Biology subjects

Koenderink, G. H.

Publications and source records attributed to Koenderink, G. H..

At least 19 recordsLinked to original sources

bioRxiv2026

Division of Synthetic Cells Using a Genomically Encoded One-Protein Divisome

Synthetic biology is emerging as a powerful tool to understand life by building minimal synthetic cells whose functions are encoded within their genome. An essential function of cells is abscission, the final step of cell division that breaks the last connection between two emerging daughter cells. Abscission is challenging to reconstitute as it is energetically unfavorable to rearrange the cell membrane. Here, we demonstrate a minimal one-component abscission machinery for synthetic cells using the bacterial protein dynamin A (DynA), which is uniquely capable of driving scission inside membrane necks. Cell-free expression of this synthetic cell module is complicated by the large size of DynA (137 kDa) and the presence of a hydrophobic lipid-binding loop within its structure. We overcome these challenges by expressing DynA inside lipid vesicles with a high fraction of negatively charged lipids. We find that DynA enriches at the bridges of dumbbell-shaped vesicles, where it drives membrane scission and full division. The ability to monitor DynA dynamics through genomic encoding further provides hints about the mechanism of abscission including cooperative enrichment at the necks and constriction of the necks. Finally, we demonstrate that the smaller (71 kDa) dynamin D1 domain alone exerts the same function, which is beneficial for future integration with other synthetic cell modules since its much smaller gene size reduces the burden on the synthetic genome. This autonomously operating abscission machinery presents the first example of a self-dividing synthetic cell, marking an important step towards constructing a synthetic cell that is able to sustainably replicate.

synthetic biology↗

bioRxiv2026

Septins promote breast cancer cell invasion in 3D collagen gels by influencing actin-based protrusion formation

Septins are cytoskeletal proteins that contribute to essential cellular processes such as cell migration and cell division through interactions with the cell membrane and the cytoskeleton. High expression of septins is correlated with breast cancer malignancy and promotes cell invasion, but the molecular complexity of septins interactions has made it challenging to dissect the underlying molecular mechanisms. Here, we used a conditional knockout approach to deplete SEPT7 in the metastatic triple-negative breast cancer cell line Hs578T and examined the role of septin in 3D-matrix invasion of breast cancer cells. We show by spheroid assays that SEPT7 deletion strongly impairs breast cancer cell invasion into collagen gels. Additional single-cell migration studies using 3D collagen gels and microfluidic pillar devices that mimic the pores present in collagen matrices showed that SEPT7 expression regulates confined cell migration through control of cell shape and actin-based protrusions.

cancer biology↗

bioRxiv2026

Quantitative biophysical analysis of human septin hexamer and octamer self-assembly on model membranes

1Septins are GTP-binding cytoskeletal proteins that shape and compartmentalize the plasma membrane. Their complex interactome has made it difficult to understand the molecular factors that govern their assembly. Moreover, it is unclear whether human septin hexamers and octamers form distinct higher-order assemblies, especially at the plasma membrane. Here, we address this question by using label-free methods to probe binding and self-assembly of recombinant human septins on supported lipid bilayers. Quartz crystal microbalance with dissipation (QCM-D) monitoring revealed that septin-membrane binding is diffusion-limited and concentration-dependent. Hexamers and octamers showed distinct viscoelastic properties, suggestive of structural differences. Imaging by atomic force microscopy (AFM) revealed that septin hexamers formed aligned nematic filamentous networks, whereas septin octamers formed aligned curved structures including spirals. QCM-D and AFM measurements both showed that septins form double-layered filament networks. However, upon C-terminal truncation of the SEPT6 and SEPT7 subunits, hexamers no longer bound the membrane while octamers formed a single-layered network of filament spirals. Our findings reveal that human septin hexamers and octamers interact differently with membranes, providing a baseline to understand their functions in the cell. 2 Significance statementO_LISeptins are cytoskeletal proteins that control cell membrane shape and stiffness. It is poorly understood how septin oligomers, the basic building blocks of septin filaments, bind and assemble on membranes. C_LIO_LIWe used label-free biophysical assays to quantitatively compare the binding kinetics and self-assembly behavior of recombinant human septin hexamers and octamers on supported lipid bilayer membranes. C_LIO_LIOur findings reveal that human septin hexamers and octamers both form organized filamentous networks on membranes, but with different structural properties that may potentially translate into different biological functions. C_LI

biophysics↗

bioRxiv2026

Lipid-conjugated DNA enables on-demand delivery of lipids and proteins to synthetic cells

