Enhancing electrospray ionization efficiency for particle transmission through an aerodynamic lens stack.

This work investigates the performance of the electrospray aerosol generator at the European X-ray Free Electron Laser (EuXFEL). This generator is, together with an aerodynamic lens stack that transports the particles into the X-ray interaction vacuum chamber, the method of choice to deliver particles for single-particle coherent diffractive imaging (SPI) experiments at the EuXFEL. For these experiments to be successful, it is necessary to achieve high transmission of particles from solution into the vacuum interaction region. Particle transmission is highly dependent on efficient neutralization of the charged aerosol generated by the electrospray mechanism as well as the geometry in the vicinity of the Taylor cone. We report absolute particle transmission values for different neutralizers and geometries while keeping the conditions suitable for SPI experiments. Our findings reveal that a vacuum ultraviolet ionizer demonstrates a transmission efficiency approximately seven times greater than the soft X-ray ionizer used previously. Combined with an optimized orifice size on the counter electrode, we achieve >40% particle transmission from solution into the X-ray interaction region. These findings offer valuable insights for optimizing electrospray aerosol generator configurations and data rates for SPI experiments.

Targeting STING with covalent small-molecule inhibitors.

Aberrant activation of innate immune pathways is associated with a variety of diseases. Progress in understanding the molecular mechanisms of innate immune pathways has led to the promise of targeted therapeutic approaches, but the development of drugs that act specifically on molecules of interest remains challenging. Here we report the discovery and characterization of highly potent and selective small-molecule antagonists of the stimulator of interferon genes (STING) protein, which is a central signalling component of the intracellular DNA sensing pathway1,2. Mechanistically, the identified compounds covalently target the predicted transmembrane cysteine residue 91 and thereby block the activation-induced palmitoylation of STING. Using these inhibitors, we show that the palmitoylation of STING is essential for its assembly into multimeric complexes at the Golgi apparatus and, in turn, for the recruitment of downstream signalling factors. The identified compounds and their derivatives reduce STING-mediated inflammatory cytokine production in both human and mouse cells. Furthermore, we show that these small-molecule antagonists attenuate pathological features of autoinflammatory disease in mice. In summary, our work uncovers a mechanism by which STING can be inhibited pharmacologically and demonstrates the potential of therapies that target STING for the treatment of autoinflammatory disease.

3D printed protein-based robotic structures actuated by molecular motor assemblies.

Upscaling motor protein activity to perform work in man-made devices has long been an ambitious goal in bionanotechnology. The use of hierarchical motor assemblies, as realized in sarcomeres, has so far been complicated by the challenges of arranging sufficiently high numbers of motor proteins with nanoscopic precision. Here, we describe an alternative approach based on actomyosin cortex-like force production, allowing low complexity motor arrangements in a contractile meshwork that can be coated onto soft objects and locally activated by ATP. The design is reminiscent of a motorized exoskeleton actuating protein-based robotic structures from the outside. It readily supports the connection and assembly of micro-three-dimensional printed modules into larger structures, thereby scaling up mechanical work. We provide an analytical model of force production in these systems and demonstrate the design flexibility by three-dimensional printed units performing complex mechanical tasks, such as microhands and microarms that can grasp and wave following light activation.

3D printed devices and infrastructure for liquid sample delivery at the European XFEL.

The Sample Environment and Characterization (SEC) group of the European X-ray Free-Electron Laser (EuXFEL) develops sample delivery systems for the various scientific instruments, including systems for the injection of liquid samples that enable serial femtosecond X-ray crystallography (SFX) and single-particle imaging (SPI) experiments, among others. For rapid prototyping of various device types and materials, sub-micrometre precision 3D printers are used to address the specific experimental conditions of SFX and SPI by providing a large number of devices with reliable performance. This work presents the current pool of 3D printed liquid sample delivery devices, based on the two-photon polymerization (2PP) technique. These devices encompass gas dynamic virtual nozzles (GDVNs), mixing-GDVNs, high-viscosity extruders (HVEs) and electrospray conical capillary tips (CCTs) with highly reproducible geometric features that are suitable for time-resolved SFX and SPI experiments at XFEL facilities. Liquid sample injection setups and infrastructure on the Single Particles, Clusters, and Biomolecules and Serial Femtosecond Crystallography (SPB/SFX) instrument are described, this being the instrument which is designated for biological structure determination at the EuXFEL.

