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Actin and microtubule filaments, with their auxiliary proteins, enable the cytoskeleton to carry out vital processes in the cell by tuning the organizational and mechanical properties of the network. Despite their critical importance and interactions in cells, we are only beginning to uncover information about the composite network. The challenge is due to the high complexity of combining actin, microtubules, and their hundreds of known associated proteins. Here, we use fluorescence microscopy, fluctuation, and cross-correlation analysis to examine the role of actin and microtubules in the presence of an antiparallel microtubule crosslinker, MAP65, and a generic, strong actin crosslinker, biotin-NeutrAvidin. For a fixed ratio of actin and microtubule filaments, we vary the amount of each crosslinker and measure the organization and fluctuations of the filaments. We find that the microtubule crosslinker plays the principle role in the organization of the system, while, actin crosslinking dictates the mobility of the filaments. We have previously demonstrated that the fluctuations of filaments are related to the mechanics, implying that actin crosslinking controls the mechanical properties of the network, independent of the microtubule-driven re-organization.

The dynamic morphology and mechanics of the cytoskeleton is determined by interacting networks of semiflexible actin filaments and rigid microtubules. Active rearrangement of networks of actin and microtubules can not only be driven by motor proteins but by changes to ionic conditions. For example, high concentrations of multivalent ions can induce bundling and crosslinking of both filaments. Yet, how cytoskeleton networks respond in real-time to changing ion concentrations, and how actin-microtubule interactions impact network response to these changing conditions remains unknown. Here, we use microfluidic perfusion chambers and two-color confocal fluorescence microscopy to show that increasing magnesium ions trigger contraction of both actin and actin-microtubule networks. Specifically, we use microfluidics to vary the Mg2+ concentration between 2 and 20 mM while simultaneously visualizing the triggered changes to the overall network size. We find that as Mg2+ concentration increases both actin and actin-microtubule networks undergo bulk contraction, which we measure as the shrinking width of each network. However, surprisingly, lowering the Mg2+concentration back to 2 mM does not stop or reverse the contraction but rather causes both networks to contract further. Further, actin networks begin to contract at lower Mg2+ concentrations and shorter times than actin-microtubule networks. In fact, actin-microtubule networks only undergo substantial contraction once the Mg2+ concentration begins to lower from 20 mM back to 2 mM. Our intriguing findings shed new light on how varying environmental conditions can dynamically tune the morphology of cytoskeleton networks and trigger active contraction without the use of motor proteins.

The cytoskeleton is able to precisely tune its structure and mechanics through interactions between semiflexible actin filaments, rigid microtubules and a suite of crosslinker proteins. However, the role that each of these components, as well as the interactions between them, plays in the dynamics of the composite cytoskeleton remains an open question. Here, we use optical tweezers microrheology and fluorescence confocal microscopy to reveal the surprising ways in which actin crosslinking tunes the viscoelasticity and mobility of actin-microtubule composites from steady-state to the highly nonlinear regime. While previous studies have shown that increasing crosslinking in actin networks increases elasticity and stiffness, we instead find that composite stiffness displays a striking non-monotonic dependence on actin crosslinking - first increasing then decreasing to a response similar to or even lower than un-linked composites. We further show that actin crosslinking has an unexpectedly strong impact on the mobility of microtubules; and it is in fact the microtubule mobility - dictated by crosslinker-driven rearrangements of actin filaments - that controls composite stiffness. This result is at odds with conventional thought that actin mobility drives cytoskeleton mechanics. More generally, our results demonstrate that - when crosslinking composite materials to confer strength and resilience - more is not always better.

