Spatiotemporal dynamics of self-organized branching in pancreas-derived organoids.

The development dynamics and self-organization of glandular branched epithelia is of utmost importance for our understanding of diverse processes ranging from normal tissue growth to the growth of cancerous tissues. Using single primary murine pancreatic ductal adenocarcinoma (PDAC) cells embedded in a collagen matrix and adapted media supplementation, we generate organoids that self-organize into highly branched structures displaying a seamless lumen connecting terminal end buds, replicating in vivo PDAC architecture. We identify distinct morphogenesis phases, each characterized by a unique pattern of cell invasion, matrix deformation, protein expression, and respective molecular dependencies. We propose a minimal theoretical model of a branching and proliferating tissue, capturing the dynamics of the first phases. Observing the interaction of morphogenesis, mechanical environment and gene expression in vitro sets a benchmark for the understanding of self-organization processes governing complex organoid structure formation processes and branching morphogenesis.

VASP localization to lipid bilayers induces polymerization driven actin bundle formation.

Actin bundles constitute important cytoskeleton structures and enable a scaffold for force transmission inside cells. Actin bundles are formed by proteins, with multiple F-actin binding domains cross-linking actin filaments to each other. Vasodilator-stimulated phosphoprotein (VASP) has mostly been reported as an actin elongator, but it has been shown to be a bundling protein as well and is found in bundled actin structures at filopodia and adhesion sites. Based on in vitro experiments, it remains unclear when and how VASP can act as an actin bundler or elongator. Here we demonstrate that VASP bound to membranes facilitates the formation of large actin bundles during polymerization. The alignment by polymerization requires the fluidity of the lipid bilayers. The mobility within the bilayer enables VASP to bind to filaments and capture and track growing barbed ends. VASP itself phase separates into a protein-enriched phase on the bilayer. This VASP-rich phase nucleates and accumulates at bundles during polymerization, which in turn leads to a reorganization of the underlying lipid bilayer. Our findings demonstrate that the nature of VASP localization is decisive for its function. The up-concentration based on VASP's affinity to actin during polymerization enables it to simultaneously fulfill the function of an elongator and a bundler.

Reversible and spatiotemporal control of colloidal structure formation.

Tuning colloidal structure formation is a powerful approach to building functional materials, as a wide range of optical and viscoelastic properties can be accessed by the choice of individual building blocks and their interactions. Precise control is achieved by DNA specificity, depletion forces, or geometric constraints and results in a variety of complex structures. Due to the lack of control and reversibility of the interactions, an autonomous oscillating system on a mesoscale without external driving was not feasible until now. Here, we show that tunable DNA reaction circuits controlling linker strand concentrations can drive the dynamic and fully reversible assembly of DNA-functionalized micron-sized particles. The versatility of this approach is demonstrated by programming colloidal interactions in sequential and spatial order to obtain an oscillatory structure formation process on a mesoscopic scale. The experimental results represent an approach for the development of active materials by using DNA reaction networks to scale up the dynamic control of colloidal self-organization.

Mechanical plasticity of collagen directs branch elongation in human mammary gland organoids.

Epithelial branch elongation is a central developmental process during branching morphogenesis in diverse organs. This fundamental growth process into large arborized epithelial networks is accompanied by structural reorganization of the surrounding extracellular matrix (ECM), well beyond its mechanical linear response regime. Here, we report that epithelial ductal elongation within human mammary organoid branches relies on the non-linear and plastic mechanical response of the surrounding collagen. Specifically, we demonstrate that collective back-and-forth motion of cells within the branches generates tension that is strong enough to induce a plastic reorganization of the surrounding collagen network which results in the formation of mechanically stable collagen cages. Such matrix encasing in turn directs further tension generation, branch outgrowth and plastic deformation of the matrix. The identified mechanical tension equilibrium sets a framework to understand how mechanical cues can direct ductal branch elongation.

Intra-bundle contractions enable extensile properties of active actin networks.

