Publications by authors named "Gijsje H Koenderink"

87 Publications

The role of cell-matrix interactions in connective tissue mechanics.

Phys Biol 2022 01 18;19(2). Epub 2022 Jan 18.

Department of Bionanoscience, Kavli Institute of Nanoscience Delft, Delft University of Technology, Van der Maasweg 9, Delft, The Netherlands.

Living tissue is able to withstand large stresses in everyday life, yet it also actively adapts to dynamic loads. This remarkable mechanical behaviour emerges from the interplay between living cells and their non-living extracellular environment. Here we review recent insights into the biophysical mechanisms involved in the reciprocal interplay between cells and the extracellular matrix and how this interplay determines tissue mechanics, with a focus on connective tissues. We first describe the roles of the main macromolecular components of the extracellular matrix in regards to tissue mechanics. We then proceed to highlight the main routes via which cells sense and respond to their biochemical and mechanical extracellular environment. Next we introduce the three main routes via which cells can modify their extracellular environment: exertion of contractile forces, secretion and deposition of matrix components, and matrix degradation. Finally we discuss how recent insights in the mechanobiology of cell-matrix interactions are furthering our understanding of the pathophysiology of connective tissue diseases and cancer, and facilitating the design of novel strategies for tissue engineering.
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http://dx.doi.org/10.1088/1478-3975/ac42b8DOI Listing
January 2022

Septin-microtubule association via a motif unique to isoform 1 of septin 9 tunes stress fibers.

J Cell Sci 2022 Jan 10;135(1). Epub 2022 Jan 10.

Centre de Recherche en Cancérologie de Marseille (CRCM), INSERM, Institut Paoli-Calmettes, Aix Marseille Univ, CNRS, 13009 Marseille, France.

Septins, a family of GTP-binding proteins that assemble into higher order structures, interface with the membrane, actin filaments and microtubules, and are thus 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 microtubule-associated protein (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 abrogate 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. This article has an associated First Person interview with the first author of the paper.
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http://dx.doi.org/10.1242/jcs.258850DOI Listing
January 2022

Molecular Structure and Surface Accumulation Dynamics of Hyaluronan at the Water-Air Interface.

Macromolecules 2021 Sep 16;54(18):8655-8663. Epub 2021 Sep 16.

Amolf, Science Park 104, 1098 XG Amsterdam, The Netherlands.

Hyaluronan is a biopolymer that is essential for many biological processes in the human body, like the regulation of tissue lubrication and inflammatory responses. Here, we study the behavior of hyaluronan at aqueous surfaces using heterodyne-detected vibrational sum-frequency generation spectroscopy (HD-VSFG). Low-molecular-weight hyaluronan (∼150 kDa) gradually covers the water-air interface within hours, leading to a negatively charged surface and a reorientation of interfacial water molecules. The rate of surface accumulation strongly increases when the bulk concentration of low-molecular-weight hyaluronan is increased. In contrast, high-molecular-weight hyaluronan (>1 MDa) cannot be detected at the surface, even hours after the addition of the polymer to the aqueous solution. The strong dependence on the polymer molecular weight can be explained by entanglements of the hyaluronan polymers. We also find that for low-molecular-weight hyaluronan the migration kinetics of hyaluronan in aqueous media shows an anomalous dependence on the pH of the solution, which can be explained from the interplay of hydrogen bonding and electrostatic interactions of hyaluronan polymers.
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http://dx.doi.org/10.1021/acs.macromol.1c00366DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8482758PMC
September 2021

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

J Cell Sci 2021 08 5;134(15). Epub 2021 Aug 5.

Institut Fresnel, CNRS UMR7249, Aix Marseille Univ, Centrale Marseille, 13013 Marseille, France.

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 subunit (also known as SEPTIN9). 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, for the first time, the 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 and microtubule cytoskeleton.
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http://dx.doi.org/10.1242/jcs.258484DOI Listing
August 2021

Optimized cDICE for Efficient Reconstitution of Biological Systems in Giant Unilamellar Vesicles.

ACS Synth Biol 2021 07 29;10(7):1690-1702. Epub 2021 Jun 29.

Department of Living Matter, AMOLF, 1098 XG Amsterdam, The Netherlands.

Giant unilamellar vesicles (GUVs) are often used to mimic biological membranes in reconstitution experiments. They are also widely used in research on synthetic cells, as they provide a mechanically responsive reaction compartment that allows for controlled exchange of reactants with the environment. However, while many methods exist to encapsulate functional biomolecules in GUVs, there is no one-size-fits-all solution and reliable GUV fabrication still remains a major experimental hurdle in the field. Here, we show that defect-free GUVs containing complex biochemical systems can be generated by optimizing a double-emulsion method for GUV formation called continuous droplet interface crossing encapsulation (cDICE). By tightly controlling environmental conditions and tuning the lipid-in-oil dispersion, we show that it is possible to significantly improve the reproducibility of high-quality GUV formation as well as the encapsulation efficiency. We demonstrate efficient encapsulation for a range of biological systems including a minimal actin cytoskeleton, membrane-anchored DNA nanostructures, and a functional PURE (protein synthesis using recombinant elements) system. Our optimized cDICE method displays promising potential to become a standard method in biophysics and bottom-up synthetic biology.
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http://dx.doi.org/10.1021/acssynbio.1c00068DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8291763PMC
July 2021

Membrane binding controls ordered self-assembly of animal septins.

