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

Bottom-up assembly of target-specific cytotoxic synthetic cells

Immune vigilance ensures body integrity by eliminating malignant cells through the complex but coordinated cooperation of highly diversified lymphocytes populations. The sheer complexity of the immune system has slowed development of immunotherapies based on top-down genetic engineering of lymphocytes. In contrast, bottom-up assembly of synthetic cell compartments has contributed novel engineering strategies to reverse engineer and understand cellular phenomena as molecularly defined systems. Towards reducing the complexity of immunological systems, herein, a bottom-up approach for controlled assembly of fully-synthetic immune-inspired cells from predefined molecular components based on giant unilamellar vesicles is described. For construction of target-specific cytotoxic immune cells, the Fas-ligand-based apoptosis-inducing immune cell module is combined with an antibody-mediated cellular cytotoxicity-inspired system. The designed immune cells identify leukemia cells by specific surface antigens. Subsequently, they form stable attachments sites and eliminate their targets by induction of apoptosis. A structural and functional characterization of the synthetic immune cells by means of microfluidics, live cell, confocal and electron microscopy, dynamic light scattering as well as flow cytometry is presented. This study demonstrates the bioinspired construction of effector immune cells from defined molecular building blocks, enabling learning-by-building approaches in synthetic immunology

Capsid protein structure, self-assembly, and processing reveal morphogenesis of the marine virophage mavirus

Virophages have the unique property of parasitizing giant viruses within unicellular hosts. Little is understood about how they form infectious virions in this tripartite interplay. We provide mechanistic insights into assembly and maturation of mavirus, a marine virophage, by combining structural and stability studies on capsomers, virus-like particles (VLPs), and native virions. We found that the mavirus protease processes the double jelly-roll (DJR) major capsid protein (MCP) at multiple C-terminal sites and that these sites are conserved among virophages. Mavirus MCP assembled in Escherichia coli in the absence and presence of penton protein, forming VLPs with defined size and shape. While quantifying VLPs in E. coli lysates, we found that full-length rather than processed MCP is the competent state for capsid assembly. Full-length MCP was thermally more labile than truncated MCP, and crystal structures of both states indicate that full-length MCP has an expanded DJR core. Thus, we propose that the MCP C-terminal domain serves as a scaffolding domain by adding strain on MCP to confer assembly competence. Mavirus protease processed MCP more efficiently after capsid assembly, which provides a regulation mechanism for timing capsid maturation. By analogy to Sputnik and adenovirus, we propose that MCP processing renders mavirus particles infection competent by loosening interactions between genome and capsid shell and destabilizing pentons for genome release into host cells. The high structural similarity of mavirus and Sputnik capsid proteins together with conservation of protease and MCP processing suggest that assembly and maturation mechanisms described here are universal for virophages

One-Pot Assembly of Complex Giant Unilamellar Vesicle-Based Synthetic Cells

Here, we introduce a one-pot method for the bottom-up assembly of complex single- and multicompartment synthetic cells. Cellular components are enclosed within giant unilamellar vesicles (GUVs), produced at the milliliter scale directly from small unilamellar vesicles (SUVs) or proteoliposomes with only basic laboratory equipment within minutes. Toward this end, we layer an aqueous solution, containing SUVs and all biocomponents, on top of an oil–surfactant mix. Manual shaking induces the spontaneous formation of surfactant-stabilized water-in-oil droplets with a spherical supported lipid bilayer at their periphery. Finally, to release GUV-based synthetic cells from the oil and the surfactant shell into the physiological environment, we add an aqueous buffer and a droplet-destabilizing agent. We prove that the obtained GUVs are unilamellar by reconstituting the pore-forming membrane protein α-hemolysin and assess the membrane quality with cryotransmission electron microscopy (cryoTEM), fluorescence recovery after photobleaching (FRAP), and zeta-potential measurements as well as confocal fluorescence imaging. We further demonstrate that our GUV formation method overcomes key challenges of standard techniques, offering high volumes, a flexible choice of lipid compositions and buffer conditions, straightforward coreconstitution of proteins, and a high encapsulation efficiency of biomolecules and even large cargo including cells. We thereby provide a simple, robust, and broadly applicable strategy to mass-produce complex multicomponent GUVs for high-throughput testing in synthetic biology and biomedicine, which can directly be implemented in laboratories around the world

A new mouse model for the slow-channel congenital myasthenic syndrome induced by the AChR εL221F mutation

We have generated a new mouse model for congenital myasthenic syndromes by inserting the missense mutation L221F into the ? subunit of the acetylcholine receptor by homologous recombination. This mutation has been identified in man to cause a mild form of slow−channel congenital myasthenic syndrome with variable penetrance. In our mouse model we observe as in human patients prolonged endplate currents. The summation of endplate potentials may account for a depolarization block at increasing stimulus frequencies, moderate reduced muscle strength and tetanic fade. Calcium and intracellular vesicle accumulation as well as junctional fold loss and organelle degeneration underlying a typical endplate myopathy, were identified. Moreover, a remodelling of neuromuscular junctions occurs in a muscle−dependent pattern expressing variable phenotypic effects. Altogether, this mouse model provides new insight into the pathophysiology of congenital myasthenia and serves as a new tool for deciphering signalling pathways induced by excitotoxicity at peripheral synapses. Key words: Neuromuscular Junction, Slow Channel Congenital Myasthenic Syndrome, Acetylcholine Receptor, mouse model, homologous recombinatio

A 192-heme electron transfer network in the hydrazine dehydrogenase complex

Anaerobic ammonium oxidation (anammox) is a major process in the biogeochemical nitrogen cycle in which nitrite and ammonium are converted to dinitrogen gas and water through the highly reactive intermediate hydrazine. So far, it is unknown how anammox organisms convert the toxic hydrazine into nitrogen and harvest the extremely low potential electrons (-750 mV) released in this process. We report the crystal structure and cryo electron microscopy structures of the responsible enzyme, hydrazine dehydrogenase, which is a 1.7 MDa multiprotein complex containing an extended electron transfer network of 192 heme groups spanning the entire complex. This unique molecular arrangement suggests a way in which the protein stores and releases the electrons obtained from hydrazine conversion, the final step in the globally important anammox process