Measuring Partition Coefficients of In Vitro Biomolecular Condensates Using Fluorescence Correlation Spectroscopy.

Liquid-liquid phase separation is a widespread organizing principle of live cells, including, for example, the spatiotemporal organization of bacterial chromatin. The biophysics of phase-separating systems is often studied in vitro to avoid the complexity of the live-cell environment and facilitate application of advanced biophysical methods. One attractive method for measuring, e.g., partition coefficients, in such systems is fluorescence correlation spectroscopy (FCS). FCS circumvents some of the limitations of the widespread confocal laser scanning microscopy image-based measurements. Here, we describe how to perform partition coefficient measurements in biological phase-separating systems. Our protocol details typical workflows for the preparation of in vitro reconstituted condensates, FCS data acquisition, and subsequent data analysis, including corrections of some common artifacts. Our recommendations should help avoid many pitfalls of partition coefficient determination in these challenging systems.

Membrane-induced 2D phase separation of the focal adhesion protein talin.

Focal adhesions form liquid-like assemblies around activated integrin receptors at the plasma membrane. How they achieve their flexible properties is not well understood. Here, we use recombinant focal adhesion proteins to reconstitute the core structural machinery in vitro. We observe liquid-liquid phase separation of the core focal adhesion proteins talin and vinculin for a spectrum of conditions and interaction partners. Intriguingly, we show that binding to PI(4,5)P2-containing membranes triggers phase separation of these proteins on the membrane surface, which in turn induces the enrichment of integrin in the clusters. We suggest a mechanism by which 2-dimensional biomolecular condensates assemble on membranes from soluble proteins in the cytoplasm: lipid-binding triggers protein activation and thus, liquid-liquid phase separation of these membrane-bound proteins. This could explain how early focal adhesions maintain a structured and force-resistant organization into the cytoplasm, while still being highly dynamic and able to quickly assemble and disassemble.

Machine learning-aided design and screening of an emergent protein function in synthetic cells.

Recently, utilization of Machine Learning (ML) has led to astonishing progress in computational protein design, bringing into reach the targeted engineering of proteins for industrial and biomedical applications. However, the design of proteins for emergent functions of core relevance to cells, such as the ability to spatiotemporally self-organize and thereby structure the cellular space, is still extremely challenging. While on the generative side conditional generative models and multi-state design are on the rise, for emergent functions there is a lack of tailored screening methods as typically needed in a protein design project, both computational and experimental. Here we describe a proof-of-principle of how such screening, in silico and in vitro, can be achieved for ML-generated variants of a protein that forms intracellular spatiotemporal patterns. For computational screening we use a structure-based divide-and-conquer approach to find the most promising candidates, while for the subsequent in vitro screening we use synthetic cell-mimics as established by Bottom-Up Synthetic Biology. We then show that the best screened candidate can indeed completely substitute the wildtype gene in Escherichia coli. These results raise great hopes for the next level of synthetic biology, where ML-designed synthetic proteins will be used to engineer cellular functions.

Protein-Assisted Large-Scale Assembly and Differential Patterning of DNA Origami Lattices.

Nanofabrication has experienced a big boost with the invention of DNA origami, enabling the production and assembly of complex nanoscale structures that may be able to unlock fully new functionalities in biology and beyond. The remarkable precision with which these structures can be designed and produced is, however, not yet matched by their assembly dynamics, which can be extremely slow, particularly when attached to biological templates, such as membranes. Here, the rapid and controlled formation of DNA origami lattices on the scale of hundreds of micrometers in as little as 30 minutes is demonstrated, utilizing active patterning by the E.coli Min protein system, thereby yielding a remarkable improvement over conventional passive diffusion-based assembly methods. Various patterns, including spots, inverse spots, mazes, and meshes can be produced at different scales, tailored through the shape and density of the assembled structures. The differential positioning accomplished by Min-induced diffusiophoresis even allows the introduction of "pseudo-colors", i.e., complex core-shell patterns, by simultaneously patterning different DNA origami species. Beyond the targeted functionalization of biological surfaces, this approach may also be promising for applications in plasmonics, catalysis, and molecular sensing.

Light-Activated Synthetic Rotary Motors in Lipid Membranes Induce Shape Changes Through Membrane Expansion.