Elife 2021 04 13;10. Epub 2021 Apr 13.

AMOLF, Department of Living Matter, Biological Soft Matter group, Amsterdam, Netherlands.

Septins are conserved cytoskeletal proteins that regulate cell cortex mechanics. The mechanisms of their interactions with the plasma membrane remain poorly understood. Here, we show by cell-free reconstitution that binding to flat lipid membranes requires electrostatic interactions of septins with anionic lipids and promotes the ordered self-assembly of fly septins into filamentous meshworks. Transmission electron microscopy reveals that both fly and mammalian septin hexamers form arrays of single and paired filaments. Atomic force microscopy and quartz crystal microbalance demonstrate that the fly filaments form mechanically rigid, 12- to 18-nm thick, double layers of septins. By contrast, C-terminally truncated septin mutants form 4-nm thin monolayers, indicating that stacking requires the C-terminal coiled coils on DSep2 and Pnut subunits. Our work shows that membrane binding is required for fly septins to form ordered arrays of single and paired filaments and provides new insights into the mechanisms by which septins may regulate cell surface mechanics.
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http://dx.doi.org/10.7554/eLife.63349DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8099429PMC
April 2021

Strong Reduction of the Chain Rigidity of Hyaluronan by Selective Binding of Ca Ions.

Macromolecules 2021 Feb 19;54(3):1137-1146. Epub 2021 Jan 19.

AMOLF, Science Park 104, 1098 XG Amsterdam, The Netherlands.

The biological functions of natural polyelectrolytes are strongly influenced by the presence of ions, which bind to the polymer chains and thereby modify their properties. Although the biological impact of such modifications is well recognized, a detailed molecular picture of the binding process and of the mechanisms that drive the subsequent structural changes in the polymer is lacking. Here, we study the molecular mechanism of the condensation of calcium, a divalent cation, on hyaluronan, a ubiquitous polymer in human tissues. By combining two-dimensional infrared spectroscopy experiments with molecular dynamics simulations, we find that calcium specifically binds to hyaluronan at millimolar concentrations. Because of its large size and charge, the calcium cation can bind simultaneously to the negatively charged carboxylate group and the amide group of adjacent saccharide units. Molecular dynamics simulations and single-chain force spectroscopy measurements provide evidence that the binding of the calcium ions weakens the intramolecular hydrogen-bond network of hyaluronan, increasing the flexibility of the polymer chain. We also observe that the binding of calcium to hyaluronan saturates at a maximum binding fraction of ∼10-15 mol %. This saturation indicates that the binding of Ca strongly reduces the probability of subsequent binding of Ca at neighboring binding sites, possibly as a result of enhanced conformational fluctuations and/or electrostatic repulsion effects. Our findings provide a detailed molecular picture of ion condensation and reveal the severe effect of a few, selective and localized electrostatic interactions on the rigidity of a polyelectrolyte chain.
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http://dx.doi.org/10.1021/acs.macromol.0c02242DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7879427PMC
February 2021

Connecting the Stimuli-Responsive Rheology of Biopolymer Hydrogels to Underlying Hydrogen-Bonding Interactions.

Macromolecules 2020 Dec 18;53(23):10503-10513. Epub 2020 Nov 18.

AMOLF, Science Park 104, Amsterdam 1098 XG, The Netherlands.

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 responses of the rheology of hyaluronan hydrogels are 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 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 because of 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 cross-linked 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.
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http://dx.doi.org/10.1021/acs.macromol.0c01742DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7735748PMC
December 2020

Effects of Diabetes Mellitus on Fibrin Clot Structure and Mechanics in a Model of Acute Neutrophil Extracellular Traps (NETs) Formation.

Int J Mol Sci 2020 Sep 26;21(19). Epub 2020 Sep 26.

Department of Hematology, Erasmus MC, University Medical Center Rotterdam, 3015 GD Rotterdam, The Netherlands.

