a synthetic mechanism for phase separation and protein sequestration, mimicking lipid- and protein-mediated domain formation in nature. The latter means opens further possibilities for re-enacting phenomena like supercomplex assembly or symmetry breaking and enriches the toolbox of bottom-up synthetic biology.

Constructing artificial respiratory chain in polymer compartments: Insights into the interplay between bo3 oxidase and the membrane.

Cytochrome bo3 ubiquinol oxidase is a transmembrane protein, which oxidizes ubiquinone and reduces oxygen, while pumping protons. Apart from its combination with F1Fo-ATPase to assemble a minimal ATP regeneration module, the utility of the proton pump can be extended to other applications in the context of synthetic cells such as transport, signaling, and control of enzymatic reactions. In parallel, polymers have been speculated to be phospholipid mimics with respect to their ability to self-assemble in compartments with increased stability. However, their usability as interfaces for complex membrane proteins has remained questionable. In the present work, we optimized a fusion/electroformation approach to reconstitute bo3 oxidase in giant unilamellar vesicles made of PDMS-g-PEO and/or phosphatidylcholine (PC). This enabled optical access, while microfluidic trapping allowed for online analysis of individual vesicles. The tight polymer membranes and the inward oriented enzyme caused 1 pH unit difference in 30 min, with an initial rate of 0.35 pH·min-1 To understand the interplay in these composite systems, we studied the relevant mechanical and rheological membrane properties. Remarkably, the proton permeability of polymer/lipid hybrids decreased after protein insertion, while the latter also led to a 20% increase of the polymer diffusion coefficient in polymersomes. In addition, PDMS-g-PEO increased the activity lifetime and the resistance to free radicals. These advantageous properties may open diverse applications, ranging from cell-free biotechnology to biomedicine. Furthermore, the presented study serves as a comprehensive road map for studying the interactions between membrane proteins and synthetic membranes, which will be fundamental for the successful engineering of such hybrid systems.

Fusion-Induced Growth of Biomimetic Polymersomes: Behavior of Poly(dimethylsiloxane)-Poly(ethylene oxide) Vesicles in Saline Solutions Under High Agitation.

Giant unilamellar vesicles serve as membrane models and primitive mockups of natural cells. With respect to the latter use, amphiphilic polymers can be used to replace phospholipids in order to introduce certain favorable properties, ultimately allowing for the creation of truly synthetic cells. These new properties also enable the employment of new preparation procedures that are incompatible with the natural amphiphiles. Whereas the growth of lipid compartments to micrometer dimensions has been well established, growth of their synthetic analogs remains underexplored. Here, the influence of experimental parameters like salt type/concentration and magnitude of agitation on the fusion of nanometer-sized vesicles made of poly(dimethylsiloxane)-poly(ethylene oxide) graft copolymer (PDMS-g-PEO) is investigated in detail. To this end, dynamic light scattering, microscopy, and membrane mixing assays are employed, and the process at different time and length scales is analyzed. This optimized method is used as an easy tool to obtain giant vesicles, equipped with membrane and cytosolic biomachinery, in the presence of salts at physiological concentrations.

Scale up of Transmembrane NADH Oxidation in Synthetic Giant Vesicles.

The transfer of electrons across and along biological membranes drives the cellular energetics. In the context of artificial cells, it can be mimicked by minimal means, while using synthetic alternatives of the phospholipid bilayer and the electron-transducing proteins. Furthermore, the scaling up to biologically relevant and optically accessible dimensions may provide further insight and allow assessment of individual events but has been rarely attempted so far. Here, we visualized the mediated transmembrane oxidation of encapsulated NADH in giant unilamellar vesicles via confocal laser scanning and time-correlated single photon counting wide-field microscopy. To this end, we first augmented phospholipid membranes with an amphiphilic copolymer in order to check its influence on the oxidation kinetics spectrophotometrically. Then, we scaled up the compartments and followed the process microscopically.

