RESUMEN
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.
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Citoesqueleto de Actina , Citoesqueleto , Actinas , Actomiosina , MiosinasRESUMEN
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.
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Citoesqueleto de Actina , Biomimética , Actinas/genética , Citoesqueleto , ADN , MicrotúbulosRESUMEN
Correction for 'Anomalous and heterogeneous DNA transport in biomimetic cytoskeleton networks' by Jonathan Garamella et al., Soft Matter, 2020, DOI: 10.1039/d0sm00544d.
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Blends of polymers of different topologies, such as ring and supercoiled, naturally occur in biology and often exhibit emergent viscoelastic properties coveted in industry. However, due to their complexity, along with the difficulty of producing polymers of different topologies, the dynamics of topological polymer blends remains poorly understood. We address this void by using both passive and active microrheology to characterize the linear and nonlinear rheological properties of blends of relaxed circular and supercoiled DNA. We characterize the dynamics as we vary the concentration from below the overlap concentration c* to above (0.5c* to 2c*). Surprisingly, despite working at the dilute-semidilute crossover, entanglement dynamics, such as elastic plateaus and multiple relaxation modes, emerge. Finally, blends exhibit an unexpected sustained elastic response to nonlinear strains not previously observed even in well-entangled linear polymer solutions.
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ADN Circular/química , ADN Superhelicoidal/química , Elasticidad , Reología , ViscosidadRESUMEN
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.
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Actinas , Microtúbulos , Citoesqueleto de Actina , CitoesqueletoRESUMEN
Optical tweezers microrheology (OTM) offers a powerful approach to probe the nonlinear response of complex soft matter systems, such as networks of entangled polymers, over wide-ranging spatiotemporal scales. OTM can also uniquely characterize the microstructural dynamics that lead to the intriguing nonlinear rheological properties that these systems exhibit. However, the strain in OTM measurements, applied by optically forcing a microprobe through the material, induces network inhomogeneities in and around the strain path, and the resultant flow field complicates the measured response of the system. Through a robust set of custom-designed OTM protocols, coupled with modeling and analytical calculations, we characterize the time-varying inhomogeneity fields induced by OTM measurements. We show that homogenization following strain does not interfere with the intrinsic stress relaxation dynamics of the system, rather it manifests as an independent component in the stress decay, even in highly nonlinear regimes such as with the microrheological large-amplitude-oscillatory-shear (MLAOS) protocols we introduce. Our specific results show that Rouse-like elastic retraction, rather than disentanglement and disengagement, dominates the nonlinear stress relaxation of entangled polymers at micro- and mesoscales. Thus, our study opens up possibilities of performing precision nonlinear microrheological measurements, such as MLAOS, on a wide range of complex macromolecular systems.
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The entanglement of ring polymers remains mysterious in many aspects. In this Letter, we use electric fields to induce self-entanglements in circular DNA molecules, which serve as a minimal system for studying chain entanglements. We show that self-threadings give rise to entanglements in ring polymers and can slow down polymer dynamics significantly. We find that strongly entangled circular molecules remain kinetically arrested in a compact state for very long times, thereby providing experimental evidence for the severe topological constraints imposed by threadings.
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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.
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Citoesqueleto de Actina/química , Actinas/química , Animales , ConejosRESUMEN
Polymer composites are ideal candidates for next generation biomimetic soft materials because of their exquisite bottom-up designability. However, the richness of behaviours comes at a price: the need for precise and extensive characterisation of material properties over a highly-dimensional parameter space, as well as a quantitative understanding of the physical principles underlying desirable features. Here we couple large-scale Molecular Dynamics simulations with optical tweezers microrheology to characterise the viscoelastic response of DNA-actin composites. We discover that the previously observed non-monotonic stress-stiffening of these composites is robust, yet tunable, in a broad range of the parameter space that spans two orders of magnitude in DNA length. Importantly, we discover that the most pronounced stiffening is achieved when the species are maximally coupled, i.e., have similar number of entanglements, and not when the number of entanglements per DNA chain is largest. We further report novel dynamical oscillations of the microstructure of the composites, alternating between mixed and bundled phases, opening the door to future investigations. The generic nature of our system renders our results applicable to the behaviour of a broad class of polymer composites.
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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.
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Citoesqueleto de Actina , Actinas , ADN , Microtúbulos , Transporte Biológico , Biomimética , Microscopía/métodosRESUMEN
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.
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Actinas/química , Reactivos de Enlaces Cruzados/química , Citoesqueleto/química , Elasticidad , Colorantes Fluorescentes/química , Cinética , Microscopía Confocal , Microtúbulos/química , Pinzas Ópticas , Estrés Mecánico , ViscosidadRESUMEN
Non-equilibrium soft materials, such as networks of actin proteins, have been intensely investigated over the past decade due to their promise for designing smart materials and understanding cell mechanics. However, current methods are unable to measure the time-dependent mechanics of such systems or map mechanics to the corresponding dynamic macromolecular properties. Here, we present an experimental approach that combines time-resolved optical tweezers microrheology with diffusion-controlled microfluidics to measure the time-evolution of microscale mechanical properties of dynamic systems during triggered activity. We use these methods to measure the viscoelastic moduli of entangled and crosslinked actin networks during chemically-triggered depolymerization and repolymerization of actin filaments. During disassembly, we find that the moduli exhibit two distinct exponential decays, with experimental time constants of â¼169 min and â¼47 min. Conversely, during reassembly, measured moduli initially exhibit power-law increase with time, after which steady-state values are achieved. We develop toy mathematical models that couple the time-evolution of filament lengths with rigidity percolation theory to shed light onto the molecular mechanisms underlying the observed mechanical transitions. The models suggest that these two distinct behaviors both arise from phase transitions between a rigidly percolated network and a non-rigid regime. Our approach and collective results can inform the general principles underlying the mechanics of a large class of dynamic, non-equilibrium systems and materials of current interest.
