Biology subjects

Schwille, P.

Publications and source records attributed to Schwille, P..

At least 19 recordsLinked to original sources

bioRxiv2026

DNA condensate-based organelles for spatially regulated gene expression and protein targeting in synthetic cells

Spatial organization of gene expression is a key feature of cells but remains a major challenge in bottom-up synthetic biology. While phase-separated DNA condensates have been used to spatially confine transcription, achieving efficient recruitment of protein-coding DNA for full protein biosynthesis within these membrane-less structures has remained a major challenge. Here, we present a modular DNA nanostructure that enables tunable and highly efficient partitioning of long client DNA into condensate-based synthetic nuclei, thereby surpassing present limitations. This serves as the starting point for a multimodal DNA organelle-based system for spatially regulated gene expression in synthetic cell environments. The flow of information includes localized transcription within this synthetic nucleus, followed by translation and product release into the surrounding cytosol. Furthermore, we extend the toolkit of spatiotemporal organization by designing protein targeting of cell-cycle protein ParR within parC-enriched orthogonal DNA condensates. Quantitative analysis reveals a trend toward higher protein yield in the condensate-based system compared to standard PURE expression, indicating a functional advantage of spatial organization even in such minimal systems. Hence, we demonstrate how protein expression can be engineered not only by molecular composition, but by spatial architecture itself. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=123 SRC="FIGDIR/small/728287v1_ufig1.gif" ALT="Figure 1"> View larger version (16K): org.highwire.dtl.DTLVardef@1db026aorg.highwire.dtl.DTLVardef@17bb05dorg.highwire.dtl.DTLVardef@177ca3aorg.highwire.dtl.DTLVardef@17dcf9f_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2026

Automated Synthetic Cell-based Screening for Designed Proteins with Emergent Functions

Designing minimal biological systems with emergent functions such as spatiotemporal self-organization is a central goal of bottom-up synthetic biology. While computational optimization and design show promises to accelerate functional protein engineering through Design-Build-Test-Learn cycles, screening libraries for complex functions remains a major challenge. Conventional screens typically lack the spatiotemporal resolution and cell-like confinement required in bottom-up synthetic biology. Here, we present PUREdrop, an automated microfluidic platform that encapsulates and expresses protein libraries in thousands of picolitre-sized synthetic cells per construct. These droplets are sorted into a 96-well plate and analyzed by time-lapse imaging, allowing parallel quantification of expression kinetics and emergent functions. To demonstrate the platforms potential, we first screened computationally re-designed variants of the bacterial cell division protein FtsZ, identifying variants with improved bundling phenotypes and faster kinetics. We then extended our screening procedure towards general protein modulators of FtsZ and identified a combination that anchors filaments to the interface, producing a ring-like phenotype. PUREdrop bridges computational protein engineering and synthetic cell research, elevating the rational engineering of complex biological function to the next level.

synthetic biology↗

bioRxiv2026

Co-transcriptional Phase Separation of Nucleic Acids at Membrane Surfaces

Transcription is usually framed as information transfer, yet it also injects a new polymer into a crowded, confined environment. Here we demonstrate how spatial confinement to surfaces in a minimal membrane-bound transcription (MBT) system displays the physical consequences of RNA synthesis. Within a dense membrane-tethered DNA network, transcription drives co-transcriptional RNA phase separation: nascent RNA oligomerizes, gels and demixes from a surrounding fluid DNA phase, generating stable spatial patterns while mechanically remodeling the DNA layer. RNA gelation sequesters T7 RNA polymerase, whereas RNA-binding and translation-associated factors reverse gelation and restore fluidity. Thus, in the absence of downstream regulatory machinery, transcription under confinement is sufficient to trigger RNA condensation and nucleic-acid phase separation. The membrane as confining interface catalyzes the onset of DNA-RNA demixing and modulates the morphology of the resulting patterns. Since such large-scale spatial unmixing may be detrimental to cellular physiology, we suggest that one fundamental role of translation is to actively prevent condensation effects created by continuous RNA production.

biochemistry↗

bioRxiv2024

Mutual dependence between membrane phase separation and bacterial division protein dynamics in synthetic cell models