Subjects with diabetes mellitus (DM) have an increased risk of arterial thrombosis, to which changes in clot structure and mechanics may contribute. Another contributing factor might be an increased formation of neutrophil extracellular traps (NETs) in DM. NETs are mainly formed during the acute phase of disease and form a network within the fibrin matrix, thereby influencing clot properties. Previous research has shown separate effects of NETs and DM on clot properties, therefore our aim was to study how DM affects clot properties in a model resembling an acute phase of disease with NETs formation. Clots were prepared from citrated plasma from subjects with and without DM with the addition of NETs, induced in neutrophils by bacteria or phorbol myristate acetate (PMA). Structural parameters were measured using scanning electron microscopy, mechanical properties using rheology, and sensitivity to lysis using a fluorescence-based fibrinolysis assay. Plasma clots from subjects with DM had significantly thicker fibers and fewer pores and branch points than clots from subjects without DM. In addition, fibrinolysis was significantly slower, while mechanical properties were similar between both groups. In conclusion, in a model of acute NETs formation, DM plasma shows prothrombotic effects on fibrin clots.
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http://dx.doi.org/10.3390/ijms21197107DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7582521PMC
September 2020

Molecular packing structure of fibrin fibers resolved by X-ray scattering and molecular modeling.

Soft Matter 2020 Sep;16(35):8272-8283

AMOLF, Biological Soft Matter Group, Amsterdam, The Netherlands and Department of Bionanoscience, Kavli Institute of Nanoscience Delft, Delft University of Technology, Van der Maasweg 9, Delft, 2629 HZ, The Netherlands.

Fibrin is the major extracellular component of blood clots and a proteinaceous hydrogel used as a versatile biomaterial. Fibrin forms branched networks built of laterally associated double-stranded protofibrils. This multiscale hierarchical structure is crucial for the extraordinary mechanical resilience of blood clots, yet the structural basis of clot mechanical properties remains largely unclear due, in part, to the unresolved molecular packing of fibrin fibers. Here the packing structure of fibrin fibers is quantitatively assessed by combining Small Angle X-ray Scattering (SAXS) measurements of fibrin reconstituted under a wide range of conditions with computational molecular modeling of fibrin protofibrils. The number, positions, and intensities of the Bragg peaks observed in the SAXS experiments were reproduced computationally based on the all-atom molecular structure of reconstructed fibrin protofibrils. Specifically, the model correctly predicts the intensities of the reflections of the 22.5 nm axial repeat, corresponding to the half-staggered longitudinal arrangement of fibrin molecules. In addition, the SAXS measurements showed that protofibrils within fibrin fibers have a partially ordered lateral arrangement with a characteristic transverse repeat distance of 13 nm, irrespective of the fiber thickness. These findings provide fundamental insights into the molecular structure of fibrin clots that underlies their biological and physical properties.
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http://dx.doi.org/10.1039/d0sm00916dDOI Listing
September 2020

Hyaluronan biopolymers release water upon pH-induced gelation.

Phys Chem Chem Phys 2020 Apr;22(16):8667-8671

AMOLF, Science Park 104, 1098 XG Amsterdam, The Netherlands.

We study the relation between the macroscopic viscoelastic properties of aqueous hyaluronan polymer solutions and the molecular-scale dynamics of water using rheology measurements, differential dynamic microscopy, and polarization-resolved infrared pump-probe spectroscopy. We observe that the addition of hyaluronan to water leads to a slowing down of the reorientation of a fraction of the water molecules. Near pH 2.4, the viscosity of the hyaluronan solution reaches a maximum, while the number of slowed down water molecules reaches a minimum. This implies that the water molecules become on average more mobile when the solution becomes more viscous. This observation indicates that the increase in viscosity involves the expulsion of hydration water from the surfaces of the hyaluronan polymers, and a bundling of the hyaluronan polymer chains.
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http://dx.doi.org/10.1039/d0cp00215aDOI Listing
April 2020

Connectivity and plasticity determine collagen network fracture.

Proc Natl Acad Sci U S A 2020 04 1;117(15):8326-8334. Epub 2020 Apr 1.

Biological Soft Matter Group, Department of Living Matter, AMOLF, 1098 XG Amsterdam, The Netherlands;

Collagen forms the structural scaffold of connective tissues in all mammals. Tissues are remarkably resistant against mechanical deformations because collagen molecules hierarchically self-assemble in fibrous networks that stiffen with increasing strain. Nevertheless, collagen networks do fracture when tissues are overloaded or subject to pathological conditions such as aneurysms. Prior studies of the role of collagen in tissue fracture have mainly focused on tendons, which contain highly aligned bundles of collagen. By contrast, little is known about fracture of the orientationally more disordered collagen networks present in many other tissues such as skin and cartilage. Here, we combine shear rheology of reconstituted collagen networks with computer simulations to investigate the primary determinants of fracture in disordered collagen networks. We show that the fracture strain is controlled by the coordination number of the network junctions, with less connected networks fracturing at larger strains. The hierarchical structure of collagen fine-tunes the fracture strain by providing structural plasticity at the network and fiber level. Our findings imply that low connectivity and plasticity provide protective mechanisms against network fracture that can optimize the strength of biological tissues.
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http://dx.doi.org/10.1073/pnas.1920062117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7165426PMC
April 2020

Charge-dependent interactions of monomeric and filamentous actin with lipid bilayers.