Membranes are the key structures to separate and spatially organize cellular systems. Their rich dynamics and transformations during the cell cycle are orchestrated by specific membrane-targeted molecular machineries, many of which operate through energy dissipation. Likewise, man-made light-activated molecular rotary motors have previously shown drastic effects on cellular systems, but their physical roles on and within lipid membranes remain largely unexplored. Here, the impact of rotary motors on well-defined biological membranes is systematically investigated. Notably, dramatic mechanical transformations are observed in these systems upon motor irradiation, indicative of motor-induced membrane expansion. The influence of several factors on this phenomenon is systematically explored, such as motor concentration and membrane composition., Membrane fluidity is found to play a crucial role in motor-induced deformations, while only minor contributions from local heating and singlet oxygen generation are observed. Most remarkably, the membrane area expansion under the influence of the motors continues as long as irradiation is maintained, and the system stays out-of-equilibrium. Overall, this research contributes to a comprehensive understanding of molecular motors interacting with biological membranes, elucidating the multifaceted factors that govern membrane responses and shape transitions in the presence of these remarkable molecular machines, thereby supporting their future applications in chemical biology.

Nanoscale structural response of biomimetic cell membranes to controlled dehydration.

Although cell membranes exist in excess of water under physiological conditions, there are a number of biochemical processes, such as adsorption of biomacromolecules or membrane fusion events, that require partial or even complete transient dehydration of lipid membranes. Even though the dehydration process is crucial for understanding all fusion events, still little is known about the structural adaptation of lipid membranes when their interfacial hydration layer is perturbed. Here, we present the study of the nanoscale structural reorganization of phase-separated, supported lipid bilayers (SLBs) under a wide range of hydration conditions. Model lipid membranes were characterised using a combination of fluorescence microscopy and atomic force microscopy and, crucially, without applying any chemical or physical modifications that have previously been considered essential for maintaining the membrane integrity upon dehydration. We revealed that decreasing the hydration state of the membrane leads to an enhanced mixing of lipids characteristic of the liquid-disordered (Ld) phase with those forming the liquid-ordered (Lo) phase. This is associated with a 2-fold decrease in the hydrophobic mismatch between the Ld and Lo lipid phases and a 3-fold decrease in the line tension for the fully desiccated membrane. Importantly, the observed changes in the hydrophobic mismatch, line tension, and lipid miscibility are fully reversible upon subsequent rehydration of the membrane. These findings provide a deeper insight into the fundamental processes, such as cell-cell fusion, that require partial dehydration at the interface of two membranes.

Dual-color DNA-PAINT single-particle tracking enables extended studies of membrane protein interactions.

DNA-PAINT based single-particle tracking (DNA-PAINT-SPT) has recently significantly enhanced observation times in in vitro SPT experiments by overcoming the constraints of fluorophore photobleaching. However, with the reported implementation, only a single target can be imaged and the technique cannot be applied straight to live cell imaging. Here we report on leveraging this technique from a proof-of-principle implementation to a useful tool for the SPT community by introducing simultaneous live cell dual-color DNA-PAINT-SPT for quantifying protein dimerization and tracking proteins in living cell membranes, demonstrating its improved performance over single-dye SPT.

Biomolecular condensate drives polymerization and bundling of the bacterial tubulin FtsZ to regulate cell division.

Cell division is spatiotemporally precisely regulated, but the underlying mechanisms are incompletely understood. In the social bacterium Myxococcus xanthus, the PomX/PomY/PomZ proteins form a single megadalton-sized complex that directly positions and stimulates cytokinetic ring formation by the tubulin homolog FtsZ. Here, we study the structure and mechanism of this complex in vitro and in vivo. We demonstrate that PomY forms liquid-like biomolecular condensates by phase separation, while PomX self-assembles into filaments generating a single large cellular structure. The PomX structure enriches PomY, thereby guaranteeing the formation of precisely one PomY condensate per cell through surface-assisted condensation. In vitro, PomY condensates selectively enrich FtsZ and nucleate GTP-dependent FtsZ polymerization and bundle FtsZ filaments, suggesting a cell division site positioning mechanism in which the single PomY condensate enriches FtsZ to guide FtsZ-ring formation and division. This mechanism shares features with microtubule nucleation by biomolecular condensates in eukaryotes, supporting this mechanism's ancient origin.