The cellular cortex is a dynamic and contractile actomyosin network modulated by actin-binding proteins. We reconstituted a minimal cortex adhered to a model cell membrane mimicking two processes mediated by the motor protein myosin: contractility and high turnover of actin monomers. Myosin reorganized these networks by extensile intra­bundle contractions leading to an altered growth mechanism. Hereby, stress within tethered bundles induced nicking of filaments followed by repair via incorporation of free monomers. This mechanism was able to break the symmetry of the previously disordered network resulting in the generation of extensile clusters, reminiscent of structures found within cells.

The dynamics of actin network turnover is self-organized by a growth-depletion feedback.

The dynamics of actin networks is modulated by a machinery consisting of actin binding proteins that control the turnover of filaments in space and time. To study this complex orchestration, in vitro reconstitution approaches strive to project actin dynamics in ideal, minimal systems. To this extent we reconstitute a self-supplying, dense network of globally treadmilling filaments. In this system we analyze growth and intrinsic turnover by means of FRAP measurements and thereby demonstrate how the depletion of monomers and actin binding partners modulate the dynamics in active actin networks. The described effects occur only in dense networks, as single filament dynamics are unable to produce depletion effects to this extent. Furthermore, we demonstrate a synergistic relationship between the nucleators formin and Arp2/3 when branched networks and formin-induced networks are colocalized. As a result, the formin-enhanced filament turnover depletes cofilin at the surface and thus protects the dense, Arp2/3 polymerized network from debranching. Ultimately, these results may be key for understanding the maintenance of the two contradicting requirements of network stability and dynamics in cells.

Transient self-organisation of DNA coated colloids directed by enzymatic reactions.

Dynamic self-organisation far from equilibrium is a key concept towards building autonomously acting materials. Here, we report the coupling of an antagonistic enzymatic reaction of RNA polymerisation and degradation to the aggregation of micron sized DNA coated colloids into fractal structures. A transient colloidal aggregation process is controlled by competing reactions of RNA synthesis of linker strands by a RNA polymerase and their degradation by a ribonuclease. By limiting the energy supply (NTP) of the enzymatic reactions, colloidal clusters form and subsequently disintegrate without the need of external stimuli. Here, the autonomous colloidal aggregation and disintegration can be modulated in terms of lifetime and cluster size. By restricting the enzyme activity locally, a directed spatial propagation of a colloidal aggregation and disintegration front is realised.

Emergence of coexisting ordered states in active matter systems.

Active systems can produce a far greater variety of ordered patterns than conventional equilibrium systems. In particular, transitions between disorder and either polar- or nematically ordered phases have been predicted and observed in two-dimensional active systems. However, coexistence between phases of different types of order has not been reported. We demonstrate the emergence of dynamic coexistence of ordered states with fluctuating nematic and polar symmetry in an actomyosin motility assay. Combining experiments with agent-based simulations, we identify sufficiently weak interactions that lack a clear alignment symmetry as a prerequisite for coexistence. Thus, the symmetry of macroscopic order becomes an emergent and dynamic property of the active system. These results provide a pathway by which living systems can express different types of order by using identical building blocks.

Reconstitution of composite actin and keratin networks in vesicles.

Although cytoskeletal networks are interpenetrating and interacting in living cells, very little is understood as to the effect their interaction has on their properties. Here, as a step towards elucidating the synergistic cellular role of these structural proteins, we investigate isolated keratin and actin composites and show how the in vitro network formation of keratin influences the properties of actin networks and vice versa. By encapsulating purified composite networks into vesicles and separating the time scales of network formation we are able to demonstrate that the actin network stabilizes keratin networks by providing an elastic resistance to their collapse in vitro.

The mechanical properties of polymer-colloid hybrid hydrogels.

The incorporation of monodisperse colloidal particles in hydrogels is a promising approach to create hybrid gels with unique structural, mechanical and functional properties. However, the colloidal structure formation within the hydrogels often remains uncontrolled, leaving behind possible mechanically synergetic effects of the polymeric and the colloidal system. Here we show that colloidal structure formation within the hybrid gels has a significant influence on the elasticity and toughness of the hybrid gels. We combine a polyacrylamide hydrogel with DNA coated colloids (DNAcc), where structure formation can be triggered independently at different points in time. Consequently, we are able to create hybrid gels that are composed of the same components, but do differ in explicit colloidal structure. While monodisperse colloids enhance the storage modulus of the gels, the yield strain is simultaneously drastically reduced. The toughness of these brittle hybrid gels is rescued by colloidal structure formation at higher polyacrylamide concentrations. The toughness is increased at lower polyacrylamide concentrations. We show that the toughness of the hydrogels at 10% (w/v) polyacrylamide and 4% (v/v) DNAcc can be increased by a factor of approx. 35, indicating that control over colloidal structure formation yields access to significant synergetic effects in polymer-colloid hybrid gels.