Light-Driven ATP Regeneration in Diblock/Grafted Hybrid Vesicles.

Light-driven ATP regeneration systems combining ATP synthase and bacteriorhodopsin have been proposed as an energy supply in the field of synthetic biology. Energy is required to power biochemical reactions within artificially created reaction compartments like protocells, which are typically based on either lipid or polymer membranes. The insertion of membrane proteins into different hybrid membranes is delicate, and studies comparing these systems with liposomes are needed. Here we present a detailed study of membrane protein functionality in different hybrid compartments made of graft polymer PDMS-g-PEO and diblock copolymer PBd-PEO. Activity of more than 90 % in lipid/polymer-based hybrid vesicles could prove an excellent biocompatibility. A significant enhancement of long-term stability (80 % remaining activity after 42 days) could be demonstrated in polymer/polymer-based hybrids.

Compartments for Synthetic Cells: Osmotically Assisted Separation of Oil from Double Emulsions in a Microfluidic Chip.

Liposomes are used in synthetic biology as cell-like compartments and their microfluidic production through double emulsions allows for efficient encapsulation of various components. However, residual oil in the membrane remains a critical bottleneck for creating pristine phospholipid bilayers. It has been discovered that osmotically driven shrinking leads to detachment of the oil drop. Separation inside a microfluidic chip has been realized to automate the procedure, which allows for controlled continuous production of monodisperse liposomes.

Polymer-Based Module for NAD+ Regeneration with Visible Light.

The regeneration of enzymatic cofactors by cell-free synthetic modules is a key step towards producing a purely synthetic cell. Herein, we demonstrate the regeneration of the enzyme cofactor NAD+ by photo-oxidation of NADH under visible-light irradiation by using metal-free conjugated polymer nanoparticles. Encapsulation of the light-active nanoparticles in the lumen of polymeric vesicles produced a fully organic module able to regenerate NAD+ in an enzyme-free system. The polymer compartment conferred physical and chemical autonomy to the module, allowing the regeneration of NAD+ to occur efficiently, even in harsh chemical environments. Moreover, we show that regeneration of NAD+ by the photocatalyst nanoparticles can oxidize a model substrate, in conjunction with the enzyme glycerol dehydrogenase. To ensure the longevity of the enzyme, we immobilized it within a protective silica matrix; this yielded enzymatic silica nanoparticles with enhanced long-term performance and compatibility with the NAD+ -regeneration system.

Transmembrane NADH Oxidation with Tetracyanoquinodimethane.

The design of efficient schemes for nicotinamide adenine dinucleotide (NAD) regeneration is essential for the development of enzymatic biotechnological processes in order to sustain continuous production. In line with our motivation for the encapsulation of redox cascades in liposomes to serve as microbioreactors, we developed a straightforward strategy for the interfacial oxidation of entrapped NADH by ferricyanide as an external electron acceptor. Instead of the commonly applied enzymatic regeneration methods, we employed a hydrophobic redox shuttle embedded in the liposome bilayer. Tetracyanoquinodimethane (TCNQ) mediated electron transfer across the membrane and thus allowed us to shortcut and emulate part of the electron transfer chain functionality without the involvement of membrane proteins. To describe the experimental system, we developed a mathematical model which allowed for the determination of rate constants and exhibited handy predictive utility.

Toward Artificial Mitochondrion: Mimicking Oxidative Phosphorylation in Polymer and Hybrid Membranes.

For energy supply to biomimetic constructs, a complex chemical energy-driven ATP-generating artificial system was built. The system was assembled with bottom-up detergent-mediated reconstitution of an ATP synthase and a terminal oxidase into two types of novel nanocontainers, built from either graft copolymer membranes or from hybrid graft copolymer/lipid membranes. The versatility and biocompatibility of the proposed nanocontainers was demonstrated through convenient system assembly and through high retained activity of both membrane-embedded enzymes. In the future, the nanocontainers might be used as a platform for the functional reconstitution of other complex membrane proteins and could considerably expedite the design of nanoreactors, biosensors, and artificial organelles.