Cell membranes in bacteria are laterally polarized to produce specific environments for membrane proteins, e.g., proteins involved in cell division which accumulate at mid-cell or the cell poles. An interesting result of such membrane-lipid interplay is the reorganization of lipid domains together with membrane-bound proteins at the onset of cell division, suggesting a functional significance of membrane compartments in the cell cycle. Here, by adopting the key bacterial division proteins MinCDE and FtsZ as an archetypal spatial patterning system, we present a simple vesicle-based in vitro model to explore the mutual dependence of protein pattern formation and membrane heterogeneity. Like many other peripheral membrane proteins, MinDE exhibit preferential binding and macro-scale pattern formation at Ld domains, which leads to altered oscillation mode selection in phase-separated membrane compartments (GUVs). Moreover, incorporating bacterial division proteins within phase-separated GUVs leads to blebbing-like membrane deformations followed by the reorganization of Lo domains aligning at the neck region of the bleb, which agrees well with the domain rearrangement in bacterial membranes immediately preceding the radial constriction process. Overall, the presented in vitro model system showcases a basic framework to better comprehend the cellular division mechanism in consideration of complex cellular lipid environments.

synthetic biology↗

bioRxiv2024

Self-organized spatial targeting of contractile actomyosin rings for synthetic cell division

One of the challenges of bottom-up synthetic biology is the engineering of a minimal module for self-division of synthetic cells. To produce the contractile forces required for the controlled excision of cell-like compartments such as giant unilamellar vesicles (GUVs), reconstituted cytokinetic rings made of actin are considered to be among the most promising structures of a potential synthetic division machinery. Although the targeting of actin rings to GUV membranes and their myosin-induced constriction have been previously demonstrated, large-scale vesicle deformation has been precluded due to the lacking spatial control of these contractile structures. Here, we show the combined in vitro reconstitution of actomyosin rings and the bacterial MinDE protein system, effective in targeting E.coli Z-rings to mid-cell, within GUVs. Incorporating this spatial positioning tool, which induces active transport of any diffusible molecule on membranes, yields self-organized assembly of actomyosin rings at the equatorial plane of vesicles. Remarkably, the synergistic effect of Min oscillations and the contractile nature of actomyosin bundles induces mid-vesicle membrane deformation and striking bleb-like protrusions, leading to shape remodeling and symmetry breaking. Our system showcases how functional machineries from various organisms may be synergistically combined in vitro, leading to the emergence of new functionality towards a synthetic division system.

synthetic biology↗

bioRxiv2024

On the annotation of Paleo- and Mesoproterozoic microfossils as fungi

How old is the crown group Fungi? Inferences from phylogenetic and fossil-based studies provided far-apart age estimates ranging between 0.75 to 2.7 billion years old. One important criterion for interpreting Paleo- and Mesoproterozoic microfossils as Fungi is their uncanny morphological resemblance with extant fungi. Here, we demonstrate that bacteria exposed to environmental conditions similar to the paleoenvironmental settings where these presumed fungi lived can spontaneously transform into their protoplasts. These protoplasts exhibit morphologies corresponding to those of presumed fungal microfossils. These observations, together with microfossil chemical composition, pose a serious challenge to interpreting Paleo- and Mesoproterozoic microfossils as fungi. Based on these results, we reiterate that morphology is not a reliable indicator of the phylogeny of microfossils older than 2.5Ga.

evolutionary biology↗

bioRxiv2023

Fine-tuning of Fgf8 morphogen gradient by heparan sulfate proteoglycans in the extracellular matrix

