Synthetic Cells Research Articles

Membrane localization of actin filaments stabilizes giant unilamellar vesicles against external deforming forces

Actin organization is crucial for establishing cell polarity, which influences processes such as directed cell motility and division. Despite its critical role in living organisms, achieving similar polarity in synthetic cells remains challenging. In this study, we employ a bottom-up approach to investigate how molecular crowders facilitate the formation of cortex-like actin networks and how these networks localize and organize based on membrane shape. Using giant unilamellar vesicles (GUVs) as models for cell membranes, we show that actin filaments can arrange along the membrane to form cortex-like structures. Notably, this organization is achieved using only actin and crowders as a minimal set of components. We utilize surface micropatterning to examine actin filament organization in deformed GUVs adhered to various pattern shapes. Our findings indicate that at the periphery of spherical GUVs, actin bundles align along the membrane. However, in highly curved regions of adhered GUVs, actin bundles avoid crossing the highly curved edges perpendicular to the adhesion site and instead remain in the lower curved regions by aligning parallel to the micropatterned surface. Furthermore, the actin bundles increase the stiffness of the GUVs, effectively counteracting strong deformations when GUVs adhere to micropatterns. This finding is corroborated by real-time deformability cytometry on GUVs with synthetic actin cortices. By precisely manipulating the shape of GUVs, our study provides a minimal system to investigate the interplay between actin structures and the membrane. Our findings provide insights into the spatial organization of actin structures within crowded environments, specifically inside GUVs that resemble the size and shape of cells. This study advances our understanding of actin network organization and functionality within cell-sized compartments.

Synthetic Condensates and Cell-Like Architectures from Amphiphilic DNA Nanostructures.

Synthetic droplets and condensates are becoming increasingly common constituents of advanced biomimetic systems and synthetic cells, where they can be used to establish compartmentalization and sustain life-like responses. Synthetic DNA nanostructures have demonstrated significant potential as condensate-forming building blocks owing to their programmable shape, chemical functionalization, and self-assembly behavior. We have recently demonstrated that amphiphilic DNA "nanostars", obtained by labeling DNA junctions with hydrophobic moieties, constitute a particularly robust and versatile solution. The resulting amphiphilic DNA condensates can be programmed to display complex, multi-compartment internal architectures, structurally respond to various external stimuli, synthesize macromolecules, capture and release payloads, undergo morphological transformations, and interact with live cells. Here, we demonstrate protocols for preparing amphiphilic DNA condensates starting from constituent DNA oligonucleotides. We will address (i) single-component systems forming uniform condensates, (ii) two-component systems forming core-shell condensates, and (iii) systems in which the condensates are modified to support in vitro transcription of RNA nanostructures.

Chemomechanical Communication between Liposomes Based on Enzyme Cascades.

Communication between cells is crucial to the survival of both uni- and multicellular organisms. The primary mode of communication involves chemical cues. There is great current interest in mimicking this behavior in synthetic cells to understand the physical basis of intercellular communication and design collective functional behavior. Using liposomal cell mimics, we demonstrate how a chemical input can elicit a mechanical response (enhanced motility). We employed a single substrate to trigger enzyme cascade-induced control of the diffusion of up to three different liposome populations. Furthermore, substrate competition allows temporal control over enhanced diffusion. The use of enzyme cascades to propagate chemical signals provides a robust and efficient mechanism for diverse populations of protocells to coordinate their motion in response to signals from each other.

Artificial Receptor in Synthetic Cells Performs Transmembrane Activation of Proteolysis.

The designof artificial, synthetic cells is a fundamentally important and fast-developing field of science. Of the diverse attributes of cellular life, artificial transmembrane signaling across the biomolecular barriers remains a high challenge with only a few documented successes. Herein, the study achieves signaling across lipid bilayers and connects an exofacial enzymatic receptor activation to an intracellular biochemical catalytic response using an artificial receptor. The mechanism of signal transduction for the artificial receptor relies on the triggered decomposition of a self-immolative linker. Receptor activation ensues its head-to-tail decomposition and the release of a secondary messenger molecule into the internal volume of the synthetic cell. Transmembrane signaling is demonstrated in synthetic cells based on liposomes and mammalian cell-sized giant unilamellar vesicles and illustrates receptor performance in cell mimics with a diverse size and composition of the lipid bilayer. In giant unilamellar vesicles, transmembrane signaling connects exofacial receptor activation with intracellular activation of proteolysis. Taken together, the results of this study take a step toward engineering receptor-mediated, responsive behavior in synthetic cells.