The microstructure and micromechanics of the tendon-bone insertion.

The exceptional mechanical properties of the load-bearing connection of tendon to bone rely on an intricate interplay of its biomolecular composition, microstructure and micromechanics. Here we identify that the Achilles tendon-bone insertion is characterized by an interface region of ∼500 µm with a distinct fibre organization and biomolecular composition. Within this region, we identify a heterogeneous mechanical response by micromechanical testing coupled with multiscale confocal microscopy. This leads to localized strains that can be larger than the remotely applied strain. The subset of fibres that sustain the majority of loading in the interface area changes with the angle of force application. Proteomic analysis detects enrichment of 22 proteins in the interfacial region that are predominantly involved in cartilage and skeletal development as well as proteoglycan metabolism. The presented mechanisms mark a guideline for further biomimetic strategies to rationally design hard-soft interfaces.

A single charge in the actin binding domain of fascin can independently tune the linear and non-linear response of an actin bundle network.

Actin binding proteins (ABPs) not only set the structure of actin filament assemblies but also mediate the frequency-dependent viscoelastic moduli of cross-linked and bundled actin networks. Point mutations in the actin binding domain of those ABPs can tune the association and dissociation dynamics of the actin/ABP bond and thus modulate the network mechanics both in the linear and non-linear response regime. We here demonstrate how the exchange of a single charged amino acid in the actin binding domain of the ABP fascin triggers such a modulation of the network rheology. Whereas the overall structure of the bundle networks is conserved, the transition point from strain-hardening to strain-weakening sensitively depends on the cross-linker off-rate and the applied shear rate. Our experimental results are consistent both with numerical simulations of a cross-linked bundle network and a theoretical description of the bundle network mechanics which is based on non-affine bending deformations and force-dependent cross-link dynamics.

Active compaction of crosslinked driven filament networks.

The contractile ability of active materials relies on the interplay of force-exerting and force-bearing structures. However, the complexity of interactions and limited parameter control of many model systems are major obstacles in advancing our understanding of the underlying fundamental principles. To shed light on these principles we introduce and analyse a minimal reconstituted system, consisting of highly concentrated actin filaments that are crosslinked by α-actinin and actively transported in the two-dimensional geometry of a motility assay. This minimal system actively compacts and evolves into highly compact fibres that exceed the length of the individual filaments by two orders of magnitude. We identify the interplay between active transport and crosslinking to be responsible for the observed active compaction. This enables us to control the structure and the length scale of active compaction.

Slow dynamics and internal stress relaxation in bundled cytoskeletal networks.

Crosslinked and bundled actin filaments form networks that are essential for the mechanical properties of living cells. Reconstituted actin networks have been extensively studied not only as a model system for the cytoskeleton, but also to understand the interplay between microscopic structure and macroscopic viscoelastic properties of network-forming soft materials. These constitute a broad class of materials with countless applications in science and industry. So far, it has been widely assumed that reconstituted actin networks represent equilibrium structures. Here, we show that fully polymerized actin/fascin bundle networks exhibit surprising age-dependent changes in their viscoelastic properties and spontaneous dynamics, a feature strongly reminiscent of out-of-equilibrium, or glassy, soft materials. Using a combination of rheology, confocal microscopy and space-resolved dynamic light scattering, we demonstrate that actin networks build up stress during their formation and then slowly relax towards equilibrium owing to the unbinding dynamics of the crosslinking molecules.

Cyclic hardening in bundled actin networks.

