Biology subjects

Koenderink, G.

Publications and source records attributed to Koenderink, G..

15 recordsLinked to original sources

bioRxiv2025

Arterial Shear Rate Determines the Structure and Mechanical Properties of Whole-Blood Clots

BackgroundArterial thrombosis arises under high shear conditions and is a leading cause of ischemic stroke and myocardial infarction. The local hemodynamic environment during the formation of the thrombus influences its final composition, which in turn influences its mechanical characteristics and the associated risk of embolization. However, how arterial shear rates determine the local mechanical properties, structure and composition of clots remains poorly understood. ObjectiveTo investigate how physiological and pathological arterial shear rates shape blood clot composition, formation dynamics, and regional mechanical properties in a newly-developed microfluidic flow model. MethodsWe used a microfluidic flow model that allows real-time imaging of blood clot formation under controlled arterial shear rates (300, 1000, and 3500s-1). We visualized platelets and fibrin deposition using confocal microscopy and quantified composition-dependent spatial heterogeneities in clot stiffness with microindentation. ResultsPathologically high (3500 s-1) arterial shear rates promoted more rapid platelet aggregation and the formation of taller platelet-rich clots. Fibrin deposition started after about 6 minutes in all shear conditions and coincided with platelet aggregate stabilization at higher shear rates. Microindentation revealed that platelet- and fibrin-rich regions were significantly stiffer than red blood cell-rich regions. Clots formed at 3500s-1 had a > 3-fold higher average stiffness than those formed at 300s-1, reflecting their higher platelet content. ConclusionsThis study demonstrates that arterial shear rate governs clot growth and composition, translating into shear-dependent microscale mechanical properties. These findings provide new mechanistic insights into clot properties that are relevant for clot stability and vulnerability to embolization.

bioengineering↗

bioRxiv2025

Data-driven traction force microscopy in 3D collagen hydrogels

Quantitative measurements of cell-generated forces in fibrillar hydrogels have traditionally modeled the extracellular matrix as a continuum. Here we present a data-driven 3D traction force microscopy (DD-TFM) approach that reconstructs discrete collagen fiber networks from second-harmonic generation images and assigns fiber mechanics calibrated by shear rheology. In silico tests confirm the robustness of an inverse force-recovery method, and application to cells in collagen reveals pulling patterns and fiber-level stress distributions.

biophysics↗

bioRxiv2025

Fine-Tuning of Material Properties via Catch Bonds

Semiflexible polymer networks are ubiquitous in biological systems, including a scaffolding structure within cells called the actin cytoskeleton. The polymers in these networks are often interconnected by transient bonds. For example, actin filaments in the cytoskeleton are physically connected via cross-linkers. The mechanical and kinetic properties of the cross-linkers significantly affect the rheological properties of the actin cytoskeleton. Here, we employed an agent-based model to elucidate how the force-dependent behaviors of the cross-linkers determine the material properties of passive networks without molecular motors and the force generation of active networks with molecular motors. The cross-linkers are assumed to behave as a slip bond whose dissociation rate is proportional to forces or as a catch-slip bond whose dissociation rate is inversely proportional to forces at low force level but proportional to forces at high force level. We found that catch-slip-bond cross-linkers can increase both stress and strain at a yield point by forming force-bearing elements that turn over continuously, which is impossible to achieve without the catch-slip bonds. In addition, we demonstrated that the catch-slip-bond cross-linkers help myosin motors generate greater internal contractile forces by reinforcing the force-bearing parts of the active network.

biophysics↗

bioRxiv2025

Anillin directly cross-links microtubules with filamentous actin

Complex morphogenetic processes such as cell division require a tight coordination of the activities of microtubules and actin filaments. There is evidence that anillin, conventionally known as an actin-binding and bundling protein, regulates microtubule-actin crosstalk during cell division. However, it is unknown whether anillin binds directly to microtubules and whether it is sufficient to establish crosslinking between microtubules and actin filaments. Here we address both questions by developing an in-vitro system to observe anillin-mediated interactions with actin filaments and dynamic microtubules using total internal reflection fluorescence microscopy. We find that anillin can interact directly with microtubules and promote microtubule bundling. We confirm that anillin binds and bundles actin filaments and find that it has a strong preference for actin bundles over individual filaments. Moreover, we show that anillin can directly crosslink microtubules and actin filaments, can cause sliding of actin filaments on the microtubule lattice, and can transport actin filaments by the growing microtubule tip. Our findings indicate that anillin can potentially serve as a direct regulator of microtubule-actin crosstalk, e.g. during cell division.