The bottom-up construction of synthetic cells based on giant unilamellar vesicles (GUVs) is a central goal in synthetic biology. Achieving targeted changes in membrane and cytoplasmic composition with temporal control remains challenging however. DNA-mediated fusion with small vesicles ([~]100 nm large unilamellar vesicles; LUVs) has been proposed as a strategy to deliver lipids and cytosolic contents in a programmable manner. However, in vitro, membrane fusion is generally found to be inefficient and poorly controllable for reasons that are poorly understood. Here, we present an approach based on lipid-conjugated DNA (LiNA) to mediate programmable fusion between LUVs and micron-sized GUVs, which we quantitatively monitor with confocal microscopy at the single-GUV level. We show that lipid and content mixing both occur with high efficiency over a wide range of LiNA concentrations, demonstrating that LiNAs indeed induce robust membrane fusion. Furthermore, we show that LiNA-mediated fusion provides a powerful tool to deliver cytosolic biomolecules, enabling control over internal activities. Our findings establish a quantitative framework for studying fusion-driven processes in synthetic cells and provide a versatile platform for the programmable delivery of lipids and cytosolic cargoes - thus advancing the development of synthetic cells that can grow and adapt through fusion-based uptake of molecular building blocks.

synthetic biology↗

bioRxiv2025

Quantification of Cholesterol Incorporation in Giant Unilamellar Vesicles Produced by a Modified cDICE Method

AbstractCholesterol is an essential component of eukaryotic cell membranes, influencing membrane packing, fluidity, and domain formation. Replicating these properties in model membranes is critical for reconstitution studies, but common emulsion-based methods for producing giant unilamellar vesicles (GUVs) fail to incorporate cholesterol efficiently. Here, we use methyl-{beta}- cyclodextrin-cholesterol (M{beta}CD-CL) complexes to deliver cholesterol into GUVs produced by the emulsion droplet interface crossing encapsulation (eDICE) method and demonstrate a convenient way to quantify the degree of cholesterol incorporation using fluorescent membrane biosensors. Spectral imaging of NR12A as well as fluorescence lifetime imaging of Flipper-TR revealed dose- dependent increases in cholesterol content for DOPC GUVs upon M{beta}CD-CL addition, consistent with increased membrane order. By calibrating these effects against GUVs with defined cholesterol contents prepared via gel-assisted swelling, we found that the cholesterol content of eDICE vesicles can be increased to at least 40 mol%. Binary mixtures of DOPC with saturated lipids (DMPC and PC(18:0-14:0)) showed a similar trend as pure DOPC GUVs. Interestingly, we could trigger liquid-ordered domain formation by adding cholesterol to DOPC:DMPC vesicles. Our findings provide a quantitative and non-disruptive method to modulate and assess cholesterol content in emulsion-based GUVs, advancing their use in bottom-up synthetic biology and membrane biophysics.