Proc Natl Acad Sci U S A 2020 03 2;117(11):5861-5872. Epub 2020 Mar 2.

Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, 9747 AG, Groningen, The Netherlands;

The cytoskeletal protein actin polymerizes into filaments that are essential for the mechanical stability of mammalian cells. In vitro experiments showed that direct interactions between actin filaments and lipid bilayers are possible and that the net charge of the bilayer as well as the presence of divalent ions in the buffer play an important role. In vivo, colocalization of actin filaments and divalent ions are suppressed, and cells rely on linker proteins to connect the plasma membrane to the actin network. Little is known, however, about why this is the case and what microscopic interactions are important. A deeper understanding is highly beneficial, first, to obtain understanding in the biological design of cells and, second, as a possible basis for the building of artificial cortices for the stabilization of synthetic cells. Here, we report the results of coarse-grained molecular dynamics simulations of monomeric and filamentous actin in the vicinity of differently charged lipid bilayers. We observe that charges on the lipid head groups strongly determine the ability of actin to adsorb to the bilayer. The inclusion of divalent ions leads to a reversal of the binding affinity. Our in silico results are validated experimentally by reconstitution assays with actin on lipid bilayer membranes and provide a molecular-level understanding of the actin-membrane interaction.
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http://dx.doi.org/10.1073/pnas.1914884117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7084070PMC
March 2020

Particle diffusion in extracellular hydrogels.

Soft Matter 2020 Feb 15;16(5):1366-1376. Epub 2020 Jan 15.

AMOLF, Department of Living Matter, Biological Soft Matter group, Science Park 104, 1098 XG Amsterdam, The Netherlands.

Hyaluronic acid is an abundant polyelectrolyte in the human body that forms extracellular hydrogels in connective tissues. It is essential for regulating tissue biomechanics and cell-cell communication, yet hyaluronan overexpression is associated with pathological situations such as cancer and multiple sclerosis. Due to its enormous molecular weight (in the range of millions of Daltons), accumulation of hyaluronan hinders transport of macromolecules including nutrients and growth factors through tissues and also hampers drug delivery. However, the exact contribution of hyaluronan to tissue penetrability is poorly understood due to the complex structure and molecular composition of tissues. Here we reconstitute biomimetic hyaluronan gels and systematically investigate the effects of gel composition and crosslinking on the diffusion of microscopic tracer particles. We combine ensemble-averaged measurements via differential dynamic microscopy with single-particle tracking. We show that the particle diffusivity depends on the particle size relative to the network pore size and also on the stress relaxation dynamics of the network. We furthermore show that addition of collagen, the other major biopolymer in tissues, causes the emergence of caged particle dynamics. Our findings are useful for understanding macromolecular transport in tissues and for designing biomimetic extracellular matrix hydrogels for drug delivery and tissue regeneration.
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http://dx.doi.org/10.1039/c9sm01837aDOI Listing
February 2020

Revealing the molecular origins of fibrin's elastomeric properties by in situ X-ray scattering.

Acta Biomater 2020 03 7;104:39-52. Epub 2020 Jan 7.

Department of Living Matter, AMOLF, Amsterdam 1098XG, the Netherlands; Department of Bionanoscience, Kavli Institute of Nanoscience Delft, Delft University of Technology, Delft 2629HZ, the Netherlands. Electronic address:

Fibrin is an elastomeric protein forming highly extensible fiber networks that provide the scaffold of blood clots. Here we reveal the molecular mechanisms that explain the large extensibility of fibrin networks by performing in situ small angle X-ray scattering measurements while applying a shear deformation. We simultaneously measure shear-induced alignment of the fibers and changes in their axially ordered molecular packing structure. We show that fibrin networks exhibit distinct structural responses that set in consecutively as the shear strain is increased. They exhibit an entropic response at small strains (<5%), followed by progressive fiber alignment (>25% strain) and finally changes in the fiber packing structure at high strain (>100%). Stretching reduces the fiber packing order and slightly increases the axial periodicity, indicative of molecular unfolding. However, the axial periodicity changes only by 0.7%, much less than the 80% length increase of the fibers, suggesting that fiber elongation mainly stems from uncoiling of the natively disordered αC-peptide linkers that laterally bond the molecules. Upon removal of the load, the network structure returns to the original isotropic state, but the fiber structure becomes more ordered and adopts a smaller packing periodicity compared to the original state. We conclude that the hierarchical packing structure of fibrin fibers, with built-in disorder, makes the fibers extensible and allows for mechanical annealing. Our results provide a basis for interpreting the molecular basis of haemostatic and thrombotic disorders associated with clotting and provide inspiration to design resilient bio-mimicking materials. STATEMENT OF SIGNIFICANCE: Fibrin provides structural integrity to blood clots and is also widely used as a scaffold for tissue engineering. To fulfill their biological functions, fibrin networks have to be simultaneously compliant like skin and resilient against rupture. Here, we unravel the structural origin underlying this remarkable mechanical behaviour. To this end, we performed in situ measurements of fibrin structure across multiple length scales by combining X-ray scattering with shear rheology. Our findings show that fibrin sustains large strains by undergoing a sequence of structural changes on different scales with increasing strain levels. This demonstrates new mechanistic aspects of an important biomaterial's structure and its mechanical function, and serves as an example in the design of biomimicking materials.
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http://dx.doi.org/10.1016/j.actbio.2020.01.002DOI Listing
March 2020