The cytoskeleton dynamically tunes its mechanical properties by altering the interactions between semiflexible actin filaments, rigid microtubules, and crosslinking proteins. Here, we use optical tweezers microrheology and confocal microscopy to characterize how varying crosslinking motifs impact the microscopic and mesoscale mechanics and mobility of actin-microtubule composites. We show that, upon subtle changes in the crosslinking pattern, composites separate into two distinct classes of force response – primarily elastic versus more viscous behavior. For example, a composite in which actin and microtubules are crosslinked to each other is markedly more elastic than one in which both filaments are crosslinked but cannot link together. Notably, this distinction only emerges at mesoscopic scales in response to nonlinear forcing, whereas varying crosslinking motifs have little impact on the microscale mechanics and steady-state mobility of composites. Our unexpected scale-dependent results not only inform the physics underlying key cytoskeleton processes and structures, but, more generally, provide valuable perspective to materials engineering endeavors focused on polymer composites.

We use optical tweezers microrheology and fluorescence microscopy to characterize the nonlinear mesoscale mechanics and mobility of in vitro co-entangled actin-microtubule composites. We create a suite of randomly oriented, well-mixed networks of actin and microtubules by co-polymerizing varying ratios of actin and tubulin in situ. To perturb each composite far from equilibrium, we use optical tweezers to displace an embedded microsphere a distance greater than the lengths of the filaments at a speed much faster than their intrinsic relaxation rates. We simultaneously measure the force the filaments exert on the bead and the subsequent force relaxation. We find that the presence of a large fraction of microtubules (>0.7) is needed to substantially increase the measured force, which is accompanied by large heterogeneities in force response. Actin minimizes these heterogeneities by reducing the mesh size of the composites and supporting microtubules against buckling. Composites also undergo a sharp transition from strain softening to stiffening when the fraction of microtubules (ϕ(T)) exceeds 0.5, which we show arises from faster poroelastic relaxation and suppressed actin bending fluctuations. The force after bead displacement relaxes via power-law decay after an initial period of minimal relaxation. The short-time relaxation profiles (t < 0.06 s) arise from poroelastic and bending contributions, whereas the long-time power-law relaxation is indicative of filaments reptating out of deformed entanglement constraints. The scaling exponents for the long-time relaxation exhibit a nonmonotonic dependence on ϕ(T), reaching a maximum for equimolar composites (ϕ(T) = 0.5), suggesting that reptation is fastest in ϕ(T) = 0.5 composites. Corresponding mobility measurements of steady-state actin and microtubules show that both filaments are indeed the most mobile in ϕ(T) = 0.5 composites. This nonmonotonic dependence of mobility on ϕ(T) demonstrates the important interplay between mesh size and filament rigidity in polymer networks and highlights the surprising emergent properties that can arise in composites.

We use optical tweezers microrheology and fluorescence microscopy to characterize the nonlinear mesoscale mechanics and mobility of in vitro co-entangled actin-microtubule composites. We create a suite of randomly-oriented, well-mixed networks of actin and microtubules by co-polymerizing varying ratios of actin and tubulin in situ. To perturb each composite far from equilibrium, we use optical tweezers to displace an embedded microsphere a distance greater than the lengths of the filaments at a speed much faster than their intrinsic relaxation rates. We simultaneously measure the resistive force the filaments exert and the subsequent force relaxation. We find that the presence of a large fraction of microtubules (>0.7) is needed to substantially increase the resistive force, which is accompanied by large heterogeneities in force response. Actin minimizes these heterogeneities by reducing the mesh size of the composites and supporting microtubules against buckling. Composites also undergo a sharp transition from stress-softening to stiffening when the fraction of microtubules (ϕT) exceeds 0.5, by microtubules suppressing actin bending fluctuations. The induced force following strain relaxes via two time-dependent power-law decays. The first decay phase, with scaling exponents that increase proportionally with the fraction of actin, signifies actin bending fluctuations. Alternatively, the second phase, with a ϕT-independent scaling exponent of ~0.4, is indicative of filaments reptating out of deformed entanglement constraints. Corresponding mobility measurements of steady-state actin and microtubules show that both filaments are more mobile in equimolar composites (ϕT=0.5) compared to networks of primarily actin or microtubules. This non-monotonic dependence of mobility on ϕT, which further demonstrates the important role mesh size plays in composites, highlights the surprising emergent properties that can arise in composites.