Nonlinear deformations can irreversibly alter the mechanical properties of materials. Most soft materials, such as rubber and living tissues, display pronounced softening when cyclically deformed. Here we show that, in contrast, reconstituted networks of crosslinked, bundled actin filaments harden when subject to cyclical shear. As a consequence, they exhibit a mechano-memory where a significant stress barrier is generated at the maximum of the cyclic shear strain. This unique response is crucially determined by the network architecture: at lower crosslinker concentrations networks do not harden, but soften showing the classic Mullins effect known from rubber-like materials. By simultaneously performing macrorheology and confocal microscopy, we show that cyclic shearing results in structural reorganization of the network constituents such that the maximum applied strain is encoded into the network architecture.

Structural and viscoelastic properties of actin/filamin networks: cross-linked versus bundled networks.

The high diversity of cytoskeletal actin structures is accomplished by myriads of actin binding proteins (ABPs). Depending on its concentration, even a single type of ABP can induce different actin microstructures. Thus, for an overall understanding of the cytoskeleton, a detailed characterization of the cross-linker's effect on structural and mechanical properties of actin networks is required for each ABP. Using confocal microscopy and macrorheology, we investigate both cross-linked and bundled actin/filamin networks and compare their microstructures as well as their viscoelastic properties in the linear and the nonlinear regime.

Cytoskeletal polymer networks: viscoelastic properties are determined by the microscopic interaction potential of cross-links.

Although the structure of cross-linking molecules mainly determines the structural organization of actin filaments and with that the static elastic properties of the cytoskeleton, it is largely unknown how the biochemical characteristics of transiently cross-linking proteins (actin-binding proteins (ABPs)) affect the viscoelasticity of actin networks. In this study, we show that the macroscopic network response of reconstituted actin networks can be traced back to the microscopic interaction potential of an individual actin/ABP bond. The viscoelastic response of cross-linked actin networks is set by the cross-linker off-rate, the binding energy, and the characteristic bond length of individual actin/ABP interactions.

Transient binding and dissipation in cross-linked actin networks.

In contrast with entangled actin solutions, transiently cross-linked actin networks can provide highly elastic properties while still allowing for local rearrangements in the microstructure-on biological relevant time scales. Here, we show that thermal unbinding of transient cross-links entails local stress relaxation and energy dissipation in an intermediate elasticity dominated frequency regime. We quantify the viscoelastic response of an isotropically cross-linked actin network by experimentally tuning the off rate of the transiently cross-linking molecules, their density, and the solvent viscosity. We reproduce the measured frequency response by a semiphenomenological model that is predicated on microscopic unbinding events.

Cross-linking molecules modify composite actin networks independently.

While cells make use of many actin binding proteins (ABPs) simultaneously to tailor the mechanical properties of the cytoskeleton, the detailed interplay of different ABPs is not understood. By a combination of macrorheological measurements and confocal microscopy, we show that the ABPs fascin and filamin modify the structural and viscoelastic properties of composite in vitro actin networks independently. The outnumbering ABP dictates the local network structure and therefore also dominates the macromechanical network response.

Helical twist controls the thickness of F-actin bundles.

In the presence of condensing agents such as nonadsorbing polymer, multivalent counter ions, and specific bundling proteins, chiral biopolymers typically form bundles with a finite thickness, rather than phase-separating into a polymer-rich phase. Although short-range repulsive interactions or geometrical frustrations are thought to force the equilibrium bundle size to be limited, the precise mechanism is yet to be resolved. The importance of the tight control of biopolymer bundle size is illustrated by the ubiquitous cytoskeletal actin filament bundles that are crucial for the proper functioning of cells. Using an in vitro model system, we show that size control relies on a mismatch between the helical structure of individual actin filaments and the geometric packing constraints within bundles. Small rigid actin-binding proteins change the twist of filamentous actin (F-actin) in a concentration-dependent manner, resulting in small, well defined bundle thickness up to approximately 20 filaments, comparable to those found in filopodia. Other F-actin cross-linking proteins can subsequently link these small, well organized bundles into larger structures of several hundred filaments, comparable to those found in, for example, Drosophila bristles. The energetic tradeoff between filament twisting and cross-linker binding within a bundle is suggested as a fundamental mechanism by which cells can precisely adjust bundle size and strength.