biophysics↗

bioRxiv2025

Plectin affects cell viscoelasticity at small and large deformations

Plectin is a giant protein of the plakin family that crosslinks the cytoskeleton of mammalian cells. It is expressed in virtually all tissues and its dysfunction is associated with various diseases such as skin blistering. There is evidence that plectin regulates the mechanical integrity of the cytoskeleton in diverse cell and tissue types. However, it is unknown how plectin modulates the mechanical response of cells depending on the frequency and amplitude of mechanical loading. Here we demonstrate the role of plectin in the viscoelastic properties of fibroblasts at small and large deformations by quantitative single-cell compression measurements. To identify the importance of plectin, we compared the mechanical properties of wild type (Plec+/+) fibroblasts and plectin knockout (Plec-/-) fibroblasts. We show that plectin knockout cells are nearly 2-fold softer than wild type cells, but their strain-stiffening behaviour is similar. Plectin deficiency also caused faster viscoelastic stress relaxation at long times. Fluorescence recovery after photobleaching experiments indicated that this was due to 3-fold faster actin turnover. Short-time poroelastic relaxation was also faster in Plec-/- cells as compared to Plec+/+ cells, suggesting a more sparse cytoskeletal network. Confocal imaging indicated that this was due to a marked change in the architecture of the vimentin network, from a fine meshwork in wild type cells to a bundled network in the plectin knockout cells. Our findings therefore indicate that plectin is an important regulator of the organization and viscoelastic properties of the cytoskeleton in fibroblasts. Our findings emphasize that mechanical integration of the different cytoskeletal networks present in cells is important for regulating the versatile mechanical properties of cells. SIGNIFICANCEMammalian cells combine superior mechanical strength with the ability to actively deform themselves. They owe this paradoxical mechanical behaviour to their cytoskeleton, an intracellular web of protein filaments that includes actin filaments and intermediate filaments. It is known that both cytoskeletal filament types contribute to cell stiffness on their own, but the impact of their mechanical integration via cytoskeletal crosslinker proteins remains unknown. Here we test the effect of crosslinking of actin and vimentin intermediate filaments by the crosslinker protein plectin in fibroblasts by single-cell compression measurements. By comparing normal cells and cells in which plectin is knocked out, we find that plectin significantly increases cell stiffness and provides a protective mechanism against actin network disruption by compressive loading.

biophysics↗

bioRxiv2025

Cancer cell deformability impacts the rate of confined migration but not decision making

Cancer cells can utilize different invasion strategies to overcome physical arrest during confined migration through tissues with small pores. Cancer cell plasticity allows switches between different migration modes and transitions between single-cell and collective migration. The biophysical parameters that guide these decisions are poorly understood. In this work we investigated the link between cell deformability and migration efficacy in constrictions of two mesenchymal cancer cell types with similar invasion strategies: HT1080 fibrosarcoma cells and MV3 melanoma cells. To this end, we designed microfluidic platforms for (1) high-throughput cell deformability measurements and (2) migration through a variety of confining geometries. We measured different deformabilities for HT1080 and MV3 cells and correlated this to their migration efficacy through confinements. However, higher deformability and improved squeezing ability did not impact decision-making at junctions of channels of different widths. Our findings show that cell deformability correlates with better squeezing abilities through confinements, but does not impact directionality decisions.

cancer biology↗

bioRxiv2025

Invasive cancer cells soften collagen networks and disrupt stress-stiffening via volume exclusion, contractility and adhesion

Collagen networks form the structural backbone of the extracellular matrix in both healthy and cancerous tissues, exhibiting nonlinear mechanical properties that crucially regulate tissue mechanics and cell behavior. Here, we investigate how the presence of invasive breast cancer cells (MDA-MB-231) influences the polymerization kinetics and mechanics of collagen networks using bulk shear rheology and rheo-confocal microscopy. We show that embedded cancer cells delay the onset of collagen polymerization due to volume exclusion effects. During polymerization, the cells (at 4% volume fraction) cause an unexpected time-dependent softening of the network. We show that this softening effect arises from active remodeling via adhesion and contractility rather than from proteolytic degradation. At higher cell volume fractions, the dominant effect of the cells shifts to volume exclusion, causing a two-fold reduction of network stiffness. Additionally, we demonstrate that cancer cells suppress the characteristic stress-stiffening response of collagen. This effect (partially) disappears when cell adhesion and contractility are inhibited, and it is absent when the cells are replaced by passive hydrogel particles. These findings provide new insights into how active inclusions modify the mechanics of fibrous networks, contributing to a better understanding of the role of cells in the mechanics of healthy and diseased tissues like invasive tumors.

biophysics↗

bioRxiv2025

Matrix stiffness affects spheroid invasion, collagen remodeling and the effective reach of stress into the ECM.