biophysics↗

bioRxiv2024

Optogenetic and chemical genetic tools for rapid repositioning of vimentin intermediate filaments

Intermediate filaments (IFs) are a key component of the cytoskeleton, essential for regulating cell mechanics, maintaining nuclear integrity, organelle positioning, and modulating cell signaling. Current insights into IF function primarily come from studies using long-term perturbations, such as protein depletion or mutation. Here, we present tools that allow rapid manipulation of vimentin IFs in the whole cytoplasm or within specific subcellular regions by inducibly coupling them to microtubule motors, either pharmacologically or using light. Rapid perinuclear clustering of vimentin had no major immediate effects on the actin or microtubule organization, cell spreading, or focal adhesion number, but reduced cell stiffness. Mitochondria and endoplasmic reticulum sheets were reorganized due to vimentin clustering, whereas lysosomes were only briefly displaced and rapidly regained their normal distribution. Keratin moved along with vimentin in some cell lines but remained intact in others. Our tools help to study the immediate and local effects of vimentin perturbation and identify direct links of vimentin to other cellular structures.

cell biology↗

bioRxiv2024

Characterizing microbubble-mediated permeabilization in a vessel-on-a-chip model

Drug transport from blood to extravascular tissue can locally be achieved by increasing the vascular permeability through ultrasound-activated microbubbles. However, the mechanism remains unknown, including whether short and long cycles of ultrasound induce the same onset rate, spatial distribution, and amount of vascular permeability increase. Accurate models are necessary for insights into the mechanism so a microvessel-on-a-chip is developed with a membrane-free extravascular space. Using these microvessels-on-a-chip, we show distinct differences between 2 MHz ultrasound treatments with 10 or 1000 cycles. The onset rate is slower for 10 than 1000 cycles, while both cycle lengths increase the permeability in spot-wise patterns without affecting cell viability. Significantly less vascular permeability increase and sonoporation are induced for 10 versus 1000 cycles at 750 kPa (i.e., highest studied peak negative acoustic pressure (PNP)). The PNP threshold for vascular permeability increases is 750 versus 550 kPa for 10 versus 1000 cycles, while this is 750 versus 220 kPa for sonoporation. Vascular permeability increases do not correlate with v{beta}3-targeted microbubble behavior, while sonoporation correlates with v{beta}3-targeted microbubble clustering. In conclusion, the further mechanistic unraveling of vascular permeability increase by ultrasound-activated microbubbles in a developed microvessel-on-a-chip model aids safe and efficient development of microbubble-mediated drug transport.

bioengineering↗

bioRxiv2024

EMT-dependent cell-matrix interactions are linked to unjamming transitions in cancer spheroid invasion

The plasticity of cancer cells allows them to switch between different migration modes, promoting their invasion into the extracellular matrix (ECM) and hence increasing the risks of metastasis. Epithelial-to-mesenchymal transitions (EMT) and unjamming transitions provide two distinct pathways for cancer cells to become invasive, but it is still unclear to what extent these pathways are connected. Here we addressed this question by performing 3D spheroid invasion assays of lung adenocarcinoma (A549, epithelial) and melanoma (MV3, mesenchymal-like) cancer cell lines in collagen-based hydrogels, where we varied both the invasive character of the cells (using Transforming Growth Factor (TGF)-{beta} to promote EMT and matrix metalloprotease (MMP) inhibition to block cell-mediated matrix degradation) and the porosity of the matrix. Using a quantitative image analysis method to track spheroid invasion, we discovered that the onset time of invasion mostly depended on the matrix porosity and corresponded with vimentin levels, while the subsequent spheroid expansion rate mostly depended on metalloprotease MMP1 levels and thus cell-matrix interaction. Morphological analysis revealed that spheroids displayed solid-like (non-invasive) behavior in small-pore hydrogels and switched to fluid-like (strand-based) or gas-like (disseminating cells) phases in large-pore hydrogels and when cells were more mesenchymal-like. Our findings are consistent with unjamming transitions as a function of cell motility and matrix confinement predicted in recent models for cancer invasion, but show that cell motility and matrix confinement are coupled via EMT-dependent matrix degradation.