Poroelasticity of (bio)polymer networks during compression: theory and experiment.

Soft Matter 2020 Feb 10;16(5):1298-1305. Epub 2020 Jan 10.

AMOLF, Theory of Biomolecular Matter, Science Park 104, 1098XG Amsterdam, The Netherlands.

Soft living tissues like cartilage can be considered as biphasic materials comprising a fibrous complex biopolymer network and a viscous background liquid. Here, we show by a combination of experiment and theoretical analysis that both the hydraulic permeability and the elastic properties of (bio)polymer networks can be determined with simple ramp compression experiments in a commercial rheometer. In our approximate closed-form solution of the poroelastic equations of motion, we find the normal force response during compression as a combination of network stress and fluid pressure. Choosing fibrin as a biopolymer model system with controllable pore size, measurements of the full time-dependent normal force during compression are found to be in excellent agreement with the theoretical calculations. The inferred elastic response of large-pore (μm) fibrin networks depends on the strain rate, suggesting a strong interplay between network elasticity and fluid flow. Phenomenologically extending the calculated normal force into the regime of nonlinear elasticity, we find strain-stiffening of small-pore (sub-μm) fibrin networks to occur at an onset average tangential stress at the gel-plate interface that depends on the polymer concentration in a power-law fashion. The inferred permeability of small-pore fibrin networks scales approximately inverse squared with the fibrin concentration, implying with a microscopic cubic lattice model that the number of protofibrils per fibrin fiber cross-section decreases with protein concentration. Our theoretical model provides a new method to obtain the hydraulic permeability and the elastic properties of biopolymer networks and hydrogels with simple compression experiments, and paves the way to study the relation between fluid flow and elasticity in biopolymer networks during dynamical compression.
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http://dx.doi.org/10.1039/c9sm01973aDOI Listing
February 2020

Colloidal Liquid Crystals Confined to Synthetic Tactoids.

Sci Rep 2019 12 31;9(1):20391. Epub 2019 Dec 31.

AMOLF, Department of Living Matter, Amsterdam, 1098XG, The Netherlands.

When a liquid crystal forming particles are confined to a spatial volume with dimensions comparable to that of their own size, they face a complex trade-off between their global tendency to align and the local constraints imposed by the boundary conditions. This interplay may lead to a non-trivial orientational patterns that strongly depend on the geometry of the confining volume. This novel regime of liquid crystalline behavior can be probed with colloidal particles that are macro-aggregates of biomolecules. Here we study director fields of filamentous fd-viruses in quasi-2D lens-shaped chambers that mimic the shape of tactoids, the nematic droplets that form during isotropic-nematic phase separation. By varying the size and aspect ratio of the chambers we force these particles into confinements that vary from circular to extremely spindle-like shapes and observe the director field using fluorescence microscopy. In the resulting phase diagram, next to configurations predicted earlier for 3D tactoids, we find a number of novel configurations. Using Monte Carlo Simulations, we show that these novel states are metastable, yet long-lived. Their multiplicity can be explained by the co-existence of multiple dynamic relaxation pathways leading to the final stable states.
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http://dx.doi.org/10.1038/s41598-019-56729-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6938498PMC
December 2019

In Vitro Reconstitution of Dynamic Co-organization of Microtubules and Actin Filaments in Emulsion Droplets.

Methods Mol Biol 2020 ;2101:53-75

Department of Bionanoscience, Kavli Institute of Nanoscience, Delft University of Technology, Delft, The Netherlands.

In vitro (or cell-free) reconstitution is a powerful tool to study the physical basis of cytoskeletal organization in eukaryotic cells. Cytoskeletal reconstitution studies have mostly been done for individual cytoskeleton systems in unconfined 3D or quasi-2D geometries, which lack complexity relative to a cellular environment. To increase the level of complexity, we present a method to study co-organization of two cytoskeletal components, namely microtubules and actin filaments, confined in cell-sized water-in-oil emulsion droplets. We show that centrosome-nucleated dynamic microtubules can be made to interact with actin filaments through a tip-tracking complex consisting of microtubule end-binding proteins and an actin-microtubule cytolinker. In addition to the protocols themselves, we discuss the optimization steps required in order to build these more complex in vitro model systems of cytoskeletal interactions.
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http://dx.doi.org/10.1007/978-1-0716-0219-5_5DOI Listing
January 2021

Revealing the assembly of filamentous proteins with scanning transmission electron microscopy.

