Enzymatic cleaving of entangled DNA rings drives scale-dependent rheological trajectories.

DNA, which naturally occurs in linear, ring, and supercoiled topologies, frequently undergoes enzyme-driven topological conversion and fragmentation in vivo, enabling it to perform a variety of functions within the cell. In vitro, highly concentrated DNA polymers form entanglements that yield viscoelastic properties dependent on the topologies and lengths of the DNA. Enzyme-driven alterations of DNA size and shape therefore offer a means of designing active materials with programmable viscoelastic properties. Here, we incorporate multi-site restriction endonucleases into dense DNA solutions to linearize and fragment circular DNA molecules. We pair optical tweezers microrheology with differential dynamic microscopy and single-molecule tracking to measure the linear and nonlinear viscoelastic response and transport properties of entangled DNA solutions over a wide range of spatiotemporal scales throughout the course of enzymatic digestion. We show that, at short timescales, relative to the relaxation timescales of the polymers, digestion of these 'topologically-active' fluids initially causes an increase in elasticity and relaxation times followed by a gradual decrease. Conversely, for long timescales, linear viscoelastic moduli exhibit signatures of increasing elasticity. DNA diffusion, likewise, becomes increasingly slowed, in direct opposition to the short-time behavior. We hypothesize that this scale-dependent rheology arises from the population of small DNA fragments, which increases as digestion proceeds, driving self-association of larger fragments via depletion interactions, giving rise to slow relaxation modes of clusters of entangled chains, interspersed among shorter unentangled fragments. While these slow modes likely dominate at long times, they are presumably frozen out in the short-time limit, which instead probes the faster relaxation modes of the unentangled population.

Design and Building of a Customizable, Single-Objective, Light-Sheet Fluorescence Microscope for the Visualization of Cytoskeleton Networks.

Reconstituted cytoskeleton composites have emerged as a valuable model system for studying non-equilibrium soft matter. The faithful capture of the dynamics of these 3D, dense networks calls for optical sectioning, which is often associated with fluorescence confocal microscopes. However, recent developments in light-sheet fluorescence microscopy (LSFM) have established it as a cost-effective and, at times, superior alternative. To make LSFM accessible to cytoskeleton researchers less familiar with optics, we present a step-by-step beginner's guide to building a versatile light-sheet fluorescence microscope from off-the-shelf components. To enable sample mounting with traditional slide samples, this LSFM follows the single-objective light-sheet (SOLS) design, which utilizes a single objective for both the excitation and emission collection. We describe the function of each component of the SOLS in sufficient detail to allow readers to modify the instrumentation and design it to fit their specific needs. Finally, we demonstrate the use of this custom SOLS instrument by visualizing asters in kinesin-driven microtubule networks.

Kinesin and myosin motors compete to drive rich multiphase dynamics in programmable cytoskeletal composites.

The cellular cytoskeleton relies on diverse populations of motors, filaments, and binding proteins acting in concert to enable nonequilibrium processes ranging from mitosis to chemotaxis. The cytoskeleton's versatile reconfigurability, programmed by interactions between its constituents, makes it a foundational active matter platform. However, current active matter endeavors are limited largely to single force-generating components acting on a single substrate-far from the composite cytoskeleton in cells. Here, we engineer actin-microtubule (MT) composites, driven by kinesin and myosin motors and tuned by crosslinkers, to ballistically restructure and flow with speeds that span three orders of magnitude depending on the composite formulation and time relative to the onset of motor activity. Differential dynamic microscopy analyses reveal that kinesin and myosin compete to delay the onset of acceleration and suppress discrete restructuring events, while passive crosslinking of either actin or MTs has an opposite effect. Our minimal advection-diffusion model and spatial correlation analyses correlate these dynamics to structure, with motor antagonism suppressing reconfiguration and demixing, while crosslinking enhances clustering. Despite the rich formulation space and emergent formulation-dependent structures, the nonequilibrium dynamics across all composites and timescales can be organized into three classes-slow isotropic reorientation, fast directional flow, and multimode restructuring. Moreover, our mathematical model demonstrates that diverse structural motifs can arise simply from the interplay between motor-driven advection and frictional drag. These general features of our platform facilitate applicability to other active matter systems and shed light on diverse ways that cytoskeletal components can cooperate or compete to enable wide-ranging cellular processes.

Cooperative Rheological State-Switching of Enzymatically-Driven Composites of Circular DNA And Dextran.