Protein-Based Patterning to Spatially Functionalize Biomimetic Membranes.

The bottom-up reconstitution of proteins for their modular engineering into synthetic cellular systems can reveal hidden protein functions in vitro. This is particularly evident for the bacterial Min proteins, a paradigm for self-organizing reaction-diffusion systems that displays an unexpected functionality of potential interest for bioengineering: the directional active transport of any diffusible cargo molecule on membranes. Here, the MinDE protein system is reported as a versatile surface patterning tool for the rational design of synthetically assembled 3D systems. Employing two-photon lithography, microswimmer-like structures coated with tailored lipid bilayers are fabricated and demonstrate that Min proteins can uniformly pattern bioactive molecules on their surface. Moreover, it is shown that the MinDE system can form stationary patterns inside lipid vesicles, which allow the targeting and distinctive clustering of higher-order protein structures on their inner leaflet. Given their facile use and robust function, Min proteins thus constitute a valuable molecular toolkit for spatially patterned functionalization of artificial biosystems like cell mimics and microcarriers.

Binding and Characterization of DNA Origami Nanostructures on Lipid Membranes.

DNA origami is an extremely versatile nanoengineering tool with widespread applicability in various fields of research, including membrane physiology and biophysics. The possibility to easily modify DNA strands with lipophilic moieties enabled the recent development of a variety of membrane-active DNA origami devices. Biological membranes, as the core barriers of the cells, display vital structural and functional roles. Therefore, lipid bilayers are widely popular targets of DNA origami nanotechnology for synthetic biology and biomedical applications. In this chapter, we summarize the typical experimental methods used to investigate the interaction of DNA origami with synthetic membrane models. Herein, we present detailed protocols for the production of lipid model membranes and characterization of membrane-targeted DNA origami nanostructures using different microscopy approaches.

Structural diversity of photoswitchable sphingolipids for optodynamic control of lipid microdomains.

Sphingolipids are a structurally diverse class of lipids predominantly found in the plasma membrane of eukaryotic cells. These lipids can laterally segregate with other rigid lipids and cholesterol into liquid-ordered domains that act as organizing centers within biomembranes. Owing the vital role of sphingolipids for lipid segregation, controlling their lateral organization is of utmost significance. Hence, we made use of the light-induced trans-cis isomerization of azobenzene-modified acyl chains to develop a set of photoswitchable sphingolipids with different headgroups (hydroxyl, galactosyl, phosphocholine) and backbones (sphingosine, phytosphingosine, tetrahydropyran-blocked sphingosine) that are able to shuttle between liquid-ordered and liquid-disordered regions of model membranes upon irradiation with UV-A (λ = 365 nm) and blue (λ = 470 nm) light, respectively. Using combined high-speed atomic force microscopy, fluorescence microscopy, and force spectroscopy, we investigated how these active sphingolipids laterally remodel supported bilayers upon photoisomerization, notably in terms of domain area changes, height mismatch, line tension, and membrane piercing. Hereby, we show that the sphingosine-based (Azo-ß-Gal-Cer, Azo-SM, Azo-Cer) and phytosphingosine-based (Azo-α-Gal-PhCer, Azo-PhCer) photoswitchable lipids promote a reduction in liquid-ordered microdomain area when in the UV-adapted cis-isoform. In contrast, azo-sphingolipids having tetrahydropyran groups that block H-bonding at the sphingosine backbone (lipids named Azo-THP-SM, Azo-THP-Cer) induce an increase in the liquid-ordered domain area when in cis, accompanied by a major rise in height mismatch and line tension. These changes were fully reversible upon blue light-triggered isomerization of the various lipids back to trans, pinpointing the role of interfacial interactions for the formation of stable liquid-ordered domains.

Insights into receptor structure and dynamics at the surface of living cells.