cancer biology↗

bioRxiv2023

Biomimetic actin cortices shape cell-sized lipid vesicles

Animal cells are shaped by a thin layer of actin filaments underneath the plasma membrane known as the actin cortex. This cortex stiffens the cell surface and thus opposes cellular deformation, yet also actively generates membrane protrusions by exerting polymerization forces. It is unclear how the interplay between these two opposing mechanical functions plays out to shape the cell surface. To answer this question, we reconstitute biomimetic actin cortices nucleated by the Arp2/3 complex inside cell-sized lipid vesicles. We show that thin Arp2/3-nucleated actin cortices strongly deform and rigidify the shapes of giant unilamellar vesicles and impart a shape memory on time scales that exceeds the time of actin turnover. In addition, actin cortices can produce finger-like membrane protrusions, showing that Arp2/3-mediated actin polymerization forces alone are sufficient to initiate protrusions in the absence of actin bundling or membrane curving proteins. Combining mathematical modeling and our experimental results reveals that the concentration of actin nucleating proteins, rather than actin polymerization speed, is crucial for protrusion formation. This is because locally concentrated actin polymerization forces can drive a positive feedback loop between recruitment of actin and its nucleators to drive membrane deformation. Our work paints a picture where the actin cortex can either drive or inhibit deformations depending on the local distribution of nucleators. Significance StatementThe cells in our body must actively change shape in order to migrate, grow and divide, but they also need to maintain their shape to withstand external forces during tissue development. Cellular shape control results from an interplay between the plasma membrane and its underlying cortex, a shell composed of crosslinked actin filaments. Using cell-free reconstitution and mathematical modelling, we show that minimal biomimetic actin cortices can mechanically rigidify lipid vesicles while at the same time driving membrane protrusion formation. Our observations suggest that the spatial distribution of actin nucleation determines whether the actin cortex drives or inhibits membrane deformations.

biophysics↗

bioRxiv2022

Reconstituted branched actin networks sense and generate micron-scale membrane curvature

The actin cortex is a complex cytoskeletal machinery which drives and responds to changes in cell shape. It must generate or adapt to plasma membrane curvature to facilitate diverse functions such as cell division, migration and phagocytosis. Due to the complex molecular makeup of the actin cortex, it remains unclear whether actin networks are inherently able to sense and generate membrane curvature, or whether they rely on their diverse binding partners to accomplish this. Here, we show that curvature sensing and generation is an inherent capability of branched actin networks nucleated by Arp2/3 and VCA. We develop a robust method to encapsulate actin inside giant unilamellar vesicles (GUVs) and assemble an actin cortex at the inner surface of the GUV membrane. We show that actin forms a uniform and thin cortical layer when present at high concentration and distinct patches that generate negative membrane curvature at low concentration. Serendipitously, we find that the GUV production method also produces dumbbell-shaped GUVs, which we explain using mathematical modelling in terms of membrane hemifusion of nested GUVs. We find that dendritic actin networks preferentially assemble at the neck of the dumbbells, which possess a micron-range convex curvature that matches the curvature generated by actin patches in spherical GUVs. Minimal dendritic actin networks can thus both generate and sense membrane curvatures, which may help mammalian cells to robustly recruit actin to curved membranes in order to facilitate diverse cellular functions such as cytokinesis and migration. SIGNIFICANCEAnimal cells move, deform and divide using their actin cortex, a thin layer of filamentous proteins that supports the plasma membrane. For these actions, actin must often assemble at curved sections of the membrane, which is widely believed to require the action of dedicated actin- or membrane-bending proteins. Here, we use a bottom-up reconstitution approach to ask whether actin networks are intrinsically able to generate and sense membrane curvature. We show that membrane-nucleated actin cortices can indeed preferentially self-assemble at concave membranes generated by hemifusion of lipid vesicles. This raises intriguing questions about how such curvature recognition works, and whether cells exploit this intrinsic capability of branched actin networks to concentrate actin in specific cortical regions.