DNA-empowered synthetic cells as minimalistic life forms.

Cells, the fundamental units of life, orchestrate intricate functions - motility, adaptation, replication, communication, and self-organization within tissues. Originating from spatiotemporally organized structures and machinery, coupled with information processing in signalling networks, cells embody the 'sensor-processor-actuator' paradigm. Can we glean insights from these processes to construct primitive artificial systems with life-like properties? Using de novo design approaches, what can we uncover about the evolutionary path of life? This Review discusses the strides made in crafting synthetic cells, utilizing the powerful toolbox of structural and dynamic DNA nanoscience. We describe how DNA can serve as a versatile tool for engineering entire synthetic cells or subcellular entities, and how DNA enables complex behaviour, including motility and information processing for adaptive and interactive processes. We chart future directions for DNA-empowered synthetic cells, envisioning interactive systems wherein synthetic cells communicate within communities and with living cells.

Soft Synthetic Cells with Mobile Membrane Ligands for Ex Vivo Expansion of Therapy-Relevant T Cell Phenotypes.

The expansion of T cells ex vivo is crucial for effective immunotherapy but currently limited by a lack of expansion approaches that closely mimic in vivo T cell activation. Taking inspiration from bottom-up synthetic biology, a new synthetic cell technology is introduced based on dispersed liquid-liquid phase-separated droplet-supported lipid bilayers (dsLBs) with tunable biochemical and biophysical characteristics, as artificial antigen presenting cells (aAPCs) for ex vivo T cell expansion. These findings obtained with the dsLB technology reveal three key insights: first, introducing laterally mobile stimulatory ligands on soft aAPCs promotes expansion of IL-4/IL-10 secreting regulatory CD8+ T cells, with a PD-1 negative phenotype, less prone to immune suppression. Second, it is demonstrated that lateral ligand mobility can mask differential T cell activation observed on substrates of varying stiffness. Third, dsLBs are applied to reveal a mechanosensitive component in bispecific Her2/CD3 T cell engager-mediated T cell activation. Based on these three insights, lateral ligand mobility, alongside receptor- and mechanosignaling, is proposed to be considered as a third crucial dimension for the design of ex vivo T cell expansion technologies.

SecYEG-mediated translocation in a model synthetic cell.

Giant unilamellar vesicles (GUVs) provide a powerful model compartment for synthetic cells. However, a key challenge is the incorporation of membrane proteins that allow for transport, energy transduction, compartment growth and division. Here, we have successfully incorporated the membrane protein complex SecYEG-the key bacterial translocase that is essential for the incorporation of newly synthesized membrane proteins-in GUVs. Our method consists of fusion of small unilamellar vesicles containing reconstituted SecYEG into GUVs, thereby forming SecGUVs. These are suitable for large-scale experiments while maintaining a high protein:lipid ratio. We demonstrate that incorporation of SecYEG into GUVs does not inhibit its translocation efficiency. Robust membrane protein functionalized proteo-GUVs are promising and flexible compartments for use in the formation and growth of synthetic cells.

Living cells and biological mechanisms as prototypes for developing chemical artificial intelligence

Artificial Intelligence (AI) is having a revolutionary impact on our societies. It is helping humans in facing the global challenges of this century. Traditionally, AI is developed in software or through neuromorphic engineering in hardware. More recently, a brand-new strategy has been proposed. It is the so-called Chemical AI (CAI), which exploits molecular, supramolecular, and systems chemistry in wetware to mimic human intelligence. In this work, two promising approaches for boosting CAI are described. One regards designing and implementing neural surrogates that can communicate through optical or chemical signals and give rise to networks for computational purposes and to develop micro/nanorobotics. The other approach concerns “bottom-up synthetic cells” that can be exploited for applications in various scenarios, including future nano-medicine. Both topics are presented at a basic level, mainly to inform the broader audience of non-specialists, and so favour the rise of interest in these frontier subjects.

Magnetic Modulation of Biochemical Synthesis in Synthetic Cells.