Fresnel lens optical fiber tweezers to evaluate the vitality of single algae cells.

Dunaliella salina algae are trapped and studied using dual-fiber optical tweezers based on nano-imprinted Fresnel lenses. Different forms of cyclic motion of living algae inside the optical trap are observed and analyzed. A characteristic periodic motion in the 0-35 Hz frequency region reflects the algal flagella activity and is used to estimate the algal vitality, by photomovement. The trap stiffness and optical forces are measured for the case of a dead algal cell. It is shown that the dual-fiber optical tweezers can be used to study the vitality (or viability) property of single cells, a property that is essential and can be scaled up to other applications, such as sperm analysis for fertility tests.

Membrane-coated 3D architectures for bottom-up synthetic biology.

One of the great challenges of bottom-up synthetic biology is to recreate the cellular geometry and surface functionality required for biological reactions. Of particular interest are lipid membrane interfaces where many protein functions take place. However, cellular 3D geometries are often complex, and custom-shaping stable lipid membranes on relevant spatial scales in the micrometer range has been hard to accomplish reproducibly. Here, we use two-photon direct laser writing to 3D print microenvironments with length scales relevant to cellular processes and reactions. We formed lipid bilayers on the surfaces of these printed structures, and we evaluated multiple combinatorial scenarios, where physiologically relevant membrane compositions were generated on several different polymer surfaces. Functional dynamic protein systems were reconstituted in vitro and their self-organization was observed in response to the 3D geometry. This method proves very useful to template biological membranes with an additional spatial dimension, and thus allows a better understanding of protein function in relation to the complex morphology of cells and organelles.

Cell-free expression of RNA encoded genes using MS2 replicase.

RNA replicases catalyse transcription and replication of viral RNA genomes. Of particular interest for in vitro studies are phage replicases due to their small number of host factors required for activity and their ability to initiate replication in the absence of any primers. However, the requirements for template recognition by most phage replicases are still only poorly understood. Here, we show that the active replicase of the archetypical RNA phage MS2 can be produced in a recombinant cell-free expression system. We find that the 3' terminal fusion of antisense RNAs with a domain derived from the reverse complement of the wild type MS2 genome generates efficient templates for transcription by the MS2 replicase. The new system enables DNA-independent gene expression both in batch reactions and in microcompartments. Finally, we demonstrate that MS2-based RNA-dependent transcription-translation reactions can be used to control DNA-dependent gene expression by encoding a viral DNA-dependent RNA polymerase on a MS2 RNA template. Our study sheds light on the template requirements of the MS2 replicase and paves the way for new in vitro applications including the design of genetic circuits combining both DNA- and RNA-encoded systems.

Shaping Giant Membrane Vesicles in 3D-Printed Protein Hydrogel Cages.

Giant unilamellar phospholipid vesicles are attractive starting points for constructing minimal living cells from the bottom-up. Their membranes are compatible with many physiologically functional modules and act as selective barriers, while retaining a high morphological flexibility. However, their spherical shape renders them rather inappropriate to study phenomena that are based on distinct cell shape and polarity, such as cell division. Here, a microscale device based on 3D printed protein hydrogel is introduced to induce pH-stimulated reversible shape changes in trapped vesicles without compromising their free-standing membranes. Deformations of spheres to at least twice their aspect ratio, but also toward unusual quadratic or triangular shapes can be accomplished. Mechanical force induced by the cages to phase-separated membrane vesicles can lead to spontaneous shape deformations, from the recurrent formation of dumbbells with curved necks between domains to full budding of membrane domains as separate vesicles. Moreover, shape-tunable vesicles are particularly desirable when reconstituting geometry-sensitive protein networks, such as reaction-diffusion systems. In particular, vesicle shape changes allow to switch between different modes of self-organized protein oscillations within, and thus, to influence reaction networks directly by external mechanical cues.

CASP1 variants influence subcellular caspase-1 localization, pyroptosome formation, pro-inflammatory cell death and macrophage deformability.