PLoS One 2019 20;14(12):e0226277. Epub 2019 Dec 20.

Department of Living Matter, AMOLF, Amsterdam, the Netherlands.

Filamentous proteins are responsible for the superior mechanical strength of our cells and tissues. The remarkable mechanical properties of protein filaments are tied to their complex molecular packing structure. However, since these filaments have widths of several to tens of nanometers, it has remained challenging to quantitatively probe their molecular mass density and three-dimensional packing order. Scanning transmission electron microscopy (STEM) is a powerful tool to perform simultaneous mass and morphology measurements on filamentous proteins at high resolution, but its applicability has been greatly limited by the lack of automated image processing methods. Here, we demonstrate a semi-automated tracking algorithm that is capable of analyzing the molecular packing density of intra- and extracellular protein filaments over a broad mass range from STEM images. We prove the wide applicability of the technique by analyzing the mass densities of two cytoskeletal proteins (actin and microtubules) and of the main protein in the extracellular matrix, collagen. The high-throughput and spatial resolution of our approach allow us to quantify the internal packing of these filaments and their polymorphism by correlating mass and morphology information. Moreover, we are able to identify periodic mass variations in collagen fibrils that reveal details of their axially ordered longitudinal self-assembly. STEM-based mass mapping coupled with our tracking algorithm is therefore a powerful technique in the characterization of a wide range of biological and synthetic filaments.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0226277PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6924676PMC
March 2020

Uncovering the dynamic precursors to motor-driven contraction of active gels.

Soft Matter 2019 Oct;15(42):8552-8565

AMOLF, Living Matter Department, 1098 XG Amsterdam, The Netherlands.

Cells and tissues have the remarkable ability to actively generate the forces required to change their shape. This active mechanical behavior is largely mediated by the actin cytoskeleton, a crosslinked network of actin filaments that is contracted by myosin motors. Experiments and active gel theories have established that the length scale over which gel contraction occurs is governed by a balance between molecular motor activity and crosslink density. By contrast, the dynamics that govern the contractile activity of the cytoskeleton remain poorly understood. Here we investigate the microscopic dynamics of reconstituted actin-myosin networks using simultaneous real-space video microscopy and Fourier-space dynamic light scattering. Light scattering reveals different regimes of microscopic dynamics as a function of sample age. We uncover two dynamical precursors that precede macroscopic gel contraction. One is characterized by a progressive acceleration of stress-induced rearrangements, while the other consists of sudden, heterogeneous rearrangements. Intriguingly, our findings suggest a qualitative analogy between self-driven rupture and collapse of active gels and the delayed rupture of passive gels observed in earlier studies of colloidal gels under external loads.
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http://dx.doi.org/10.1039/c9sm01172bDOI Listing
October 2019

Cytolinker Gas2L1 regulates axon morphology through microtubule-modulated actin stabilization.

EMBO Rep 2019 11 5;20(11):e47732. Epub 2019 Sep 5.

Department of Biology, Cell Biology, Faculty of Science, Utrecht University, Utrecht, The Netherlands.

Crosstalk between the actin and microtubule cytoskeletons underlies cellular morphogenesis. Interactions between actin filaments and microtubules are particularly important for establishing the complex polarized morphology of neurons. Here, we characterized the neuronal function of growth arrest-specific 2-like 1 (Gas2L1), a protein that can directly bind to actin, microtubules and microtubule plus-end-tracking end binding proteins. We found that Gas2L1 promotes axon branching, but restricts axon elongation in cultured rat hippocampal neurons. Using pull-down experiments and in vitro reconstitution assays, in which purified Gas2L1 was combined with actin and dynamic microtubules, we demonstrated that Gas2L1 is autoinhibited. This autoinhibition is relieved by simultaneous binding to actin filaments and microtubules. In neurons, Gas2L1 primarily localizes to the actin cytoskeleton and functions as an actin stabilizer. The microtubule-binding tail region of Gas2L1 directs its actin-stabilizing activity towards the axon. We propose that Gas2L1 acts as an actin regulator, the function of which is spatially modulated by microtubules.
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http://dx.doi.org/10.15252/embr.201947732DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6831992PMC
November 2019

Direct Observation of Intrachain Hydrogen Bonds in Aqueous Hyaluronan.

J Phys Chem A 2019 Sep 12;123(38):8220-8225. Epub 2019 Sep 12.

AMOLF , Science Park 104 , 1098 XG Amsterdam , The Netherlands.