Synthetic cells can be constructed from diverse molecular components, without the design constraints associated with modifying 'living' biological systems. This can be exploited to generate cells with abiotic components, creating functionalities absent in biology. One example is magnetic responsiveness, the activation and modulation of encapsulated biochemical processes using a magnetic field, which is absent from existing synthetic cell designs. This is a critical oversight, as magnetic fields are uniquely bio-orthogonal, noninvasive, and highly penetrative. Here, we address this by producing artificial magneto-responsive organelles by coupling thermoresponsive membranes with hyperthermic Fe3O4 nanoparticles and embedding them in synthetic cells. Combining these systems enables synthetic cell microreactors to be built using a nested vesicle architecture, which can respond to alternating magnetic fields through in situ enzymatic catalysis. We also demonstrate the modulation of biochemical reactions by using different magnetic field strengths and the potential to tune the system using different lipid compositions. This platform could unlock a wide range of applications for synthetic cells as programmable micromachines in biomedicine and biotechnology.

Abstract 141: Lesion Site-specific Mass Spectrometry Analysis Of Human Carotid Plaques Reveals Strongest Association Of The Necrotic Core And Fibrous Cap Proteome With Plaque Stability

The spatial impact of the plaque proteome on atherosclerotic plaque stability remains largely unknown. In this study, we performed mass spectrometry-based proteomics of various carotid plaque subregions (media, necrotic core, fibrous cap), using guided laser capture micro-dissections (LCM) of FFPE tissue in a matched cohort of 112 patients undergoing carotid endarterectomy (CEA) to study the impact of the plaque proteome, not only on lesion stability, but also on subregional changes of expression. Further, we performed a histological analysis according to the AHA classification and combined the data with clinical information on risk factors, comorbidities, and patient symptoms. Our LCM mass spectrometry analysis yielded over 3000 proteins for each plaque subregion. Gene set enrichment analysis revealed a high abundance of extracellular proteins and vice versa for cellular proteins. Subregion analysis identified the necrotic core and the fibrous cap to display the most significant changes in proteins associated with the stability status, whereas the media proteome remained largely unaffected. Primary and secondary enrichment analysis revealed synthetic vascular smooth muscle cells as the major source of these proteins. In addition, we analyzed the serum proteome of more than 500 patients (including non-athero control patients) with overlap from the tissue cohort. We combined these with histological evaluation and a complete set of clinical data. We used these two datasets for developing tissue and serological markers associated with plaque stability. In summary, our approach utilized LCM mass spectrometry, histological evaluation, and clinical data to analyze the association of different lesion subregions on plaque stability. We are able to demonstrate a high through-put method to identify potential protein markers relevant to atherosclerotic plaque stability.

Double Cell-Like Objects Created from Synthetic DNA (Human Placenta) Crown Cells Using Monolaurin

Synthetic DNA crown cells (artificial cells) can be prepared in vitro using sphingosine (Sph)-DNA-adenosine-monolaurin compounds. These DNA crown cells can proliferate within egg whites in vivo. Synthetic DNA (E. coli) crown cells formed assemblies and cells proliferate after the addition of monolaurin and egg white and could be cultured on agar plates. In this study, it was examined whether such phenomena were observed in synthetic DNA (Human placenta) crown cells. In synthetic DNA (Human placenta) crown cells using monolaurin, several kinds of objects that may differ from original cells, were observed on the agar plates. Here, the microscopic characteristics of double cell-like objects are described.

Synthetic protein circuits for programmable control of mammalian cell death

Natural cell death pathways such as apoptosis and pyroptosis play dual roles: they eliminate harmful cells and modulate the immune system by dampening or stimulating inflammation. Synthetic protein circuits capable of triggering specific death programs in target cells could similarly remove harmful cells while appropriately modulating immune responses. However, cells actively influence their death modes in response to natural signals, making it challenging to control death modes. Here, we introduce naturally inspired "synpoptosis" circuits that proteolytically regulate engineered executioner proteins and mammalian cell death. These circuits direct cell death modes, respond to combinations of protease inputs, and selectively eliminate target cells. Furthermore, synpoptosis circuits can be transmitted intercellularly, offering a foundation for engineering synthetic killer cells that induce desired death programs in target cells without self-destruction. Together, these results lay the groundwork for programmable control of mammalian cell death.