CASP1 variants result in reduced enzymatic activity of procaspase-1 and impaired IL-1ß release. Despite this, affected individuals can develop systemic autoinflammatory disease. These seemingly contradictory observations have only partially been explained by increased NF-κB activation through prolonged interaction of variant procaspase-1 with RIP2. To identify further disease underlying pathomechanisms, we established an in vitro model using shRNA-directed knock-down of procaspase-1 followed by viral transduction of human monocytes (THP-1) with plasmids encoding for wild-type procaspase-1, disease-associated CASP1 variants (p.L265S, p.R240Q) or a missense mutation in the active center of procaspase-1 (p.C285A). THP1-derived macrophages carrying CASP1 variants exhibited mutation-specific molecular alterations. We here provide in vitro evidence for abnormal pyroptosome formation (p.C285A, p.240Q, p.L265S), impaired nuclear (pro)caspase-1 localization (p.L265S), reduced pro-inflammatory cell death (p.C285A) and changes in macrophage deformability that may contribute to disease pathophysiology of patients with CASP1 variants. This offers previously unknown molecular pathomechanisms in patients with systemic autoinflammatory disease.

Reconstitution and Coupling of DNA Replication and Segregation in a Biomimetic System.

A biomimetic system capable of replication and segregation of genetic material constitutes an essential component for the future design of a minimal synthetic cell. Here we have used the simple T7 bacteriophage system and the plasmid-derived ParMRC system to establish in vitro DNA replication and DNA segregation, respectively. These processes were incorporated into biomimetic compartments providing an enclosed reaction space. The functional lifetime of the encapsulated segregation system could be prolonged by equipping it with ATP-regenerating and oxygen-scavenging systems. Finally, we showed that DNA replication and segregation processes could be coupled in vitro by using condensed DNA nanoparticles resulting from DNA replication. ParM spindles extended over tens of micrometers and could thus be used for segregation in compartments that are significantly longer than bacterial cell size. Overall, this work demonstrates the successful bottom-up assembly and coupling of molecular machines that mediate replication and segregation, thus providing an important step towards the development of a fully functional minimal cell.

Light-Induced Printing of Protein Structures on Membranes in Vitro.

Reconstituting functional modules of biological systems in vitro is an important yet challenging goal of bottom-up synthetic biology, in particular with respect to their precise spatiotemporal regulation. One of the most desirable external control parameters for the engineering of biological systems is visible light, owing to its specificity and ease of defined application in space and time. Here we engineered the PhyB-PIF6 system to spatiotemporally target proteins by light onto model membranes and thus sequentially guide protein pattern formation and structural assembly in vitro from the bottom up. We show that complex micrometer-sized protein patterns can be printed on time scales of seconds, and the pattern density can be precisely controlled by protein concentration, laser power, and activation time. Moreover, when printing self-assembling proteins such as the bacterial cytoskeleton protein FtsZ, the targeted assembly into filaments and large-scale structures such as artificial rings can be accomplished. Thus, light mediated sequential protein assembly in cell-free systems represents a promising approach to hierarchically building up the next level of complexity toward a minimal cell.

Direct characterization of the evanescent field in objective-type total internal reflection fluorescence microscopy.

Total internal reflection fluorescence (TIRF) microscopy is a commonly used method for studying fluorescently labeled molecules in close proximity to a surface. Usually, the TIRF axial excitation profile is assumed to be single-exponential with a characteristic penetration depth, governed by the incident angle of the excitation laser beam towards the optical axis. However, in practice, the excitation profile does not only comprise the theoretically predicted single-exponential evanescent field, but also an additional non-evanescent contribution, supposedly caused by scattering within the optical path or optical aberrations. We developed a calibration slide to directly characterize the TIRF excitation field. Our slide features ten height steps ranging from 25 to 550 nanometers, fabricated from a polymer with a refractive index matching that of water. Fluorophores in aqueous solution above the polymer step layers sample the excitation profile at different heights. The obtained excitation profiles confirm the theoretically predicted exponential decay over increasing step heights as well as the presence of a non-evanescent contribution.

Spontaneous motion in hierarchically assembled active matter.

