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.

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.

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.

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.