Embryonic development is orchestrated by the action of morphogens, which spread out from a local source and activate, in a field of target cells, different cellular programs based on their concentration gradient. Fibroblast growth factor 8 (Fgf8) is a morphogen with important functions in embryonic organizing centers. It forms a gradient in the extracellular space by free diffusion, interaction with the extracellular matrix (ECM) and receptor-mediated endocytosis. However, morphogen gradient regulation by ECM is still poorly understood. Here we show that specific Heparan Sulfate Proteoglycans (HSPGs) bind Fgf8 directly in the ECM of living zebrafish embryos, thus affecting its diffusion and signaling. Using single-molecule Fluorescence Correlation Spectroscopy, we quantify this binding in vivo, and find two different modes of interaction. First, reducing or increasing the concentration of specific HSPGs in the extracellular space alters Fgf8 diffusion, and thus, its gradient shape. Second, ternary complex formation of Fgf8 ligand with Fgf-receptors and HSPGs at the cell surface requires HSPG attachment to the cell membrane. Together, our results show that graded Fgf8 morphogen distribution is achieved by constraining free Fgf8 diffusion through successive interactions with HSPGs at the cell surface and in ECM space. Statement of significanceFgf8 is a secreted morphogen signaling molecule that instructs neighboring arrays of undifferentiated cells about their position and cellular identity in tissue. Fgf8 and other morphogens are often distributed in a graded fashion, and can typically work at very low concentrations. To reproducibly generate information in developing tissue, mechanisms have evolved to carefully control distribution and concentration of Fgf8 morphogen. We show that freely diffusing Fgf8 morphogen moves through interstitial cell spaces on its way to target cells, and while doing so, interacts with ECM molecules in these spaces and at cell surfaces via low affinity, reversible binding. These interactions are important tuning mechanisms that contribute to forming the Fgf8 morphogen gradient and to cell surface receptor binding, and thus, to controlling cell type identity. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=166 SRC="FIGDIR/small/565243v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@b6d073org.highwire.dtl.DTLVardef@2f64c1org.highwire.dtl.DTLVardef@17fbf19org.highwire.dtl.DTLVardef@1db334a_HPS_FORMAT_FIGEXP M_FIG C_FIG Graphical abstractTo generate a gradient, the morphogen Fgf8 shuttles between fast free diffusion through extracellular space (i), slow diffusion or immobility (ii, iii) when bound to Heparan Sulfate Proteoglycans (HSPGs), in the extracellular matrix (ECM) and at cell surface receptors (iv), as revealed by single molecule studies in living zebrafish embryos.

developmental biology↗

bioRxiv2023

Nanoscale structural response of biomimetic cell membranes to controlled dehydration.

Although cell membranes in physiological conditions exist in excess of water, there is 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 the lipid membranes when their interfacial hydration layer is perturbed. Here, we introduce the study on the nanoscale structural reorganization of the phase-separated, supported lipid bilayers (SLBs) under a wide range of hydration conditions. Model lipid membranes were characterized with the combination of fluorescence microscopy and atomic force microscopy, and crucially, without applying any chemical or physical modifications, that so far have been considered to be indispensable for maintaining the membrane integrity upon dehydration. We revealed that decreasing hydration state of the membrane leads to an enhanced mixing of lipids characteristic for the liquid-disordered (Ld) phase with those forming 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 of line tension for the completely desiccated membrane. Importantly, the observed changes in the hydrophobic mismatch, line tension, and miscibility of lipids are fully reversible upon subsequent rehydration of the membrane. These findings provide deeper insights into the fundamental processes such as cell-cell fusion that require partial dehydration at the interface of two membranes.

biophysics↗

bioRxiv2023

Membrane-induced 2D phase separation of focal adhesion proteins

Focal adhesions form liquid-like assemblies around activated integrin receptors at the plasma membrane. Made up of hundreds of proteins, focal adhesions are dynamic structures which can assemble and disassemble quickly, withstand strong actomyosin-applied forces, and form highly stable complexes. How they achieve these flexible characteristics is not well understood. Here, we use recombinant focal adhesion proteins to reconstitute the core structural machinery in vitro, with the goal of understanding the underlying protein dynamics and interactions. We observe liquid-liquid phase separation of the core focal adhesion proteins talin and vinculin for a spectrum of conditions and in combination with several interaction partners. Intriguingly, we show that membrane binding triggers phase separation of these proteins on the membrane, which in turn induces the enrichment of integrin in the clusters. We also introduce a novel experimental setup to probe talin-membrane interactions down to the single protein level. Our results suggest that membrane composition triggers condensate assembly at the membrane, a regulatory mechanism which could widely apply to membrane-localized biomolecular condensates and provide a pathway of how spatial organization of lipids within the membrane can couple into the cytosol.

cell biology↗

bioRxiv2023

Designing a protein with emergent function by combined in silico, in vitro and in vivo screening

Recently, utilization of machine learning (ML) based methods has led to astonishing progress in protein design and, thus, the design of new biological functionality. However, emergent functions that require higher-order molecular interactions, such as the ability to self-organize, are still extremely challenging to implement. Here, we describe a comprehensive in silico, in vitro, and in vitro screening pipeline (i3-screening) to develop and validate ML-designed artificial homologs of a bacterial protein that confers its role in cell division through the emergent function of spatiotemporal pattern formation. Moreover, we present complete substitution of a wildtype gene by an ML-designed artificial homolog 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.