Polymer topology, which plays a principal role in the rheology of polymeric fluids, and non-equilibrium materials, which exhibit time-varying rheological properties, are topics of intense investigation. Here, composites of circular DNA and dextran are pushed out-of-equilibrium via enzymatic digestion of DNA rings to linear fragments. These time-resolved rheology measurements reveal discrete state-switching, with composites undergoing abrupt transitions between dissipative and elastic-like states. The gating time and lifetime of the elastic-like states, and the magnitude and sharpness of the transitions, are surprisingly decorrelated from digestion rates and non-monotonically depend on the DNA fraction. These results are modeled using sigmoidal two-state functions to show that bulk state-switching can arise from continuous molecular-level activity due to the necessity for cooperative percolation of entanglements to support macroscopic stresses. This platform, coupling the tunability of topological composites with the power of enzymatic reactions, may be leveraged for diverse material applications from wound-healing to self-repairing infrastructure.

Crowding and confinement act in concert to slow DNA diffusion within cell-sized droplets.

Dynamics of biological macromolecules, such as DNA, in crowded and confined environments are critical to understanding cellular processes such as transcription, infection, and replication. However, the combined effects of cellular confinement and crowding on macromolecular dynamics remain poorly understood. Here, we use differential dynamic microscopy to investigate the diffusion of large DNA molecules confined in cell-sized droplets and crowded by dextran polymers. We show that confined and crowded DNA molecules exhibit universal anomalous subdiffusion with scaling that is insensitive to the degree of confinement and crowding. However, effective DNA diffusion coefficients D e f f decrease up to 2 orders of magnitude as droplet size decreases-an effect that is enhanced by increased crowding. We mathematically model the coupling of crowding and confinement by combining polymer scaling theories with confinement-induced depletion effects. The generality and tunability of our system and models render them applicable to elucidating wide-ranging crowded and confined systems.

Reconstituting and Characterizing Actin-Microtubule Composites with Tunable Motor-Driven Dynamics and Mechanics.

The composite cytoskeleton, comprising interacting networks of semiflexible actin filaments and rigid microtubules, restructures and generates forces using motor proteins such as myosin II and kinesin to drive key processes such as migration, cytokinesis, adhesion, and mechanosensing. While actin-microtubule interactions are key to the cytoskeleton's versatility and adaptability, an understanding of their interplay with myosin and kinesin activity is still nascent. This work describes how to engineer tunable three-dimensional composite networks of co-entangled actin filaments and microtubules that undergo active restructuring and ballistic motion, driven by myosin II and kinesin motors, and are tuned by the relative concentrations of actin, microtubules, motor proteins, and passive crosslinkers. Protocols for fluorescence labeling of the microtubules and actin filaments to most effectively visualize composite restructuring and motion using multi-spectral confocal imaging are also detailed. Finally, the results of data analysis methods that can be used to quantitatively characterize non-equilibrium structure, dynamics, and mechanics are presented. Recreating and investigating this tunable biomimetic platform provides valuable insight into how coupled motor activity, composite mechanics, and filament dynamics can lead to myriad cellular processes from mitosis to polarization to mechano-sensation.

Optical-Tweezers-integrating-Differential-Dynamic-Microscopy maps the spatiotemporal propagation of nonlinear strains in polymer blends and composites.

How local stresses propagate through polymeric fluids, and, more generally, how macromolecular dynamics give rise to viscoelasticity are open questions vital to wide-ranging scientific and industrial fields. Here, to unambiguously connect polymer dynamics to force response, and map the deformation fields that arise in macromolecular materials, we present Optical-Tweezers-integrating-Differential -Dynamic-Microscopy (OpTiDMM) that simultaneously imposes local strains, measures resistive forces, and analyzes the motion of the surrounding polymers. Our measurements with blends of ring and linear polymers (DNA) and their composites with stiff polymers (microtubules) uncover an unexpected resonant response, in which strain alignment, superdiffusivity, and elasticity are maximized when the strain rate is comparable to the entanglement rate. Microtubules suppress this resonance, while substantially increasing elastic storage, due to varying degrees to which the polymers buildup, stretch and flow along the strain path, and configurationally relax induced stress. More broadly, the rich multi-scale coupling of mechanics and dynamics afforded by OpTiDDM, empowers its interdisciplinary use to elucidate non-trivial phenomena that sculpt stress propagation dynamics-critical to commercial applications and cell mechanics alike.

Quantifying Cytoskeleton Dynamics Using Differential Dynamic Microscopy.