The extracellular matrix (ECM) provides structural support to cells thereby forming a functional tissue. In cancer, the growth of the tumor creates an internal mechanical stress which, together with remodeling activity of tumor cells and fibroblasts, alters the ECM structure leading to an increased stiffness of the pathological ECM. The enhanced ECM stiffness in turn stimulates tumor growth, activates tumor-promoting fibroblasts and tumor cell migration, leading to metastasis and an increased therapy resistance. The connection between internal tumor stress, ECM stiffness, ECM remodeling, and cell migration are unresolved. Here we used 3D ECM-embedded spheroids and hydrogel-particle stress sensors, to quantify and correlate internal tumor-spheroid pressure, ECM stiffness, ECM remodeling, and tumor cell migration. 4T1 breast cancer spheroids and SV80 fibroblast spheroids showed increased invasion - described by area, complexity, number of branches and branch area - in a stiffer, cross-linked ECM. On the other hand, changing the ECM stiffness only minimally changed the radial alignment of fibers but highly changed the amount of fibers.. For both cell types, the pressure measured in spheroids gradually decreased as the distance into the ECM increased. For 4T1 spheroids, increased ECM stiffness resulted in a further reach of mechanical stress into the ECM which, together with the invasive phenotype, was reduced by inhibition of ROCK-mediated contractility. By contrast, such correlation between ECM stiffness and stress-reach was not observed for SV80 spheroids. Our findings connect ECM stiffness with tumor invasion, ECM remodeling, and the reach of tumor-induced mechanical stress into the ECM. Such mechanical connections between tumor and ECM are expected to drive early steps in cancer metastasis.

biophysics↗

bioRxiv2025

Actin and vimentin jointly control cell viscoelasticity and compression stiffening

AbstractThe mechanical properties of cells are governed by the cytoskeleton, a dynamic network of actin filaments, intermediate filaments, and microtubules. Understanding the individual and collective mechanical contributions of these three different cytoskeletal elements is essential to elucidate how cells maintain mechanical integrity during deformation. Here we use a custom single-cell rheometer to identify the distinct contributions of actin and vimentin to the viscoelastic and nonlinear elastic response of cells to uniaxial compression. We used mouse embryonic fibroblasts (MEFs) isolated from wild type (WT) and vimentin knockout (vim -/-) mice in combination with chemical treatments to manipulate actin polymerization and contractility. We show through small amplitude oscillatory measurements and strain ramp tests that vimentin, often overlooked in cellular mechanics, plays a role comparable to actin in maintaining cell stiffness and resisting large compressive forces. However, actin appears to be more important than vimentin in determining cellular energy dissipation. Finally we show by comparing wild type and enucleated cells that compression stiffening originates from the actin and vimentin cytoskeleton, while the nucleus appears to play little role in this. Our findings provide insight into how cytoskeletal networks collectively determine the mechanical properties of cells, providing a basis to understand the role of the cytoskeleton in the ability of cells to resist external as well as internal forces. Significance statementO_LIA cells response to mechanical stress is largely governed by the actin and vimentin cytoskeletal networks, but their relative contribution to cell viscoelasticity and response to large deformations are poorly characterized. C_LIO_LIWe reveal that actin and vimentin networks have an almost equal contribution to cellular stiffness and the cells ability to strain-stiffen under uniaxial compression. C_LIO_LIThis work underscores the cytoskeletons central role in cellular mechanics and the mechanical synergy between the cytoskeletal networks, providing a framework for understanding how cellular components coordinate to maintain structural integrity and respond to different mechanical environments. C_LI

