Biology subjects

Koenderink, G. H.

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

28 records · Page 2Linked to original sources

bioRxiv2021

Cross-linkers at growing microtubule ends generate forces that drive actin transport

The actin and microtubule cytoskeletons form active networks in the cell that can contract and remodel, resulting in vital cellular processes as cell division and motility. Motor proteins play an important role in generating the forces required for these processes, but more recently the concept of passive cross-linkers being able to generate forces has emerged. So far, these passive cross-linkers have been studied in the context of separate actin and microtubule systems. Here, we show that crosslinkers also allow actin and microtubules to exert forces on each other. More specifically, we study single actin filaments that are cross-linked to growing microtubule ends, using in vitro reconstitution, computer simulations, and a minimal theoretical model. We show that microtubules can transport actin filaments over large (micrometer-range) distances, and find that this transport results from two antagonistic forces arising from the binding of cross-linkers to the overlap between the actin and microtubule filaments. The cross-linkers attempt to maximize the overlap between the actin and the tip of the growing microtubules, creating an affinity-driven forward condensation force, and simultaneously create a competing friction force along the microtubule lattice. We predict and verify experimentally how the average transport time depends on the actin filament length and the microtubule growth velocity, confirming the competition between a forward condensation force and a backward friction force. In addition, we theoretically predict and experimentally verify that the condensation force is of the order of 0.1 pN. Thus, our results reveal a new mechanism for local actin remodeling by growing microtubules. Significance StatementComplex cellular processes such as cell migration require coordinated remodeling of both the actin and the microtubule cytoskeleton. The two networks for instance exert forces on each other via active motor proteins. Here we show that, surprisingly, coupling via passive cross-linkers can also result in force generation. We specifically study the transport of actin filaments by growing microtubule ends. We show by cell-free reconstitution experiments, computer simulations, and theoretical modeling that this transport is driven by the affinity of the cross-linker for the chemically distinct microtubule tip region. Our work predicts that growing microtubules could potentially rapidly relocate newly nucleated actin filaments to the leading edge of the cell and thus boost migration.

biophysics↗

bioRxiv2021

The molecular structure and surface accumulation dynamics of hyaluronan at the water/air interface

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). We find that high-molecular weight hyaluronan (>1 MDa) does not come to the surface, even hours after addition of the polymer to the aqueous solution. In contrast, low-molecular weight hyaluronan (~150 kDa) gradually covers the water-air interface within hours, leading to a negatively charged surface and a reorientation of the interfacial water molecules. This strong dependence on the polymer molecular weight can be explainend from entanglements of the hyaluronan polymers. We also find that 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 the hyaluronan polymers.

biophysics↗

bioRxiv2021

Optimized cDICE for efficient reconstitution of biological systems in giant unilamellar vesicles

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 minimal 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.

synthetic biology↗

bioRxiv2020

Membrane binding controls ordered self-assembly of animal septins

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 membrane binding 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 septins 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.

cell biology↗

bioRxiv2020

Strong reduction of the chain rigidity of hyaluronan by selective binding of Ca2+ ions

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 intra-molecular 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 Ca2+ strongly reduces the probability of subsequent binding of Ca2+ 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. TOC O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=74 SRC="FIGDIR/small/277194v1_ufig1.gif" ALT="Figure 1"> View larger version (56K): org.highwire.dtl.DTLVardef@d0e95borg.highwire.dtl.DTLVardef@1886d60org.highwire.dtl.DTLVardef@1c4c4aborg.highwire.dtl.DTLVardef@14bcf0d_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2020

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

Fibrin is the major extracellular component of blood clots and a proteinaceous hydrogel used as a versatile biomaterial. Fibrin forms branched networks of polymeric fibers, 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 structure of fibrin fibers. Here we quantitatively assess the packing structure of fibrin fibers by combining Small Angle X-ray Scattering (SAXS) measurements of fibrin networks reconstituted under a wide range of conditions with computational molecular modeling of fibrin oligomers. 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.

biophysics↗

bioRxiv2019

Revealing the Assembly of Filamentous Proteins with Scanning Transmission Electron Microscopy

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.

biophysics↗

bioRxiv2018

Frustrated binding of biopolymer crosslinkers

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 networks stress relaxation timescale using rheology and the lifetime of bound crosslinkers using fluorescence recovery after photobleaching. lntruigingly, 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 double bound ones. We attribute the increased unbinding rate of doubly bound crosslinkers to the high stiffness of biopolymers, which frustrates crosslinker binding.

biophysics↗

bioRxiv2018

Automated Tracking of Biopolymer Growth and Network Deformation with TSOAX

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.

cell biology↗

bioRxiv2018

Polarity sorting drives remodeling of actin-myosin networks

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 leads to polar actin aster formation, 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.

biophysics↗