Cells can crawl, self-heal, and tune their stiffness due to their remarkably dynamic cytoskeleton. As such, reconstituting networks of cytoskeletal biopolymers may lead to a host of active and adaptable materials. However, engineering such materials with precisely tuned properties requires measuring how the dynamics depend on the network composition and synthesis methods. Quantifying such dynamics is challenged by variations across the time, space, and formulation space of composite networks. The protocol here describes how the Fourier analysis technique, differential dynamic microscopy (DDM), can quantify the dynamics of biopolymer networks and is particularly well suited for studies of cytoskeleton networks. DDM works on time sequences of images acquired using a range of microscopy modalities, including laser-scanning confocal, widefield fluorescence, and brightfield imaging. From such image sequences, one can extract characteristic decorrelation times of density fluctuations across a span of wave vectors. A user-friendly, open-source Python package to perform DDM analysis is also developed. With this package, one can measure the dynamics of labeled cytoskeleton components or of embedded tracer particles, as demonstrated here with data of intermediate filament (vimentin) networks and active actin-microtubule networks. Users with no prior programming or image processing experience will be able to perform DDM using this software package and associated documentation.

Motor-driven advection competes with crowding to drive spatiotemporally heterogeneous transport in cytoskeleton composites.

The cytoskeleton-a composite network of biopolymers, molecular motors, and associated binding proteins-is a paradigmatic example of active matter. Particle transport through the cytoskeleton can range from anomalous and heterogeneous subdiffusion to superdiffusion and advection. Yet, recapitulating and understanding these properties-ubiquitous to the cytoskeleton and other out-of-equilibrium soft matter systems-remains challenging. Here, we combine light sheet microscopy with differential dynamic microscopy and single-particle tracking to elucidate anomalous and advective transport in actomyosin-microtubule composites. We show that particles exhibit multi-mode transport that transitions from pronounced subdiffusion to superdiffusion at tunable crossover timescales. Surprisingly, while higher actomyosin content increases the range of timescales over which transport is superdiffusive, it also markedly increases the degree of subdiffusion at short timescales and generally slows transport. Corresponding displacement distributions display unique combinations of non-Gaussianity, asymmetry, and non-zero modes, indicative of directed advection coupled with caged diffusion and hopping. At larger spatiotemporal scales, particles in active composites exhibit superdiffusive dynamics with scaling exponents that are robust to changing actomyosin fractions, in contrast to normal, yet faster, diffusion in networks without actomyosin. Our specific results shed important new light on the interplay between non-equilibrium processes, crowding and heterogeneity in active cytoskeletal systems. More generally, our approach is broadly applicable to active matter systems to elucidate transport and dynamics across scales.

Active cytoskeletal composites display emergent tunable contractility and restructuring.

The cytoskeleton is a model active matter system that controls processes as diverse as cell motility and mechanosensing. While both active actomyosin dynamics and actin-microtubule interactions are key to the cytoskeleton's versatility and adaptability, an understanding of their interplay is lacking. Here, we couple microscale experiments with mechanistic modeling to elucidate how connectivity, rigidity, and force-generation affect emergent material properties in composite networks of actin, tubulin, and myosin. We use multi-spectral imaging, time-resolved differential dynamic microscopy and spatial image autocorrelation to show that ballistic contraction occurs in composites with sufficient flexibility and motor density, but that a critical fraction of microtubules is necessary to sustain controlled dynamics. The active double-network models we develop, which recapitulate our experimental findings, reveal that while percolated actomyosin networks are essential for contraction, only composites with comparable actin and microtubule densities can simultaneously resist mechanical stresses while supporting substantial restructuring. The comprehensive phase map we present not only provides important insight into the different routes the cytoskeleton can use to alter its dynamics and structure, but also serves as a much-needed blueprint for designing cytoskeleton-inspired materials that couple tunability with resilience and adaptability for diverse applications ranging from wound healing to soft robotics.

Core-shell droplets and microcapsules formed through liquid-liquid phase separation of a colloid-polymer mixture.

Microcapsules allow for the controlled containment, transport, and release of cargoes ranging from pharmaceuticals to fragrances. Given the interest from a variety of industries in microcapsules and other core-shell structures, a multitude of fabrication strategies exist. Here, we report on a method relying on a mixture of temperature-responsive microgel particles, poly(N-isopropylacrylamide) (pNIPAM), and a polymer which undergo fluid-fluid phase separation. At room temperature this mixture separates into colloid-rich (liquid) and colloid-poor (gas) fluids. By heating the sample above a critical temperature where the microgel particles shrink dramatically and develop a more deeply attractive interparticle potential, the droplets of the colloid-rich phase become gel-like. As the temperature is lowered back to room temperature, these droplets of gelled colloidal particles reliquefy and phase separation within the droplet occurs. This phase separation leads to colloid-poor droplets within the colloid-rich droplets surrounded by a continuous colloid-poor phase. The gas/liquid/gas all-aqueous double emulsion lasts only a few minutes before a majority of the inner droplets escape. However, the colloid-rich shell of the core-shell droplets can solidify with the addition of salt. That this method creates core-shell structures with a shell composed of stimuli-sensitive microgel colloidal particles using only aqueous components makes it attractive for encapsulating biological materials and making capsules that respond to changes in, for example, temperature, salt concentration, or pH.