We use optical tweezers microrheology and fluorescence microscopy to apply nonlinear microscale strains to entangled and cross-linked actin networks, and measure the resulting stress and actin filament deformations. We couple nonlinear stress response and relaxation to the velocities and displacements of individual fluorescent-labeled actin segments, at varying times throughout the strain and varying distances from the strain path, to determine the underlying molecular dynamics that give rise to the debated nonlinear response and stress propagation of cross-linked and entangled actin networks at the microscale. We show that initial stress stiffening arises from acceleration of strained filaments due to molecular extension along the strain, while softening and yielding is coupled to filament deceleration, halting, and recoil. We also demonstrate a surprising nonmonotonic dependence of filament deformation on cross-linker concentration. Namely, networks with no cross-links or substantial cross-links both exhibit fast initial filament velocities and reduced molecular recoil while intermediate cross-linker concentrations display reduced velocities and increased recoil. We show that these collective results are due to a balance of network elasticity and force-induced cross-linker unbinding and rebinding. We further show that cross-links dominate entanglement dynamics when the length between cross-linkers becomes smaller than the length between entanglements. In accord with recent simulations, we demonstrate that post-strain stress can be long-lived in cross-linked networks by distributing stress to a small fraction of highly strained connected filaments that span the network and sustain the load, thereby allowing the rest of the network to recoil and relax.

Double-stranded DNA offers a robust platform for investigating fundamental questions regarding the dynamics of entangled polymer solutions. The exceptional monodispersity and multiple naturally occurring topologies of DNA, as well as a wide range of tunable lengths and concentrations that encompass the entanglement regime, enable direct testing of molecular-level entanglement theories and corresponding scaling laws. DNA is also amenable to a wide range of techniques from passive to nonlinear measurements and from single-molecule to bulk macroscopic experiments. Over the past two decades, researchers have developed methods to directly visualize and manipulate single entangled DNA molecules in steady-state and stressed conditions using fluorescence microscopy, particle tracking and optical tweezers. Developments in microfluidics, microrheology and bulk rheology have also enabled characterization of the viscoelastic response of entangled DNA from molecular levels to macroscopic scales and over timescales that span from linear to nonlinear regimes. Experiments using DNA have uniquely elucidated the debated entanglement properties of circular polymers and blends of linear and circular polymers. Experiments have also revealed important lengthscale and timescale dependent entanglement dynamics not predicted by classical tube models, both validating and refuting new proposed extensions and alternatives to tube theory and motivating further theoretical work to describe the rich dynamics exhibited in entangled polymer systems.

The high concentrations of proteins crowding cells greatly influence intracellular DNA dynamics. These crowders, ranging from small mobile proteins to large cytoskeletal filaments such as semiflexible actin and rigid microtubules, can hinder diffusion and induce conformational changes in DNA. While previous studies have mainly focused on the effect of small mobile crowders on DNA transport, we examine the impact of crowding by actin filaments and microtubules. Further, because actin filaments and microtubules are formed by polymerization of actin monomers and tubulin dimers, respectively, we also investigate the role that the polymerization state of each protein plays in DNA transport and in the time-varying conformational changes of single DNA molecules diffusing in in vitro networks of polymerized and monomeric actin and tubulin. We find that crowding by actin monomers slows DNA diffusion while tubulin crowding actually increases diffusion coefficients. Monomeric actin crowding DNA diffusion, more than when actin is polymerized, while crowding by tubulin dimers increases DNA diffusion more than when tubulin is polymerized (microtubules). Further, we find unexpected relationships between DNA coil size and diffusion when crowded. All crowding conditions lead to some degree of DNA compaction, but less compaction enables faster dynamics.