We use two-dimensional infrared spectroscopy to study the interactions between the amide and carboxylate anion groups of hyaluronan polymers at neutral pH. The spectra reveal the presence of intrachain hydrogen bonds between the amide and carboxylate anion groups in aqueous solution. We determine the relative orientation of the amide and carboxylate anion groups when forming this hydrogen bond and quantify the fraction of amide groups that participate in hydrogen bonding. We find that a variation of the pH and/or temperature has a negligible effect on this fraction, whereas the persistence length of the hyaluronan chains and the associated viscosity of hyaluronan solutions are known to change significantly. We conclude that the hydrogen bonding between the amide and carboxylate anion groups does not significantly contribute to the chain rigidity of hyaluronan polymers.
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http://dx.doi.org/10.1021/acs.jpca.9b06462DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6767362PMC
September 2019

Stiffening and inelastic fluidization in vimentin intermediate filament networks.

Soft Matter 2019 Sep;15(36):7127-7136

Living Matter Department, AMOLF, 1098 XG Amsterdam, The Netherlands.

Intermediate filaments are cytoskeletal proteins that are key regulators of cell mechanics, a role which is intrinsically tied to their hierarchical structure and their unique ability to accommodate large axial strains. However, how the single-filament response to applied strains translates to networks remains unclear, particularly with regards to the crosslinking role played by the filaments' disordered "tail" domains. Here we test the role of these noncovalent crosslinks in the nonlinear rheology of reconstituted networks of the intermediate filament protein vimentin, probing their stress- and rate-dependent mechanics. Similarly to previous studies we observe elastic stress-stiffening but unlike previous work we identify a characteristic yield stress σ*, above which the networks exhibit rate-dependent softening of the network, referred to as inelastic fluidization. By investigating networks formed from tail-truncated vimentin, in which noncovalent crosslinking is suppressed, and glutaraldehyde-treated vimentin, in which crosslinking is made permanent, we show that rate-dependent inelastic fluidization is a direct consequence of vimentin's transient crosslinking. Surprisingly, although the tail-tail crosslinks are individually weak, the effective timescale for stress relaxation of the network exceeds 1000 s at σ*. Vimentin networks can therefore maintain their integrity over a large range of strains (up to ∼1000%) and loading rates (10-3 to 10-1 s-1). Our results provide insight into how the hierarchical structure of vimentin networks contributes to the cell's ability to be deformable yet strong.
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http://dx.doi.org/10.1039/c9sm00590kDOI Listing
September 2019

Origin of Slow Stress Relaxation in the Cytoskeleton.

Phys Rev Lett 2019 May;122(21):218102

Living Matter Department, AMOLF, Science Park 104, 1098 XG Amsterdam, The Netherlands.

Dynamically cross-linked semiflexible biopolymers such as the actin cytoskeleton govern the mechanical behavior of living cells. Semiflexible biopolymers nonlinearly stiffen in response to mechanical loads, whereas the cross-linker dynamics allow for stress relaxation over time. Here we show, through rheology and theoretical modeling, that the combined nonlinearity in time and stress leads to an unexpectedly slow stress relaxation, similar to the dynamics of disordered systems close to the glass transition. Our work suggests that transient cross-linking combined with internal stress can explain prior reports of soft glassy rheology of cells, in which the shear modulus increases weakly with frequency.
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http://dx.doi.org/10.1103/PhysRevLett.122.218102DOI Listing
May 2019

Response of an actin network in vesicles under electric pulses.

Sci Rep 2019 05 31;9(1):8151. Epub 2019 May 31.

Department of Chemical Engineering, Delft University of Technology, Delft, The Netherlands.

We study the role of a biomimetic actin network 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 network provide a step towards understanding the discrepancies between the electroporation mechanism of a living cell and its simplified model of the empty GUV.
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http://dx.doi.org/10.1038/s41598-019-44613-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6544639PMC
May 2019

Shape and Size Control of Artificial Cells for Bottom-Up Biology.

ACS Nano 2019 05 16;13(5):5439-5450. Epub 2019 May 16.

Department of Bionanoscience, Kavli Institute of Nanoscience Delft , Delft University of Technology , Van der Maasweg 9 , 2629 HZ Delft , The Netherlands.

Bottom-up biology is an expanding research field that aims to understand the mechanisms underlying biological processes via in vitro assembly of their essential components in synthetic cells. As encapsulation and controlled manipulation of these elements is a crucial step in the recreation of such cell-like objects, microfluidics is increasingly used for the production of minimal artificial containers such as single-emulsion droplets, double-emulsion droplets, and liposomes. Despite the importance of cell morphology on cellular dynamics, current synthetic-cell studies mainly use spherical containers, and methods to actively shape manipulate these have been lacking. In this paper, we describe a microfluidic platform to deform the shape of artificial cells into a variety of shapes (rods and discs) with adjustable cell-like dimensions below 5 μm, thereby mimicking realistic cell morphologies. To illustrate the potential of our method, we reconstitute three biologically relevant protein systems (FtsZ, microtubules, collagen) inside rod-shaped containers and study the arrangement of the protein networks inside these synthetic containers with physiologically relevant morphologies resembling those found in living cells.
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http://dx.doi.org/10.1021/acsnano.9b00220DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6543616PMC
May 2019

Frustrated binding of biopolymer crosslinkers.