Two-color differential dynamic microscopy for capturing fast dynamics.

Differential dynamic microscopy (DDM) is increasingly used in the fields of soft matter physics and biophysics to extract the dynamics of microscopic objects across a range of wavevectors by optical microscopy. Standard DDM is limited to detecting dynamics no faster than the camera frame rate. We report on an extension to DDM where we sequentially illuminate the sample with spectrally distinct light and image with a color camera. By pulsing blue and then red light separated by a lag time much smaller than the camera's exposure time, we are able to use this two-color DDM method to measure dynamics occurring much faster than the camera frame rate.

Myosin-driven actin-microtubule networks exhibit self-organized contractile dynamics.

The cytoskeleton is a dynamic network of proteins, including actin, microtubules, and their associated motor proteins, that enables essential cellular processes such as motility, division, and growth. While actomyosin networks are extensively studied, how interactions between actin and microtubules, ubiquitous in the cytoskeleton, influence actomyosin activity remains an open question. Here, we create a network of co-entangled actin and microtubules driven by myosin II. We combine dynamic differential microscopy, particle image velocimetry, and particle tracking to show that both actin and microtubules undergo ballistic contraction with unexpectedly indistinguishable characteristics. This contractility is distinct from faster disordered motion and rupturing that active actin networks exhibit. Our results suggest that microtubules enable self-organized myosin-driven contraction by providing flexural rigidity and enhanced connectivity to actin networks. Beyond the immediate relevance to cytoskeletal dynamics, our results shed light on the design of active materials that can be precisely tuned by the network composition.

Correction: Anomalous and heterogeneous DNA transport in biomimetic cytoskeleton networks.

Correction for 'Anomalous and heterogeneous DNA transport in biomimetic cytoskeleton networks' by Jonathan Garamella et al., Soft Matter, 2020, DOI: 10.1039/d0sm00544d.

Anomalous and heterogeneous DNA transport in biomimetic cytoskeleton networks.

The cytoskeleton, a complex network of protein filaments and crosslinking proteins, dictates diverse cellular processes ranging from division to cargo transport. Yet, the role the cytoskeleton plays in the intracellular transport of DNA and other macromolecules remains poorly understood. Here, using single-molecule conformational tracking, we measure the transport and conformational dynamics of linear and relaxed circular (ring) DNA in composite networks of actin and microtubules with variable types of crosslinking. While both linear and ring DNA undergo anomalous, non-Gaussian, and non-ergodic subdiffusion, the detailed dynamics are controlled by both DNA topology (linear vs. ring) and crosslinking motif. Ring DNA swells, exhibiting heterogeneous subdiffusion controlled via threading by cytoskeleton filaments, while linear DNA compacts, exhibiting transport via caging and hopping. Importantly, while the crosslinking motif has little effect on ring DNA, linear DNA in networks with actin-microtubule crosslinking is significantly less ergodic and shows more heterogeneous transport than with actin-actin or microtubule-microtubule crosslinking.

Topology-dependent anomalous dynamics of ring and linear DNA are sensitive to cytoskeleton crosslinking.

Cytoskeletal crowding plays a key role in the diffusion of DNA molecules through the cell, acting as a barrier to effective intracellular transport and conformational stability required for processes such as transfection, viral infection, and gene therapy. Here, we elucidate the transport properties and conformational dynamics of linear and ring DNA molecules diffusing through entangled and crosslinked composite networks of actin and microtubules. We couple single-molecule conformational tracking with differential dynamic microscopy to reveal that ring and linear DNA exhibit unexpectedly distinct transport properties that are influenced differently by cytoskeleton crosslinking. Ring DNA coils are swollen and undergo heterogeneous and biphasic subdiffusion that is hindered by crosslinking. Conversely, crosslinking actually facilitates the single-mode subdiffusion that compacted linear chains exhibit. Our collective results demonstrate that transient threading by cytoskeleton filaments plays a key role in the dynamics of ring DNA, whereas the mobility of the cytoskeleton dictates transport of linear DNA.