Designer peptide–DNA cytoskeletons regulate the function of synthetic cells

The bottom-up engineering of artificial cells requires a reconfigurable cytoskeleton that can organize at distinct locations and dynamically modulate its structural and mechanical properties. Here, inspired by the vast array of actin-binding proteins and their ability to reversibly crosslink or bundle filaments, we have designed a library of peptide–DNA crosslinkers varying in length, valency and geometry. Peptide filaments conjoint through DNA hybridization give rise to tactoid-shaped bundles with tunable aspect ratios and mechanics. When confined in cell-sized water-in-oil droplets, the DNA crosslinker design guides the localization of cytoskeletal structures at the cortex or within the lumen of the synthetic cells. The tunable spatial arrangement regulates the passive diffusion of payloads within the droplets and complementary DNA handles allow for the reversible recruitment and release of payloads on and off the cytoskeleton. Heat-induced reconfiguration of peptide–DNA architectures triggers shape deformations of droplets, regulated by DNA melting temperatures. Altogether, the modular design of peptide–DNA architectures is a powerful strategy towards the bottom-up assembly of synthetic cells.

Cell-free Protein Synthesis System for Building Synthetic Cells.

The Cell-Free Protein Synthesis (CFPS) system has been widely employed to facilitate the bottom-up assembly of synthetic cells. It serves as the host for the core machinery of the Central Dogma, standing as an optimal chassis for the integration and assembly of diverse artificial cellular mimicry systems. Despite its frequent use in the fabrication of synthetic cells, establishing a tailored and robust CFPS system for a specific application remains a nontrivial challenge. In this methods paper, we present a comprehensive protocol for the CFPS system, routinely employed in constructing synthetic cells. This protocol encompasses key stages in the preparation of the CFPS system, including the cell extract, template preparation, and routine expression optimization utilizing a fluorescent reporter protein. Additionally, we show representative results by encapsulating the CFPS system within various micro-compartments, such as monolayer droplets, double-emulsion vesicles, and chambers situated atop supported lipid bilayers. Finally, we elucidate the critical steps and conditions necessary for the successful assembly of these CFPS systems in distinct environments. We expect that our approach will facilitate the establishment of good working practices among various laboratories within the continuously expanding synthetic cell community, thereby accelerating progress in the field of synthetic cell development.

Synthetic Vesicles for Sustainable Energy Recycling and Delivery of Building Blocks for Lipid Biosynthesis†.

ATP is a universal energy currency that is essential for life. l-Arginine degradation via deamination is an elegant way to generate ATP in synthetic cells, which is currently limited by a slow l-arginine/l-ornithine exchange. We are now implementing a new antiporter with better kinetics to obtain faster ATP recycling. We use l-arginine-dependent ATP formation for the continuous synthesis and export of glycerol 3-phosphate by including glycerol kinase and the glycerol 3-phosphate/Pi antiporter. Exported glycerol 3-phosphate serves as a precursor for the biosynthesis of phospholipids in a second set of vesicles, which forms the basis for the expansion of the cell membrane. We have therefore developed an out-of-equilibrium metabolic network for ATP recycling, which has been coupled to lipid synthesis. This feeder-utilizer system serves as a proof-of-principle for the systematic buildup of synthetic cells, but the vesicles can also be used to study the individual reaction networks in confinement.

Leveraging Active Learning to Establish Efficient In Vitro Transcription and Translation from Bacterial Chromosomal DNA.

Gene expression is a fundamental aspect in the construction of a minimal synthetic cell, and the use of chromosomes will be crucial for the integration and regulation of complex modules. Expression from chromosomes in vitro transcription and translation (IVTT) systems presents limitations, as their large size and low concentration make them far less suitable for standard IVTT reactions. Here, we addressed these challenges by optimizing lysate-based IVTT systems at low template concentrations. We then applied an active learning tool to adapt IVTT to chromosomes as template DNA. Further insights into the dynamic data set led us to adjust the previous protocol for chromosome isolation and revealed unforeseen trends pointing at limiting transcription kinetics in our system. The resulting IVTT conditions allowed a high template DNA efficiency for the chromosomes. In conclusion, our system shows a protein-to-chromosome ratio that moves closer to in vivo biology and represents an advancement toward chromosome-based synthetic cells.

Programmable Enzymatic Reaction Network in Artificial Cell-Like Polymersomes.