biophysics↗

bioRxiv2021

Septin-microtubule association requires a MAP-like motif unique to Sept9 isoform 1 embedded into septin octamers

Septins, a family of GTP-binding proteins assembling into higher order structures, interface with the membrane, actin filaments and microtubules, which positions them as important regulators of cytoarchitecture. Septin 9 (Sept9), which is frequently overexpressed in tumors and mutated in hereditary neuralgic amyotrophy (HNA), mediates the binding of septins to microtubules, but the molecular determinants of this interaction remained uncertain. We demonstrate that a short MAP-like motif unique to Sept9 isoform 1 (Sept9_i1) drives septin octamer-microtubule interaction in cells and in vitro reconstitutions. Septin-microtubule association requires polymerizable septin octamers harboring Sept9_i1. Although outside of the MAP-like motif, HNA mutations abrogates this association, identifying a putative regulatory domain. Removal of this domain from Sept9_i1 sequesters septins on microtubules, promotes microtubule stability and alters actomyosin fiber distribution and tension. Thus, we identify key molecular determinants and potential regulatory roles of septin-microtubule interaction, paving the way to deciphering the mechanisms underlying septin associated pathologies.

cell biology↗

bioRxiv2021

Insights into animal septins using recombinant human septin octamers with distinct SEPT9 isoforms

Septin GTP-binding proteins contribute essential biological functions that range from the establishment of cell polarity to animal tissue morphogenesis. Human septins in cells form hetero-octameric septin complexes containing the ubiquitously expressed SEPT9. Despite the established role of SEPT9 in mammalian development and human pathophysiology, biochemical and biophysical studies have relied on monomeric SEPT9 thus not recapitulating its native assembly into hetero-octameric complexes. We established a protocol that enabled the first-time isolation of recombinant human septin octamers containing distinct SEPT9 isoforms. A combination of biochemical and biophysical assays confirmed the octameric nature of the isolated complexes in solution. Reconstitution studies showed that octamers with either a long or a short SEPT9 isoform form filament assemblies, and can directly bind and cross-link actin filaments, raising the possibility that septin-decorated actin structures in cells reflect direct actin-septin interactions. Recombinant SEPT9-containing octamers will make it possible to design cell-free assays to dissect the complex interactions of septins with cell membranes and the actin/microtubule cytoskeleton. SummaryHuman septins in cells form hetero-octameric complexes containing the ubiquitously expressed SEPT9. Iv et al. describe the first-time isolation of recombinant human septin octamers with distinct SEPT9 isoforms. Reconstitution studies show that octamers with either a long or a short SEPT9 isoform form higher-order filament assemblies and directly bind and cross-link actin filaments.

cell biology↗

bioRxiv2020

Weak catch bonds make strong networks

Molecular catch bonds are ubiquitous in biology and well-studied in the context of leukocyte extravasion1, cellular mechanosensing2,3, and urinary tract infection4. Unlike normal (slip) bonds, catch bonds strengthen under tension. The current paradigm is that this remarkable ability enables cells to increase their adhesion in fast fluid flows1,4, and hence provides strength-on-demand. Recently, cytoskeletal crosslinkers have been discovered that also display catch bonding5-8. It has been suggested that they strengthen cells, following the strength-on-demand paradigm9,10. However, catch bonds tend to be weaker compared to regular (slip) bonds because they have cryptic binding sites that are often inactive11-13. Therefore, the role of catch bonding in the cytoskeleton remains unclear. Here we reconstitute cytoskeletal actin networks to show that catch bonds render them both stronger and more deformable than slip bonds, even though the bonds themselves are weaker. We develop a model to show that weak binding allows the catch bonds to mitigate crack initiation by moving from low- to high-tension areas in response to mechanical loading. By contrast, slip bonds remain trapped in stress-free areas. We therefore propose that the mechanism of catch bonding is typified by dissociation-on-demand rather than strength-on-demand. Dissociation-on-demand can explain how both cytolinkers5-8,10,14,15 and adhesins1,2,4,12,16-20 exploit continuous redistribution to combine mechanical strength with the adaptability required for movement and proliferation21. Our findings provide a new perspective on diseases where catch bonding is compromised11,12 such as kidney focal segmental glomerulosclerosis22,23, caused by the -actinin-4 mutant studied here. Moreover, catch bonds provide a route towards creating life-like materials that combine strength with deformability24.