synthetic biology↗

bioRxiv2023

Crosslinking by ZapD drives the assembly of short, discontinuous FtsZ filaments into ring-like structures in solution

Bacterial cell division relies on the Z ring, a cytoskeletal structure that acts as a scaffold for the assembly of the divisome. To date, the detailed mechanisms underlying the assembly and stabilization of the Z ring remain elusive. This study highlights the role of the FtsZ-associated protein (Zap) ZapD in the assembly and stabilization of Z-ring-like structures via filament crosslinking. Using cryo-electron tomography and biochemical analysis, we show that, at equimolar concentrations of ZapD and FtsZ, ZapD induces the formation of toroidal structures composed of short, curved FtsZ filaments that are crosslinked vertically, but also laterally and diagonally. At higher concentrations of ZapD, regularly spaced ZapD dimers crosslink FtsZ filaments from above, resulting in the formation of straight bundles. Despite the simplicity of this reconstituted system, these findings provide valuable insights into the structural organization and stabilization of the Z ring by Zap proteins in bacterial cells, revealing the key role of optimal crosslinking density and geometry in enabling filament curvature and ring formation.

biochemistry↗

bioRxiv2022

A phase-separated biomolecular condensate nucleates polymerization of the tubulin homolog FtsZ to spatiotemporally regulate bacterial cell division

Cell division is spatiotemporally precisely regulated, but the underlying mechanisms are incompletely understood. In the social, predatory bacterium Myxococcus xanthus, the PomX/PomY/PomZ proteins form a single large megadalton-sized complex that directly positions and stimulates cytokinetic ring formation by the tubulin homolog FtsZ. Here, we studied 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, suggesting a novel cell division site positioning mechanism in which the single PomY condensate enriches FtsZ to guide FtsZ-ring formation and division. PomY-nucleated FtsZ polymerization shares features with microtubule nucleation by biomolecular condensates in eukaryotes, supporting this mechanisms ancient origin.

cell biology↗

bioRxiv2022

DNA-PAINT single-particle tracking (DNA-PAINT-SPT) enables extended single-molecule 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-colour DNA-PAINT-SPT for quantifying protein dimerisation and tracking proteins in living cell membranes, demonstrating its improved performance over single-dye SPT.

biophysics↗

bioRxiv2022

One-pot synthesis of prenylated proteins utilizing E. coli cell-free expression

Bottom-up synthetic biology is a powerful tool for uncovering the mechanisms underlying vital biological processes, such as signaling and cell polarization. The core principle of reconstituting cellular functions in their minimal forms can be achieved through modular protein design. However, assembling multiple purified proteins into a functional and synchronized system remains a technical challenge. The fact that many regulatory proteins show direct or indirect membrane interactions further exacerbates the complications. Here, we introduce the Cell-Free prenylated Protein Synthesis (CFpPS) system which enables the production of prenylated proteins in a single reaction mix, through reconstituted prenylation machinery. Not only does the CFpPS system offer a fast and reliable method for producing solubilized prenylated proteins, but it can also produce the protein of interest directly in the vicinity of biomimetic membranes, thus enabling microscopy-based functional assessment. As proof of principle, we demonstrate synthesis and solubilization of various important signaling proteins from the Ras superfamily, as well as membrane binding and extraction of the key polarity regulator Cdc42. Furthermore, our method can be used to confer membrane affinity to any protein, simply by adding a 4-peptide motif to the C-terminus of the protein. In sum, the CFpPS system offers a versatile and effective platform for designing peripheral membrane proteins for synthetic biology applications.

synthetic biology↗

bioRxiv2022

Mass-Sensitive Particle Tracking to Characterize Membrane-Associated Macromolecule Dynamics

Short-lived or transient interactions of macromolecules at and with lipid membranes, an interface where a multitude of essential biological reactions take place, are inherently difficult to assess with standard biophysical methods. The introduction of mass-sensitive particle tracking (MSPT) constitutes an important step towards a thorough quantitative characterization of such processes. Technically, this was made possible through the advent of interferometric scattering microscopy (iSCAT)-based mass photometry (MP). When the background removal strategy is optimized to reveal the two-dimensional motion of membrane-associated particles, this technique allows the real-time analysis of both diffusion and molecular mass of unlabeled macromolecules on biological membranes. Here, a detailed protocol to perform and analyze mass-sensitive particle tracking of membrane-associated systems is described. Measurements performed on a commercial mass photometer achieve time resolution in the millisecond regime and, depending on the MP system, a mass detection limit down to 50 kDa. To showcase the potential of MSPT for the in-depth analysis of membrane-catalyzed macromolecule dynamics in general, results obtained for exemplary protein systems such as the native membrane interactor annexin V are presented. SUMMARYThis protocol describes an iSCAT-based image processing and single-particle tracking approach that enables the simultaneous investigation of the molecular mass and the diffusive behavior of macromolecules interacting with lipid membranes. Step-by-step instructions for sample preparation, mass-to-contrast conversion, movie acquisition, and post-processing are provided along-side directions to prevent potential pitfalls.