The ability to precisely control in vitro enzymatic reactions in synthetic cells plays a crucial role in the bottom-up design of artificial cell models that can recapitulate the key cellular features and functions such as metabolism. However, integration of enzymatic reactions has been limited to bulk or microfluidic emulsions without a membrane, lacking the ability to design more sophisticated higher-order artificial cell communities for reconstituting spatiotemporal biological information at multiple length scales. Herein, droplet microfluidics is utilized to synthesize artificial cell-like polymersomes with distinct molecular permeability for spatiotemporal control of enzymatic reactions driven by external signals and fuels. The presence of a competing reverse enzymatic reaction that depletes the active substrates is shown to enable demonstration of fuel-driven formation of sub-microcompartments within polymersomes as well as realization of out-of-equilibrium systems. In addition, the different permeability characteristics of polymersome membranes are exploited to successfully construct a programmable enzymatic reaction network that mimics cellular communication within a heterogeneous cell community through selective molecular transport.

Sonogenetics-controlled synthetic designer cells for cancer therapy in tumor mouse models

Bacteria-based therapies are powerful strategies for cancer therapy, yet their clinical application is limited by a lack of tunable genetic switches to safely regulate the local expression and release of therapeutic cargoes. Rapid advances in remote-control technologies have enabled precise control of biological processes in time and space. We developed therapeutically active engineered bacteria mediated by a sono-activatable integrated gene circuit based on the thermosensitive transcriptional repressor TlpA39. Through promoter engineering and ribosome binding site screening, we achieved ultrasound (US)-induced protein expression and secretion in engineered bacteria with minimal noise and high induction efficiency. Specifically, delivered either intratumorally or intravenously, engineered bacteria colonizing tumors suppressed tumor growth through US-irradiation-induced release of the apoptotic protein azurin and an immune checkpoint inhibitor, a nanobody targeting programmed death-ligand 1, in different tumor mouse models. Beyond developing safe and high-performance designer bacteria for tumor therapy, our study illustrates a sonogenetics-controlled therapeutic platform that can be harnessed for bacteria-based precision medicine.

Microfluidically Produced Microcapsules with Amphiphilic Polymer Conetwork Shells

AbstractMicrocapsules with an aqueous core can be conveniently prepared by water‐in‐oil‐in‐water double emulsion microfluidics. However, conventional shell materials are based on hydrophobic polymers or colloidal particles. Thus, these microcapsules feature a hydrophobic shell impermeable to water‐soluble compounds. Capsules with semipermeable hydrogel shells have been demonstrated but may exhibit poor mechanical properties. Here, amphiphilic polymer conetworks (APCNs) based on poly(2‐hydroxyethyl acrylate)‐linked by‐polydimethylsiloxane (PHEA‐l‐PDMS) are introduced as a new class of wall materials in double emulsion microcapsules. These APCNs are mechanically robust silicone hydrogels that are swellable and permeable to water and are soft and elastic when dry or swollen. Therefore, the microcapsules can be dried and rehydrated multiple times or shrunken in sodium chloride salt solutions without getting damaged. Moreover, the APCNs are permeable for hydrophilic organic compounds and impermeable for macromolecules. Thus, they can be loaded with macromolecules or nanoparticles during microfluidic formation and with organic molecules after capsule synthesis. The microcapsules serve as microreactors for catalytically active platinum nanoparticles that decompose hydrogen peroxide. Finally, the surface of the APCN microcapsules can be selectively functionalized with a cholesterol‐based linker. Concluding, APCN microcapsules could find applications for the controlled delivery of drugs, as microreactors for synthesis, or as scaffolds for synthetic cells.

Building Synthetic Cells─From the Technology Infrastructure to Cellular Entities.

The de novo construction of a living organism is a compelling vision. Despite the astonishing technologies developed to modify living cells, building a functioning cell "from scratch" has yet to be accomplished. The pursuit of this goal alone has─and will─yield scientific insights affecting fields as diverse as cell biology, biotechnology, medicine, and astrobiology. Multiple approaches have aimed to create biochemical systems manifesting common characteristics of life, such as compartmentalization, metabolism, and replication and the derived features, evolution, responsiveness to stimuli, and directed movement. Significant achievements in synthesizing each of these criteria have been made, individually and in limited combinations. Here, we review these efforts, distinguish different approaches, and highlight bottlenecks in the current research. We look ahead at what work remains to be accomplished and propose a "roadmap" with key milestones to achieve the vision of building cells from molecular parts.