With remarkable precision and reproducibility, cells orchestrate the cooperative action of thousands of nanometre-sized molecular motors to carry out mechanical tasks at much larger length scales, such as cell motility, division and replication. Besides their biological importance, such inherently non-equilibrium processes suggest approaches for developing biomimetic active materials from microscopic components that consume energy to generate continuous motion. Being actively driven, these materials are not constrained by the laws of equilibrium statistical mechanics and can thus exhibit sought-after properties such as autonomous motility, internally generated flows and self-organized beating. Here, starting from extensile microtubule bundles, we hierarchically assemble far-from-equilibrium analogues of conventional polymer gels, liquid crystals and emulsions. At high enough concentration, the microtubules form a percolating active network characterized by internally driven chaotic flows, hydrodynamic instabilities, enhanced transport and fluid mixing. When confined to emulsion droplets, three-dimensional networks spontaneously adsorb onto the droplet surfaces to produce highly active two-dimensional nematic liquid crystals whose streaming flows are controlled by internally generated fractures and self-healing, as well as unbinding and annihilation of oppositely charged disclination defects. The resulting active emulsions exhibit unexpected properties, such as autonomous motility, which are not observed in their passive analogues. Taken together, these observations exemplify how assemblages of animate microscopic objects exhibit collective biomimetic properties that are very different from those found in materials assembled from inanimate building blocks, challenging us to develop a theoretical framework that would allow for a systematic engineering of their far-from-equilibrium material properties.

Beating Vesicles: Encapsulated Protein Oscillations Cause Dynamic Membrane Deformations.

The bacterial Min protein system was encapsulated in giant unilamellar vesicles (GUVs). Using confocal fluorescence microscopy, we identified several distinct modes of spatiotemporal patterns inside spherical GUVs. For osmotically deflated GUVs, the vesicle shape actively changed in concert with the Min oscillations. The periodic relocation of Min proteins from the vesicle lumen to the membrane and back is accompanied by drastic changes in the mechanical properties of the lipid bilayer. In particular, two types of oscillating membrane-shape changes are highlighted: 1) GUVs that repeatedly undergo fission into two connected compartments and fusion of these compartments back into a dumbbell shape and 2) GUVs that show periodic budding and subsequent merging of the buds with the mother vesicle, accompanied by an overall shape change of the vesicle reminiscent of a bouncing ball. These findings demonstrate how reaction-diffusion-based protein self-organization can directly yield visible mechanical effects on membrane compartments, even up to autonomous division, without the need for coupling to cytoskeletal elements.

Enzymatically Inactive Procaspase 1 stabilizes the ASC Pyroptosome and Supports Pyroptosome Spreading during Cell Division.

Caspase-1 is a key player during the initiation of pro-inflammatory innate immune responses, activating pro-IL-1ß in so-called inflammasomes. A subset of patients with recurrent febrile episodes and systemic inflammation of unknown origin harbor mutations in CASP1 encoding caspase-1. CASP1 variants result in reduced enzymatic activity of caspase-1 and impaired IL-1ß secretion. The apparent paradox of reduced IL-1ß secretion but systemic inflammation led to the hypothesis that CASP1 mutations may result in variable protein interaction clusters, thus activating alternative signaling pathways. To test this hypothesis, we established and characterized an in vitro system of transduced immortalized murine macrophages expressing either WT or enzymatically inactive (p.C284A) procaspase-1 fusion reporter proteins. Macrophages with variant p.C284A caspase-1 did not secrete IL-1ß and exhibited reduced inflammatory cell death, referred to as pyroptosis. Caspase-1 and apoptosis-associated speck-like protein containing a CARD (ASC) formed cytosolic macromolecular complexes (so-called pyroptosomes) that were significantly increased in number and size in cells carrying the p.C284A caspase-1 variant compared with WT caspase-1. Furthermore, enzymatically inactive caspase-1 interacted with ASC longer and with increased intensity compared with WT caspase-1. Applying live cell imaging, we documented for the first time that pyroptosomes containing enzymatically inactive variant p.C284A caspase-1 spread during cell division. In conclusion, variant p.C284A caspase-1 stabilizes pyroptosome formation, potentially enhancing inflammation by two IL-1ß-independent mechanisms: pyroptosomes convey an enhanced inflammatory stimulus through the recruitment of additional proteins (such as RIP2, receptor interacting protein kinase 2), which is further amplified through pyroptosome and cell division.

Post-sample aperture for low background diffraction experiments at X-ray free-electron lasers.

The success of diffraction experiments from weakly scattering samples strongly depends on achieving an optimal signal-to-noise ratio. This is particularly important in single-particle imaging experiments where diffraction signals are typically very weak and the experiments are often accompanied by significant background scattering. A simple way to tremendously reduce background scattering by placing an aperture downstream of the sample has been developed and its application in a single-particle X-ray imaging experiment at FLASH is demonstrated. Using the concept of a post-sample aperture it was possible to reduce the background scattering levels by two orders of magnitude.

Testing Turing's theory of morphogenesis in chemical cells.