biophysics↗

bioRxiv2024

Early Fibrin Biofilm Development in Cardiovascular Infections

The single most common microbe causing cardiovascular infections is Staphylococcus aureus (S. aureus). S. aureus produces coagulase that converts fibrinogen to fibrin, which is incorporated into biofilms. This process aids in adherence to intravascular structures, defense against the host immune system, and resistance to antimicrobial treatment. Despite its significance, fibrin formation in S. aureus biofilms remains poorly understood. Therefore, this study aimed to elucidate the early development of cardiovascular biofilms. Clinically isolated coagulase-positive S. aureus and coagulase-negative Streptococcus gordonii (S. gordonii) from patients with cardiovascular infections, and a coagulase mutant S. aureus {Delta}coa, were grown in tryptic soy broth (TSB), Iscoves Modified Dulbeccos Medium (IMDM), and pooled human plasma, with or without porcine heart valves. Bacterial growth, metabolic activity, and bacterial fibrinogen utilization were measured over 24 hr at 37 {degrees}C. Time-lapse confocal microscopy was used to visualize and track biofilm development. S. aureus exhibited more growth in TSB and human plasma than S. gordonii and S. aureus {Delta}coa, but showed similar growth as S. aureus {Delta}coa in IMDM. Peak metabolic activity for all isolates was highest in TSB and lowest in human plasma. The presence of porcine valves caused strain-dependent alterations in time to peak metabolic activity. Confocal imaging revealed fibrin-based biofilm development exclusively in the coagulase-producing S. aureus strains. Between 2 and 6 hr of biofilm development, 74.9% (p=0.034) of the fibrinogen from the medium was converted to fibrin. Variations in fibrin network porosity and density were observed among different coagulase-producing S. aureus strains. Fibrin formation is mediated by S. aureus coagulase and first strands occurred within 3 hr for clinical strains after exposure to human plasma. This study stresses the importance of experimental design given the bacterial changes due to different media and substrates and provides insights into the early pathogenesis of S. aureus cardiovascular biofilms. HighlightsO_LIBacterial growth and activity are medium and substrate dependent C_LIO_LICoagulase is necessary for Staphylococcus aureus fibrin biofilm development C_LIO_LIFibrin strands begin forming in Staphylococcus aureus biofilms within 3 hours C_LI

microbiology↗

bioRxiv2023

Reconstitution of cytolinker-mediated crosstalk between actin and vimentin

Cell shape and motility are determined by the cytoskeleton, an interpenetrating network of actin filaments, microtubules, and intermediate filaments. The biophysical properties of each filament type individually have been studied extensively by cell-free reconstitution. By contrast, the interactions between the three cytoskeletal networks are relatively unexplored. They are coupled via crosslinkers of the plakin family such as plectin. These are challenging proteins for reconstitution because of their giant size and multidomain structure. Here we engineer a recombinant actin-vimentin crosslinker protein called ACTIF that provides a minimal model system for plectin, recapitulating its modular design with actin-binding and intermediate filament-binding domains separated by a coiled-coil linker for dimerisation. We show by fluorescence and electron microscopy that ACTIF has a high binding affinity for vimentin and actin and creates mixed actin-vimentin bundles. Rheology measurements show that ACTIF-mediated crosslinking strongly stiffens actin-vimentin composites. Finally, we demonstrate the modularity of this approach by creating an ACTIF variant with the intermediate filament binding domain of Adenomatous Polyposis Coli. Our protein engineering approach provides a new cell-free system for the biophysical characterization of intermediate filament-binding crosslinkers and for understanding the mechanical synergy between actin and vimentin in mesenchymal cells. HIGHLIGHTSO_LIEngineering of a recombinant actin-vimentin crosslinker called ACTIF with the plectin intermediate filament binding domain (IFBD), calponin homology domains that mediate actin binding, and a coiled-coil linker. C_LIO_LIACTIF crosslinks F-actin and vimentin and mediates their co-localization in vitro. C_LIO_LIACTIF has a binding affinity for vimentin that is about 500 times higher than for F-actin. C_LIO_LIACTIF forms composite bundles of F-actin and vimentin filaments. C_LIO_LIComposite F-actin/vimentin networks stiffen upon crosslinking with ACTIF. C_LIO_LIThe design of actin-vimentin crosslinker is modular, as other IFBDs like APCn2 can also be used. C_LI

