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Abstract The cytoskeleton is an active composite of filamentous proteins that dictates diverse mechanical properties and processes in eukaryotic cells by generating forces and autonomously restructuring itself. Enzymatic motors that act on the comprising filaments play key roles in this activity, driving spatiotemporally heterogeneous mechanical responses that are critical to cellular multifunctionality, but also render mechanical characterization challenging. Here, we couple optical tweezers microrheology and fluorescence microscopy with simulations and mathematical modeling to robustly characterize the mechanics of active composites of actin filaments and microtubules restructured by kinesin motors. It is discovered that composites exhibit a rich ensemble of force response behaviors–elastic, yielding, and stiffening–with their propensity and properties tuned by motor concentration and strain rate. Moreover, intermediate kinesin concentrations elicit emergent mechanical stiffness and resistance while higher and lower concentrations exhibit softer, more viscous dissipation. It is further shown that composites transition from well‐mixed interpenetrating double‐networks of actin and microtubules to de‐mixed states of microtubule‐rich aggregates surrounded by relatively undisturbed actin phases. It is this de‐mixing that leads to the emergent mechanical response, offering an alternate route that composites can leverage to achieve enhanced stiffness through coupling of structure and mechanics. 

The transport of macromolecules, such as DNA, through the cytoskeleton is critical to wide-ranging cellular processes from cytoplasmic streaming to transcription. The rigidity and steric hindrances imparted by the network of filaments comprising the cytoskeleton often leads to anomalous subdiffusion, while active processes such as motor-driven restructuring can induce athermal superdiffusion. Understanding the interplay between these seemingly antagonistic contributions to intracellular dynamics remains a grand challenge. Here, we use single-molecule tracking to show that the transport of large linear and relaxed circular DNA through motor-driven microtubule networks can be non-Gaussian and multimodal, with the degree and spatiotemporal scales over which these features manifest depending nontrivially on the state of activity and DNA topology. For example, active network restructuring increases caging and non-Gaussian transport modes of linear DNA, while dampening these mechanisms for circular DNA. We further discover that circular DNA molecules exhibit either markedly enhanced subdiffusion or superdiffusion compared to their linear counterparts, in the absence or presence of kinesin activity, indicative of microtubules threading circular DNA. This strong coupling leads to both stalling and directed transport, providing a direct route towards parsing distinct contributions to transport and determining the impact of coupling on the transport signatures. More generally, leveraging macromolecular topology as a route to programming molecular interactions and transport dynamics is an elegant yet largely overlooked mechanism that cells may exploit for intracellular trafficking, streaming, and compartmentalization. This mechanism could be harnessed for the design of self-regulating, sensing, and reconfigurable biomimetic matter. Published by the American Physical Society2025 

The unique mechanical behaviors of actin–vimentin composites in both linear and nonlinear regimes are shaped by the complex interactions among actin entanglements, vimentin crosslinking, and poroelastic properties. 

Abstract 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. 

Abstract 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. 

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. 

Polymer architecture plays critical roles in both bulk rheological properties and microscale macromolecular dynamics in entangled polymer solutions and composites. Ring polymers, in particular, have been the topic of much debate due to the inability of the celebrated reptation model to capture their observed dynamics. Macrorheology and differential dynamic microscopy (DDM) are powerful methods to determine entangled polymer dynamics across scales; yet, they typically require different samples under different conditions, preventing direct coupling of bulk rheological properties to the underlying macromolecular dynamics. Here, we perform macrorheology on composites of highly overlapping DNA and dextran polymers, focusing on the role of DNA topology (rings versus linear chains) as well as the relative volume fractions of DNA and dextran. On the same samples under the same conditions, we perform DDM and single-molecule tracking on embedded fluorescent-labeled DNA molecules immediately before and after bulk measurements. We show DNA-dextran composites exhibit unexpected nonmonotonic dependences of bulk viscoelasticity and molecular-level transport properties on the fraction of DNA comprising the composites, with characteristics that are strongly dependent on the DNA topology. We rationalize our results as arising from stretching and bundling of linear DNA versus compaction, swelling, and threading of rings driven by dextran-mediated depletion interactions.