Soft Matter 2019 Apr;15(14):3036-3042

Living Matter Department, AMOLF, Science Park 104, 1098 XG Amsterdam, The Netherlands.

Transiently crosslinked actin filament networks allow cells to combine elastic rigidity with the ability to deform viscoelastically. Theoretical models of semiflexible polymer networks predict that the crosslinker unbinding rate governs the timescale beyond which viscoelastic flow occurs. However a direct comparison between network and crosslinker dynamics is lacking. Here we measure the network's stress relaxation timescale using rheology and the lifetime of bound crosslinkers using fluorescence recovery after photobleaching (FRAP). Intriguingly, we observe that the crosslinker unbinding rate measured by FRAP is more than an order of magnitude slower than the rate measured by rheology. We rationalize this difference with a three-state model where crosslinkers are bound to either 0, 1 or 2 filaments, which allows us to extract crosslinker transition rates that are otherwise difficult to access. We find that the unbinding rate of singly bound crosslinkers is nearly two orders of magnitude slower than for doubly bound ones. We attribute the increased unbinding rate of doubly bound crosslinkers to the high stiffness of biopolymers, which frustrates crosslinker binding.
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http://dx.doi.org/10.1039/c8sm02429dDOI Listing
April 2019

Molecular Origin of the Elastic State of Aqueous Hyaluronic Acid.

J Phys Chem B 2019 04 28;123(14):3043-3049. Epub 2019 Mar 28.

AMOLF , Science Park 104 , 1098 XG Amsterdam , The Netherlands.

The macroscopic mechanical properties of biological hydrogels are broadly studied and successfully mimicked in synthetic materials, but little is known about the molecular interactions that mediate these properties. Here, we use two-dimensional infrared spectroscopy to study the pH-induced gelation of hyaluronic acid, a ubiquitous biopolymer, which undergoes a transition from a viscous to an elastic state in a narrow pH range around 2.5. We find that the gelation originates from the enhanced formation of strong interchain connections, consisting of a double amide-COOH hydrogen bond and an N-D-COO hydrogen bond on the adjacent sugars of the hyaluronan disaccharide unit. We confirm the enhanced interchain connectivity in the elastic state by atomic force microscopy imaging.
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http://dx.doi.org/10.1021/acs.jpcb.9b00982DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6466474PMC
April 2019

Automated Tracking of Biopolymer Growth and Network Deformation with TSOAX.

Sci Rep 2019 02 8;9(1):1717. Epub 2019 Feb 8.

Department of Physics, Lehigh University, Bethlehem, PA, 18015, USA.

Studies of how individual semi-flexible biopolymers and their network assemblies change over time reveal dynamical and mechanical properties important to the understanding of their function in tissues and living cells. Automatic tracking of biopolymer networks from fluorescence microscopy time-lapse sequences facilitates such quantitative studies. We present an open source software tool that combines a global and local correspondence algorithm to track biopolymer networks in 2D and 3D, using stretching open active contours. We demonstrate its application in fully automated tracking of elongating and intersecting actin filaments, detection of loop formation and constriction of tilted contractile rings in live cells, and tracking of network deformation under shear deformation.
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http://dx.doi.org/10.1038/s41598-018-37182-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6368602PMC
February 2019

Polarity sorting drives remodeling of actin-myosin networks.

J Cell Sci 2018 12 13;132(4). Epub 2018 Dec 13.

Department of Living Matter, AMOLF, Science Park 104, 1098 XG Amsterdam, The Netherlands

Cytoskeletal networks of actin filaments and myosin motors drive many dynamic cell processes. A key characteristic of these networks is their contractility. Despite intense experimental and theoretical efforts, it is not clear what mechanism favors network contraction over expansion. Recent work points to a dominant role for the nonlinear mechanical response of actin filaments, which can withstand stretching but buckle upon compression. Here, we present an alternative mechanism. We study how interactions between actin and myosin-2 at the single-filament level translate into contraction at the network scale by performing time-lapse imaging on reconstituted quasi-2D networks mimicking the cell cortex. We observe myosin end-dwelling after it runs processively along actin filaments. This leads to transport and clustering of actin filament ends and the formation of transiently stable bipolar structures. Further, we show that myosin-driven polarity sorting produces polar actin asters, which act as contractile nodes that drive contraction in crosslinked networks. Computer simulations comparing the roles of the end-dwelling mechanism and a buckling-dependent mechanism show that the relative contribution of end-dwelling contraction increases as the network mesh-size decreases.
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http://dx.doi.org/10.1242/jcs.219717DOI Listing
December 2018
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