Evaluating protein structures in living cells remains a challenge. Here, we investigate Interleukin-4 receptor alpha (IL-4Rα) into which the non-canonical amino acid bicyclo[6.1.0]nonyne-lysine (BCNK) is incorporated by genetic code expansion. Bioorthogonal click labeling is performed with tetrazine-conjugated dyes. To quantify the reaction yield in situ, we develop brightness-calibrated ratiometric imaging, a protocol where fluorescent signals in confocal multi-color images are ascribed to local concentrations. Screening receptor mutants bearing BCNK in the extracellular domain uncovered site-specific variations of both click efficiency and Interleukin-4 binding affinity, indicating subtle well-defined structural perturbations. Molecular dynamics and continuum electrostatics calculations suggest solvent polarization to determine site-specific variations of BCNK reactivity. Strikingly, signatures of differential click efficiency, measured for IL-4Rα in ligand-bound and free form, mirror sub-angstrom deformations of the protein backbone at corresponding locations. Thus, click efficiency by itself represents a remarkably informative readout linked to protein structure and dynamics in the native plasma membrane.

Periodic temperature changes drive the proliferation of self-replicating RNAs in vesicle populations.

Growth and division of biological cells are based on the complex orchestration of spatiotemporally controlled reactions driven by highly evolved proteins. In contrast, it remains unknown how their primordial predecessors could achieve a stable inheritance of cytosolic components before the advent of translation. An attractive scenario assumes that periodic changes of environmental conditions acted as pacemakers for the proliferation of early protocells. Using catalytic RNA (ribozymes) as models for primitive biocatalytic molecules, we demonstrate that the repeated freezing and thawing of aqueous solutions enables the assembly of active ribozymes from inactive precursors encapsulated in separate lipid vesicle populations. Furthermore, we show that encapsulated ribozyme replicators can overcome freezing-induced content loss and successive dilution by freeze-thaw driven propagation in feedstock vesicles. Thus, cyclic freezing and melting of aqueous solvents - a plausible physicochemical driver likely present on early Earth - provides a simple scenario that uncouples compartment growth and division from RNA self-replication, while maintaining the propagation of these replicators inside new vesicle populations.

Spatiotemporal Propagation of a Minimal Catalytic RNA Network in GUV Protocells by Temperature Cycling and Phase Separation.

Compartmentalization is key to many cellular processes and a critical bottleneck of any minimal life approach. In cells, a complex chemistry is responsible for bringing together or separating biomolecules at the right place at the right time. Lipids, nucleic acids and proteins self-organize, thereby creating boundaries, interfaces and specialized microenvironments. Exploiting reversible RNA-based liquid-liquid phase separation (LLPS) inside giant unilamellar vesicles (GUVs), we present an efficient system capable of propagating an RNA-based enzymatic reaction across a population of GUVs upon freezing-thawing (FT) temperature cycles. We report that compartmentalization in the condensed RNA-rich phase can accelerate such an enzymatic reaction. In the decondensed state, RNA substrates become homogeneously dispersed, enabling content exchange between vesicles during freeze-thawing. This work explores how a minimal reversible phase separation system in lipid vesicles could help to implement spatiotemporal control in cyclic processes, as required for minimal cells.

Reconstitution of a Reversible Membrane Switch via Prenylation by One-Pot Cell-Free Expression.

Reversible membrane targeting of proteins is one of the key regulators of cellular interaction networks, for example, for signaling and polarization. So-called "membrane switches" are thus highly attractive targets for the design of minimal cells but have so far been tricky to reconstitute in vitro. Here, we introduce cell-free prenylated protein synthesis (CFpPS), which enables the synthesis and membrane targeting of proteins in a single reaction mix including the prenylation machinery. CFpPS can confer membrane affinity to any protein via addition of a 4-peptide motif to its C-terminus and offers robust production of prenylated proteins not only in their soluble forms but also in the direct vicinity of biomimetic membranes. Thus, CFpPS enabled us to reconstitute the prenylated polarity hub Cdc42 and its regulatory protein in vitro, implementing a key membrane switch. We propose CFpPS to be a versatile and effective platform for engineering complex features, such as polarity induction, in synthetic cells.

Asgard ESCRT-III and VPS4 reveal conserved chromatin binding properties of the ESCRT machinery.