Binding kinetics and multi-bond: Finding correlations by synthesizing interactions between ligand-coated bionanoparticles and receptor surfaces.

The number of bonds formed between one single bionanoparticle and many surface receptors is an important subject to be studied but is seldom quantitatively investigated. A new evaluation of the correlation between binding kinetics and number of bonds is presented by varying ligand density and receptor density. An experimental system was developed using measurements with surface plasmon resonance spectroscopy. A corresponding multi-site adsorption model elucidated the correlation. The results show that with the increase of the receptor density, the adsorption rate first decreased when the number of bonds was below a maximum value and then increased when the number of bonds stayed at this maximum value. The investigation on ligand density variation suggests that the coating density on top of the bionanoparticle surface may have a particular value below which more ligand will accelerate the adsorption rate. The ratio of ligand amount bound by the receptors to the total ligand amount associated with a single bionanoparticle will remain constant even if one attaches more ligands to a bionanoparticle. We envision that the bionanoparticle desorption will not depend on density changes from either ligand or receptor when the number of bonds reaches a specific efficient value.

Bottom-Up Synthesis of Artificial Cells: Recent Highlights and Future Challenges.

The bottom-up approach in synthetic biology aims to create molecular ensembles that reproduce the organization and functions of living organisms and strives to integrate them in a modular and hierarchical fashion toward the basic unit of life-the cell-and beyond. This young field stands on the shoulders of fundamental research in molecular biology and biochemistry, next to synthetic chemistry, and, augmented by an engineering framework, has seen tremendous progress in recent years thanks to multiple technological and scientific advancements. In this timely review of the research over the past decade, we focus on three essential features of living cells: the ability to self-reproduce via recursive cycles of growth and division, the harnessing of energy to drive cellular processes, and the assembly of metabolic pathways. In addition, we cover the increasing efforts to establish multicellular systems via different communication strategies and critically evaluate the potential applications.

En route to dynamic life processes by SNARE-mediated fusion of polymer and hybrid membranes.

A variety of artificial cells springs from the functionalization of liposomes with proteins. However, these models suffer from low durability without repair and replenishment mechanisms, which can be partly addressed by replacing the lipids with polymers. Yet natural membranes are also dynamically remodeled in multiple cellular processes. Here, we show that synthetic amphiphile membranes also undergo fusion, mediated by the protein machinery for synaptic secretion. We integrated fusogenic SNAREs in polymer and hybrid vesicles and observed efficient membrane and content mixing. We determined bending rigidity and pore edge tension as key parameters for fusion and described its plausible progression through cryo-EM snapshots. These findings demonstrate that dynamic membrane phenomena can be reconstituted in synthetic materials, thereby providing new tools for the assembly of synthetic protocells.

Light-Powered Reactivation of Flagella and Contraction of Microtubule Networks: Toward Building an Artificial Cell.

Artificial systems capable of self-sustained movement with self-sufficient energy are of high interest with respect to the development of many challenging applications, including medical treatments, but also technical applications. The bottom-up assembly of such systems in the context of synthetic biology is still a challenging task. In this work, we demonstrate the biocompatibility and efficiency of an artificial light-driven energy module and a motility functional unit by integrating light-switchable photosynthetic vesicles with demembranated flagella. The flagellar propulsion is coupled to the beating frequency, and dynamic ATP synthesis in response to illumination allows us to control beating frequency of flagella in a light-dependent manner. In addition, we verified the functionality of light-powered synthetic vesicles in in vitro motility assays by encapsulating microtubules assembled with force-generating kinesin-1 motors and the energy module to investigate the dynamics of a contractile filamentous network in cell-like compartments by optical stimulation. Integration of this photosynthetic system with various biological building blocks such as cytoskeletal filaments and molecular motors may contribute to the bottom-up synthesis of artificial cells that are able to undergo motor-driven morphological deformations and exhibit directional motion in a light-controllable fashion.