Alan Turing, in "The Chemical Basis of Morphogenesis" [Turing AM (1952) Philos Trans R Soc Lond 237(641):37-72], described how, in circular arrays of identical biological cells, diffusion can interact with chemical reactions to generate up to six periodic spatiotemporal chemical structures. Turing proposed that one of these structures, a stationary pattern with a chemically determined wavelength, is responsible for differentiation. We quantitatively test Turing's ideas in a cellular chemical system consisting of an emulsion of aqueous droplets containing the Belousov-Zhabotinsky oscillatory chemical reactants, dispersed in oil, and demonstrate that reaction-diffusion processes lead to chemical differentiation, which drives physical morphogenesis in chemical cells. We observe five of the six structures predicted by Turing. In 2D hexagonal arrays, a seventh structure emerges, incompatible with Turing's original model, which we explain by modifying the theory to include heterogeneity.

Three-dimensional-printed gas dynamic virtual nozzles for x-ray laser sample delivery.

Reliable sample delivery is essential to biological imaging using X-ray Free Electron Lasers (XFELs). Continuous injection using the Gas Dynamic Virtual Nozzle (GDVN) has proven valuable, particularly for time-resolved studies. However, many important aspects of GDVN functionality have yet to be thoroughly understood and/or refined due to fabrication limitations. We report the application of 2-photon polymerization as a form of high-resolution 3D printing to fabricate high-fidelity GDVNs with submicron resolution. This technique allows rapid prototyping of a wide range of different types of nozzles from standard CAD drawings and optimization of crucial dimensions for optimal performance. Three nozzles were tested with pure water to determine general nozzle performance and reproducibility, with nearly reproducible off-axis jetting being the result. X-ray tomography and index matching were successfully used to evaluate the interior nozzle structures and identify the cause of off-axis jetting. Subsequent refinements to fabrication resulted in straight jetting. A performance test of printed nozzles at an XFEL provided high quality femtosecond diffraction patterns.

Human procaspase-1 variants with decreased enzymatic activity are associated with febrile episodes and may contribute to inflammation via RIP2 and NF-κB signaling.

The proinflammatory enzyme caspase-1 plays an important role in the innate immune system and is involved in a variety of inflammatory conditions. Rare naturally occurring human variants of the caspase-1 gene (CASP1) lead to different protein expression and structure and to decreased or absent enzymatic activity. Paradoxically, a significant number of patients with such variants suffer from febrile episodes despite decreased IL-1ß production and secretion. In this study, we investigate how variant (pro)caspase-1 can possibly contribute to inflammation. In a transfection model, such variant procaspase-1 binds receptor interacting protein kinase 2 (RIP2) via Caspase activation and recruitment domain (CARD)/CARD interaction and thereby activates NF-κB, whereas wild-type procaspase-1 reduces intracellular RIP2 levels by enzymatic cleavage and release into the supernatant. We approach the protein interactions by coimmunoprecipitation and confocal microscopy and show that NF-κB activation is inhibited by anti-RIP2-short hairpin RNA and by the expression of a RIP2 CARD-only protein. In conclusion, variant procaspase-1 binds RIP2 and thereby activates NF-κB. This pathway could possibly contribute to proinflammatory signaling.

ATP Consumption of Eukaryotic Flagella Measured at a Single-Cell Level.

The motility of cilia and flagella is driven by thousands of dynein motors that hydrolyze adenosine triphosphate (ATP). Despite decades of genetic, biochemical, structural, and biophysical studies, some aspects of ciliary motility remain elusive, such as the regulation of beating patterns and the energetic efficiency of these nanomachines. In this study, we introduce an experimental method to measure ATP consumption of actively beating axonemes on a single-cell level. We encapsulated individual sea urchin sperm with demembranated flagellum inside water-in-oil emulsion droplets and measured the axoneme's ATP consumption by monitoring fluorescence intensity of a fluorophore-coupled reporter system for ATP turnover in the droplet. Concomitant phase contrast imaging allowed us to extract a linear dependence between the ATP consumption rate and the flagellar beating frequency, with ∼2.3 × 10(5) ATP molecules consumed per beat of a demembranated flagellum. Increasing the viscosity of the aqueous medium led to modified beating waveforms of the axonemes and to higher energy consumption per beat cycle. Our single-cell experimental platform provides both new insights, to our knowledge, into the beating mechanism of flagella and a powerful tool for future studies.