biophysics↗

bioRxiv2020

Connecting the stimuli-responsive rheology of biopolymer hydrogels to underlying hydrogen-bonding interactions

Many biopolymer hydrogels are environmentally responsive because they are held together by physical associations that depend on pH and temperature. Here we investigate how the pH and temperature response of the rheology of hyaluronan hydrogels is connected to the underlying molecular interactions. Hyaluronan is an essential structural biopolymer in the human body with many applications in biomedicine. Using two-dimensional infrared (2DIR) spectroscopy, we show that hyaluronan chains become connected by hydrogen bonds when the pH is changed from 7.0 to 2.5, and that the bond density at pH 2.5 is independent of temperature. Temperature-dependent rheology measurements show that due to this hydrogen bonding the stress relaxation at pH 2.5 is strongly slowed down in comparison to pH 7.0, consistent with the sticky reptation model of associative polymers. From the flow activation energy we conclude that each polymer is crosslinked by multiple (5-15) hydrogen bonds to others, causing slow macroscopic stress relaxation, despite the short time scale of breaking and reformation of each individual hydrogen bond. Our findings can aid the design of stimuli-responsive hydrogels with tailored viscoelastic properties for biomedical applications.

biophysics↗

bioRxiv2019

Mechanisms of motor-independent membrane remodeling driven by dynamic microtubules

Microtubule-dependent organization of membranous organelles, such as the endoplasmic reticulum, occurs through motor-based pulling and by coupling microtubule dynamics to membrane remodeling. How highly transient protein-protein interactions occurring at growing microtubule tips can induce load-bearing processive motion is currently unclear. Here, we reconstituted membrane tubulation in a minimal system with giant unilamellar vesicles, dynamic microtubules, End-Binding (EB) proteins and a membrane-targeted protein that interacts with EBs and microtubules. We showed that these components are sufficient to drive membrane remodeling by three mechanisms: membrane tubulation by growing microtubule ends, motor-independent membrane sliding along microtubule shafts and pulling by shrinking microtubules. Experiments and modeling demonstrated that the first two mechanisms can be explained by adhesion-driven biased membrane spreading on microtubules. Force spectroscopy revealed that attachments to growing and shrinking microtubule ends can sustain forces of [~]0.5 and [~]5 pN, respectively. Rapidly exchanging molecules that connect membranes to dynamic microtubules can thus bear sufficient load to induce membrane deformation and motility.

cell biology↗

bioRxiv2018

Unravelling the response of the actin cortex to electric pulses in vesicles

We study the role of a biomimetic actin cortex during the application of electric pulses that induce electroporation or electropermeabilization, using giant unilamellar vesicles (GUVs) as a model system. The actin cortex, a subjacently attached interconnected network of actin filaments, regulates the shape and mechanical properties of the plasma membrane of mammalian cells, and is a major factor influencing the mechanical response of the cell to external physical cues. We demonstrate that the presence of an actin shell inhibits the formation of macropores in the electroporated GUVs. Additionally, experiments on the uptake of dye molecules after electroporation show that the actin network slows down the resealing process of the permeabilized membrane. We further analyze the stability of the actin network inside the GUVs exposed to high electric pulses. We find disruption of the actin layer that is likely due to the electrophoretic forces acting on the actin filaments during the permeabilization of the GUVs. Our findings on the GUVs containing a biomimetic cortex provide a step towards understanding the discrepancies between the electroporation mechanism of a living cell and its simplified model of the empty GUV.

biophysics↗