biophysics↗

bioRxiv2021

On the reproductive mechanism of Gram-negative protocells

Bacterial protoplasts are known to reproduce independently of canonical molecular biological processes. Their reproduction is shown to be mediated entirely by the physicochemical properties of cell constituents. However, the physiochemical properties of the cell constituents are influenced by the environmental conditions like salinity, salt composition, and mechanical stresses experienced by a cell in natural environments. The influence of such environmental conditions on protoplast reproduction is seldom investigated. Here, we studied protoplast reproduction in their native environmental conditions. Contrary to the previous perceptions of protoplasts reproducing in an erratic manner, cells in our study reproduced in a defined sequence of steps. The process of their reproduction can be explained by an interplay between intracellular metabolism, the physicochemical properties of cell constituents, and the nature of cations in the growth media. We observed a minimal leakage of intracellular constituents during protoplast reproduction, suggesting an efficient reproduction. However, the efficiency of reproduction is determined by the environmental conditions. Under favorable environmental conditions, protoplasts reproduce with nearly similar efficiency to cells that possess a cell wall. In short, here we demonstrate the simplest method of cellular reproduction and the influence of environmental conditions on this process.

biophysics↗

bioRxiv2021

On the reproductive mechanisms of Gram-positive protocells

Prokaryotes are hypothesized to have evolved from more primitive protocells. Unlike present-day cells, protocells are thought to have been devoid of complex molecular biological processes. They are believed to have mediated reproduction entirely by biophysical forces under favorable environmental conditions. Despite this proposition, little is known about the actual mechanism of their reproduction. To understand the reproduction process of protocells in their native habitat, here we used a top-down approach to transform bacterial cells into a primitive lipid vesicle-like state. Given that environmental conditions are thought to have played an essential role in mediating protocell reproduction, we then studied these cells under the presumed environmental conditions of Archaean Eon Earth. Even in the absence of functioning biological processes, cells in our study reproduced in a defined sequence of steps, always leading to the formation of viable daughter cells. Their reproduction mechanism can be explained by the interaction between intracellular metabolism, physicochemical properties of cell constituents, and, most importantly, environmental conditions. Given the simplicity of this reproduction mechanism and its suitability to environmental conditions of early Earth, we propose that protocells reproduced by this process. Moreover, this method of reproduction is also in tune with the earlier theoretical propositions on protocells, the results of the top- down approach of building a minimal cell, and the paleontological record of the Achaean Eon. Our study is the first to bridge the gap between non-living systems like lipid vesicles, living cells, and the paleontology of the Archaean Eon.

biophysics↗

bioRxiv2021

Structural Diversity of Photoswitchable Sphingolipids for Optodynamic Control of Lipid Raft 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 saturated lipids and cholesterol into lipid rafts; liquid-ordered (Lo) microdomains that act as organizing centers within biomembranes. Owing the vital role of sphingolipids for lipid segregation, controlling their lateral localization 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 (THP)-blocked sphingosine), able to shuttle between liquid-ordered (Lo) and liquid-disordered (Ld) regions of model membranes upon irradiation with UV-A ({lambda} = 365 nm) and blue ({lambda} = 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 photo-isomerization, notably in terms of domain area changes, height mismatch, line tension, and membrane piercing. Hereby, we show that all sphingosine-(Azo-{beta}-Gal-Cer, Azo-SM, Azo-Cer) and phytosphingosine-based (Azo--Gal-PhCer, Azo-PhCer) photolipids behave similarly, promoting a reduction in Lo domain area when in the UV-adapted cis-isoform. In contrast, azo-sphingolipids having THP groups that block H-bonding at the sphingosine backbone (Azo-THP-SM, Azo-THP-Cer) induce an increase in the Lo 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 Lo lipid raft domains.

biophysics↗