Directed Growth of Biomimetic Microcompartments.

Contemporary biological cells are sophisticated and highly compartmentalized. Compartmentalization is an essential principle of prebiotic life as well as a key feature in bottom-up synthetic biology research. In this review, the dynamic growth of compartments as an essential prerequisite for enabling self-reproduction as a fundamental life process is discussed. The micrometer-sized compartments are focused on due to their cellular dimensions. Two types of compartments are considered, membraneless droplets and membrane-bound microcompartments. Growth mechanisms of aqueous droplets such as protein (condensates) or macromolecule-rich droplets (aqueous two phase systems) and coacervates are discussed, for which growth occurs via Ostwald ripening or coalescence. For membrane-bound compartments, vesicles are considered, which are composed of fatty acids, lipids, or polymers, where directed growth can occur via fusion or uptake of material from the surrounding. The development of novel approaches for growth of biomimetic microcompartments can eventually be utilized to construct new synthetic cells.

Artificial Organelles for Energy Regeneration.

One of the critical steps in sustaining life-mimicking processes in synthetic cells is energy, i.e., adenosine triphosphate (ATP) regeneration. Previous studies have shown that the simple addition of ATP or ATP regeneration systems, which do not regenerate ATP directly from ADP and Pi , have no or only limited success due to accumulation of ATP hydrolysis products. In general, ATP regeneration can be achieved by converting light or chemical energy into ATP, which may also involve redox transformations of cofactors. The present contribution provides an overview of the existing ATP regeneration strategies and the related nicotinamide adenine dinucleotide (NAD+ ) redox cycling, with a focus on compartmentalized systems. Special attention is being paid to those approaches where so-called artificial organelles are developed. They comprise a semipermeable membrane functionalized by biological or man-made components and employ external energy in the form of light or nutrients in order to generate a transmembrane proton gradient, which is further utilized for ATP synthesis.

Determination of cluster composition in heteroaggregation of binary particle systems by flow cytometry.

Cluster composition in aggregation processes of multiple particle species can be dynamically determined by flow cytometry if particle populations are fluorescently labeled. By flow cytometric single particle analysis, aggregates can be characterized according to the exact amount of constituent particles, allowing the detailed and separate quantification of homo- and heteroaggregation. This contribution demonstrates the application of flow cytometry for the experimental detection of heteroaggregation in a binary particle mixture of oppositely charged polystyrene (PS) particles and Rhodamine-B labeled melamine-formaldehyde (MF-RhB) particles. Experiments with different particle concentration, temperature, mixing mode, ionic strength and particle mixing ratio are presented. Aggregation kinetics are enhanced with increasing particle concentration and temperature as well as by increased shear of mixing. These results represent well-known behavior published in previous investigations and validate the performance of flow cytometry for probing heteroaggregation processes. Physical insight with a novel level of detail is gained by the quantification of de- and restabilization phenomena. At low ionic strength, "raspberry"-type aggregates with PS cores are formed by primary heteroaggregation. At moderate particle number ratios, these aggregates are electrostatically destabilized and form more complex aggregates in a secondary heteroaggregation process. At high particle number ratios (> or =50:1), the raspberry-type aggregates are electrostatically restabilized and secondary heteroaggregation is prevented. The dynamic change of aggregate charge was verified by zeta-potential measurements. The elevation of salt concentration over several orders of magnitude retards aggregation dynamics, since attractive interparticle forces are diminished by an electrostatic double layer. This indicates that heteroaggregation induced by attractive interparticle forces is faster than aggregation due to random Brownian motion. Destabilization at high ionic strength is facilitated by charged ions and no longer by MF-RhB coverage. This results in a species independent one step aggregation process.