biochemistry↗

bioRxiv2023

Uncovering the hidden mechanisms of giant unilamellar vesicle formation in cDICE

Giant unilamellar vesicles (GUVs) are widely used as in vitro model membranes in biophysics and as cell-sized containers in synthetic biology. Despite their ubiquitous use, there is no one-size-fits-all method for their production. Numerous methods have been developed to meet the demanding requirements of reproducibility, reliability, and high yield, while simultaneously achieving robust encapsulation. Emulsion-based methods are often praised for their apparent simplicity and good yields; hence, methods like continuous droplet interface crossing encapsulation (cDICE) that make use of this principle, have gained popularity. However, the underlying physical principles governing the formation of GUVs in cDICE and related methods remain poorly understood. To this end, we have developed a high-speed microscopy setup that allows us to visualize GUV formation in real-time. Our experiments reveal a complex droplet formation process occurring at the capillary orifice, generating both larger droplets and, likely, GUV-sized satellite droplets. According to existing theoretical models, the oil-water interface should allow for crossing of all droplets, but based on our observations and theoretical modelling of the fluid dynamics within the system, we find a size-selective crossing of GUV-sized droplets only. Finally, we demonstrate that proteins in the inner solution affect GUV formation by increasing the viscosity and altering lipid adsorption kinetics. These results will not only contribute to a better understanding of GUV formation processes in cDICE, but ultimately also aid the development of more reliable and efficient methods for GUV production. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=98 SRC="FIGDIR/small/562183v2_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@9764cforg.highwire.dtl.DTLVardef@15b63c7org.highwire.dtl.DTLVardef@f39e83org.highwire.dtl.DTLVardef@2bb63a_HPS_FORMAT_FIGEXP M_FIG C_FIG We developed a high-speed microscopy setup to study giant unilamellar vesicle formation in cDICE, revealing a complex droplet formation process occurring at the capillary orifice and size-selectivity at the oil-water interface.

synthetic biology↗

bioRxiv2023

The Impact of the Local Mechanical Environment on Cell Shape and Chondrogenesis of Mesenchymal Stromal Cells in 3D Biomimetic Composite Materials

Efforts to model and repair connective tissue through engineered tissue constructs have generated great interest in culturing cells in 3d polymer network environments. It has been shown that the polymer environment is influential in determining cellular responses such as differentiation, migration and morphology. Hydrogels are used to mimic the cellular microenvironment, but in most cases hydrogels consisting of one polymeric component are used whereas tissues are composites of different polymers. A clear understanding of how different extracellular components and their mechanical characteristics influence cell behaviour is lacking. Here we developed and characterised composite hydrogels of hyaluronan and fibrin and evaluated their use for cartilage tissue engineering. We demonstrate that these cartilage-mimicking composites have a higher stiffness relative to the individual constituents. Next, we cultured human mesenchymal stromal cells in these 3D hydrogels with chondrogenic media and revealed marked differences in cell morphology, gene expression and cartilage-like matrix deposition depending on the specific extracellular composition. We found that, despite evidence for strong adhesion of the cells to fibrin networks in 2D systems, in 3D systems the primary determinant of cellular morphology is the significantly denser hyaluronan network. Dense hyaluronan hydrogels cause local cell confinement evidenced by rounder cell morphologies, independent of the presence of fibrin. While the composite fibrin-hyaluronan hydrogels led to lower expression of chondrogenic genes than hyaluronan alone, the larger linear modulus and resistance to cell-mediated contraction due to the composite nature of the matrix provides a strong advantage in terms of macroscopic mechanical stability. These findings highlight the potential of multi-component hydrogels for controlling cellular behaviour and bulk mechanical properties of cell-hydrogel constructs independently, therefore opening avenues for better understanding the complex interplay between cells and their extracellular environment and thus improve the biofabrication of connective tissues for disease modelling and tissue regeneration. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=127 SRC="FIGDIR/small/555478v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@151962org.highwire.dtl.DTLVardef@1358890org.highwire.dtl.DTLVardef@198dcedorg.highwire.dtl.DTLVardef@d04f80_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2023

Elucidating the role of water in collagen self assembly by isotopically modulating collagen hydration

Water is known to play an important role in collagen self assembly, but it is still largely unclear how water-collagen interactions influence the assembly process and determine the fibril network properties. Here, we use the H2O/D2O isotope effect on the hydrogen-bond strength in water to investigate the role of hydration in collagen self assembly. We dissolve collagen in H2O and D2O, and compare the growth kinetics and the structure of the collagen assemblies formed in these water isotopomers. Surprisingly, collagen assembly occurs ten times faster in D2O than in H2O, and collagen in D2O self assembles into much thinner fibrils, that form a more inhomogeneous and softer network, with a fourfold reduction in elastic modulus compared to H2O. Combining spectroscopic measurements with atomistic simulations, we show that collagen in D2O is less hydrated than in H2O. This partial dehydration lowers the enthalpic penalty for water removal and reorganization at the collagen-water interface, increasing the self assembly rate and the number of nucleation centers, leading to thinner fibrils and a softer network. Coarse-grained simulations show that the acceleration in the initial nucleation rate can be reproduced by the enhancement of electrostatic interactions, which appear to be crucial in determining the acceleration of the initial nucleation rate. These results show that water acts as a mediator between collagen monomers, by moderating their interactions so as to optimize the assembly process and, thus, the final network properties. We believe that isotopically modulating the hydration of proteins can be a valuable method to investigate the role of water in protein structural dynamics and protein self assembly.