The archaeal Asgard superphylum currently stands as the most promising prokaryotic candidate, from which eukaryotic cells emerged. This unique superphylum encodes for eukaryotic signature proteins (ESP) that could shed light on the origin of eukaryotes, but the properties and function of these proteins is largely unresolved. Here, we set to understand the function of an Asgard archaeal protein family, namely the ESCRT machinery, that is conserved across all domains of life and executes basic cellular eukaryotic functions, including membrane constriction during cell division. We find that ESCRT proteins encoded in Loki archaea, express in mammalian and yeast cells, and that the Loki ESCRT-III protein, CHMP4-7, resides in the eukaryotic nucleus in both organisms. Moreover, Loki ESCRT-III proteins associated with chromatin, recruited their AAA-ATPase VPS4 counterpart to organize in discrete foci in the mammalian nucleus, and directly bind DNA. The human ESCRT-III protein, CHMP1B, exhibited similar nuclear properties and recruited both human and Asgard VPS4s to nuclear foci, indicating interspecies interactions. Mutation analysis revealed a role for the N terminal region of ESCRT-III in mediating these phenotypes in both human and Asgard ESCRTs. These findings suggest that ESCRT proteins hold chromatin binding properties that were highly preserved through the billion years of evolution separating Asgard archaea and humans. The conserved chromatin binding properties of the ESCRT membrane remodeling machinery, reported here, may have important implications for the origin of eukaryogenesis.

MipZ caps the plus-end of FtsZ polymers to promote their rapid disassembly.

The spatiotemporal regulation of cell division is a fundamental issue in cell biology. Bacteria have evolved a variety of different systems to achieve proper division site placement. In many cases, the underlying molecular mechanisms are still incompletely understood. In this study, we investigate the function of the cell division regulator MipZ from Caulobacter crescentus, a P-loop ATPase that inhibits the polymerization of the treadmilling tubulin homolog FtsZ near the cell poles, thereby limiting the assembly of the cytokinetic Z ring to the midcell region. We show that MipZ interacts with FtsZ in both its monomeric and polymeric forms and induces the disassembly of FtsZ polymers in a manner that is not dependent but enhanced by the FtsZ GTPase activity. Using a combination of biochemical and genetic approaches, we then map the MipZ-FtsZ interaction interface. Our results reveal that MipZ employs a patch of surface-exposed hydrophobic residues to interact with the C-terminal region of the FtsZ core domain. In doing so, it sequesters FtsZ monomers and caps the (+)-end of FtsZ polymers, thereby promoting their rapid disassembly. We further show that MipZ influences the conformational dynamics of interacting FtsZ molecules, which could potentially contribute to modulating their assembly kinetics. Together, our findings show that MipZ uses a combination of mechanisms to control FtsZ polymerization, which may be required to robustly regulate the spatiotemporal dynamics of Z ring assembly within the cell.

Prebiotic Foam Environments to Oligomerize and Accumulate RNA.

When water interacts with porous rocks, its wetting and surface tension properties create air bubbles in large number. To probe their relevance as a setting for the emergence of life, we microfluidically created foams that were stabilized with lipids. A persistent non-equilibrium setting was provided by a thermal gradient. The foam's large surface area triggers capillary flows and wet-dry reactions that accumulate, aggregate and oligomerize RNA, offering a compelling habitat for RNA-based early life as it offers both wet and dry conditions in direct neighborhood. Lipids were screened to stabilize the foams. The prebiotically more probable myristic acid stabilized foams over many hours. The capillary flow created by the evaporation at the water-air interface provided an attractive force for molecule localization and selection for molecule size. For example, self-binding oligonucleotide sequences accumulated and formed micrometer-sized aggregates which were shuttled between gas bubbles. The wet-dry cycles at the foam bubble interfaces triggered a non-enzymatic RNA oligomerization from 2',3'-cyclic CMP and GMP which despite the small dry reaction volume was superior to the corresponding dry reaction. The found characteristics make heated foams an interesting, localized setting for early molecular evolution.

Regulating DNA-Hybridization Using a Chemically Fueled Reaction Cycle.

Molecular machines, such as ATPases or motor proteins, couple the catalysis of a chemical reaction, most commonly hydrolysis of nucleotide triphosphates, to their conformational change. In essence, they continuously convert a chemical fuel to drive their motion. An outstanding goal of nanotechnology remains to synthesize a nanomachine with similar functions, precision, and speed. The field of DNA nanotechnology has given rise to the engineering precision required for such a device. Simultaneously, the field of systems chemistry developed fast chemical reaction cycles that convert fuel to change the function of molecules. In this work, we thus combined a chemical reaction cycle with the precision of DNA nanotechnology to yield kinetic control over the conformational state of a DNA hairpin. Future work on such systems will result in out-of-equilibrium DNA nanodevices with precise functions.