Before the breach: Interactions between colloidal particles and liquid interfaces at nanoscale separations.

Particles bound to fluid-fluid interfaces are widely used to study self-assembly and to make materials such as Pickering emulsions. In both contexts, the lateral interactions between such particles have been studied extensively. However, much less is known about the normal interactions between a particle and the interface prior to contact. We use digital holographic microscopy to measure the dynamics of individual micrometer-size colloidal particles as they approach an interface between an aqueous phase and oil. Our measurements show that the interaction between the particle and interface changes nonmonotonically as a function of salt concentration, from repulsive at 1 mM to attractive at tens of mM to negligible at 100 mM and attractive again above 200 mM. In the attractive regimes, the particles can bind to the interface at nanometer-scale separation without breaching it. Classical Derjaguin-Landau-Verwey-Overbeek theory does not explain these observations. However, a theory that accounts for nonlinear screening and correlations between the ions does predict the nonmonotonic dependence on salt concentration and produces trajectories that agree with experimental data. We further show that the normal interactions determine the lateral interactions between particles that are bound to the interface. Because the interactions we observe occur at salt concentrations used to make Pickering emulsions and other particle-laden interfaces, our results suggest that particle arrangements at the interface are likely out of equilibrium on experimental timescales.

Filament Rigidity Vies with Mesh Size in Determining Anomalous Diffusion in Cytoskeleton.

The diffusion of microscopic particles through the cell, important to processes such as viral infection, gene delivery, and vesicle transport, is largely controlled by the complex cytoskeletal network, comprised of semiflexible actin filaments and rigid microtubules, that pervades the cytoplasm. By varying the relative concentrations of actin and microtubules, the cytoskeleton can display a host of different structural and dynamic properties that, in turn, impact the diffusion of particles through the composite network. Here, we couple single-particle tracking with differential dynamic microscopy to characterize the transport of microsphere tracers diffusing through composite in vitro networks with varying ratios of actin and microtubules. We analyze multiple complementary metrics for anomalous transport to show that particles exhibit anomalous subdiffusion in all networks, which our data suggest arises from caging by networks. Further, subdiffusive characteristics are markedly more pronounced in actin-rich networks, which exhibit similarly more prominent viscoelastic properties compared to microtubule-rich composites. While the smaller mesh size of actin-rich composites compared to microtubule-rich composites plays an important role in these results, the rigidity of the filaments comprising the network also influences the anomalous characteristics that we observe. Our results suggest that as microtubules in our composites are replaced with actin filaments, the decreasing filament rigidity competes with increasing network connectivity to drive anomalous transport.

Measuring capillary wave dynamics using differential dynamic microscopy.

The interface between two fluids is roughened by thermally excited capillary waves. By using colloid-polymer systems which exhibit liquid-gas phase separation, the time and length scales of capillary waves become accessible to optical microscopy methods. Here, we study such a system using bright-field optical microscopy combined with a novel extension of differential dynamic microscopy. With differential dynamic microscopy, we analyze images in order to determine the decay time of interfacial fluctuations spanning wavevectors from 0.1 to 1 µm-1. We find capillary velocities on the order of 0.1 µm s-1 that depend on the sample composition in expected ways and that match values from the literature. This work demonstrates the first application of differential dynamic microscopy to the study of interfacial dynamics.

Bridging the spatiotemporal scales of macromolecular transport in crowded biomimetic systems.

Crowding plays a key role in the transport and conformations of biological macromolecules. Gene therapy, viral infection, and transfection require DNA to traverse the crowded cytoplasm, including the cytoskeletal network of filamentous proteins. Given the complexity of cellular crowding, the dynamics of biological molecules can be highly dependent on the spatiotemporal scale probed. We present a powerful platform that spans molecular and cellular scales by coupling single-molecule conformational tracking (SMCT) and selective-plane illumination differential dynamic microscopy (SPIDDM). We elucidate the transport and conformational properties of large DNA, crowded by custom-designed networks of actin and microtubules, to link single-molecule conformations with ensemble DNA transport and cytoskeleton structure. We show that actin crowding leads to DNA compaction and suppression of fluctuations, combined with subdiffusion and heterogeneous transport, whereas microtubules have much more subdued impact across all scales. In composite networks of both filaments, scale-dependent effects emerge such that actin dictates ensemble DNA transport while microtubules influence single-molecule dynamics. We show that these intriguing results arise from a complex interplay between network rigidity, mesh size, filament concentration, and DNA size.