biophysics↗

bioRxiv2022

A systematic review and comparison of automated tools for quantification of fibrous networks

Fibrous networks are essential structural components of biological and engineered materials. Accordingly, many approaches have been developed to quantify their structural properties, which define their material properties. However, a comprehensive overview and comparison of methods is lacking. Therefore, we systematically searched for automated tools quantifying network characteristics in confocal, stimulated emission depletion (STED) or scanning electron microscopy (SEM) images and compared these tools by applying them to fibrin, a prototypical fibrous network in thrombi. Structural properties of fibrin such as fiber diameter and alignment are clinically relevant, since they influence the risk of thrombosis. Based on a systematic comparison of the automated tools with each other, manual measurements, and simulated networks, we provide guidance to choose appropriate tools for fibrous network quantification depending on imaging modality and structural parameter. These tools are often able to reliably measure relative changes in network characteristics, but absolute numbers should be interpreted with care.

molecular biology↗

bioRxiv2022

Human septins in cells organize as octamer-based filaments mediating actin-membrane anchoring

Septins are cytoskeletal proteins conserved from algae and protists to mammals. Septin knock-out animals have established that septins are essential for animal physiology, but their molecular function remains elusive. A unique feature of septins is their presence as heteromeric complexes that polymerize into filaments in solution and on lipid membranes. Although animal septins associate extensively with actin-based structures in cells, whether actin-decorating septins organize as filaments and if septin organization impacts septin function is not known. Customizing a tripartite split-GFP complementation assay for probing the presence and composition of septin filaments in situ in cells, we show that all septins decorating actin stress fibers are present as filaments whose integrity depends on octameric septin protomers. Atomic force microscopy nanoindentation measurements on cells confirmed that cell stiffness depends on the presence of octamer-containing septin filaments. Super-resolution structured illumination microscopy revealed septin fibers with widths compatible with their organization as paired septin filaments. Nanometer-resolved distance measurements and single-protein tracking further showed that actin-associated septin filaments are membrane-bound and largely immobilized. Finally, reconstitution assays on supported lipid bilayers showed that septin filaments mediate actin-membrane anchoring. We propose that septin organization as octamer-based filaments is essential for septin function in anchoring and stabilizing actin fibers at the plasma membrane.

cell biology↗

bioRxiv2022

DisGUVery: a versatile open-source software for high-throughput image analysis of Giant Unilamellar Vesicles

Giant Unilamellar Vesicles (GUVs) are cell-sized aqueous compartments enclosed by a phospholipid bilayer. Due to their cell-mimicking properties, GUVs have become a widespread experimental tool in synthetic biology to study membrane properties and cellular processes. In stark contrast to the experimental progress, quantitative analysis of GUV microscopy images has received much less attention. Currently, most analysis is performed either manually or with custom-made scripts, which makes analysis time-consuming and results difficult to compare across studies. To make quantitative GUV analysis accessible and fast, we present DisGUVery, an open-source, versatile software that encapsulates multiple algorithms for automated detection and analysis of GUVs in microscopy images. With a performance analysis, we demonstrate that DisGUVerys three vesicle detection modules successfully identify GUVs in images obtained with a wide range of imaging sources, in various typical GUV experiments. Multiple pre-defined analysis modules allow the user to extract properties such as membrane fluorescence, vesicle shape and internal fluorescence from large populations. A new membrane segmentation algorithm facilitates spatial fluorescence analysis of non-spherical vesicles. Altogether, DisGUVery provides an accessible tool to enable high-throughput automated analysis of GUVs, and thereby to promote quantitative data analysis in GUV research.

biophysics↗