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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 (ÏT) 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 ÏT, reaching a maximum for equimolar composites (ÏT = 0.5), suggesting that reptation is fastest in ÏT = 0.5 composites. Corresponding mobility measurements of steady-state actin and microtubules show that both filaments are indeed the most mobile in ÏT = 0.5 composites. This nonmonotonic dependence of mobility on ÏT demonstrates the important interplay between mesh size and filament rigidity in polymer networks and highlights the surprising emergent properties that can arise in composites.
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Actinas/metabolismo , Microtúbulos/metabolismo , Estrés Mecánico , Animales , Fenómenos Biomecánicos , ConejosRESUMEN
Composites of flexible and rigid polymers are ubiquitous in biology and industry alike, yet the physical principles determining their mechanical properties are far from understood. Here, we couple force spectroscopy with large-scale Brownian dynamics simulations to elucidate the unique viscoelastic properties of custom-engineered blends of entangled flexible DNA molecules and semiflexible actin filaments. We show that composites exhibit enhanced stress stiffening and prolonged mechanomemory compared to systems of actin or DNA alone, and that these nonlinear features display a surprising nonmonotonic dependence on the fraction of actin in the composite. Simulations reveal that these counterintuitive results arise from synergistic microscale interactions between the two biopolymers. Namely, DNA entropically drives actin filaments to form bundles that stiffen the network but reduce the entanglement density, while a uniform well-connected actin network is required to reinforce the DNA network against yielding and flow. The competition between bundling and connectivity triggers an unexpected stress response that leads equal mass DNA-actin composites to exhibit the most pronounced stress stiffening and the most long-lived entanglements.
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Citoesqueleto de Actina/química , ADN/química , Fenómenos Mecánicos , Microscopía de Fuerza Atómica/métodos , Microesferas , Simulación de Dinámica Molecular , Pinzas Ópticas , Reología/métodos , Sustancias Viscoelásticas/químicaRESUMEN
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.
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Citoesqueleto de Actina/metabolismo , Animales , Fenómenos Biomecánicos , Elasticidad , Microfluídica , Microscopía Confocal , Microscopía Fluorescente , Músculo Esquelético , Dinámicas no Lineales , Pinzas Ópticas , Conejos , ViscosidadRESUMEN
Using light-sheet microscopy combined with digital Fourier methods we probe the dynamics of colloidal samples and DNA molecules. This combination, referred to as selective-plane illumination differential dynamic microscopy (SPIDDM), has the benefit of optical sectioning to study, with minimal photobleaching, thick samples allowing us to measure the diffusivity of colloidal particles at high volume fractions. Further, SPIDDM exploits the inherent spatially-varying thickness of Gaussian light-sheets. Where the excitation sheet is most focused, we capture high spatial frequency dynamics as the signal-to-background is high. In thicker regions, we capture the slower dynamics as diffusion out of the sheet takes longer.
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Despite the ubiquity of molecular crowding in living cells, the effects of crowding on the dynamics of genome-sized DNA are poorly understood. Here, we track single, fluorescent-labeled large DNA molecules (11, 115 kbp) diffusing in dextran solutions that mimic intracellular crowding conditions (0-40%), and determine the effects of crowding on both DNA mobility and conformation. Both DNAs exhibit ergodic Brownian motion and comparable mobility reduction in all conditions; however, crowder size (10 vs. 500 kDa) plays a critical role in the underlying diffusive mechanisms and dependence on crowder concentration. Surprisingly, in 10-kDa dextran, crowder influence saturates at â¼20% with an â¼5× drop in DNA diffusion, in stark contrast to exponentially retarded mobility, coupled to weak anomalous subdiffusion, with increasing concentration of 500-kDa dextran. Both DNAs elongate into lower-entropy states (compared to random coil conformations) when crowded, with elongation states that are gamma distributed and fluctuate in time. However, the broadness of the distribution of states and the time-dependence and length scale of elongation length fluctuations depend on both DNA and crowder size with concentration having surprisingly little impact. Results collectively show that mobility reduction and coil elongation of large crowded DNAs are due to a complex interplay between entropic effects and crowder mobility. Although elongation and initial mobility retardation are driven by depletion interactions, subdiffusive dynamics, and the drastic exponential slowing of DNA, up to â¼300×, arise from the reduced mobility of larger crowders. Our results elucidate the highly important and widely debated effects of cellular crowding on genome-sized DNA.
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ADN Bacteriano/química , Entropía , Dextranos/química , Difusión , Movimiento (Física) , Soluciones/químicaRESUMEN
Using single-molecule fluorescence microscopy and particle-tracking techniques, we elucidate the role DNA topology plays in the diffusion and conformational dynamics of crowded DNA molecules. We focus on large (115 kbp), double-stranded ring and linear DNA crowded by varying concentrations (0-40%) of dextran (10, 500 kDa) that mimic cellular conditions. By tracking the center-of-mass and measuring the lengths of the major and minor axes of single DNA molecules, we characterize both DNA mobility reduction as well as crowding-induced conformational changes (from random spherical coils). We reveal novel topology-dependent conformations, with single ring molecules undergoing compaction to ordered spherical configurations â¼20% smaller than dilute random coils, while linear DNA elongates by â¼2-fold. Surprisingly, these highly different conformations result in nearly identical exponential mobility reduction dependent solely on crowder volume fraction Φ, revealing a universal critical crowding concentration of Φcâ 2.3. Beyond Φc DNA exhibits topology-independent conformational relaxation dynamics despite highly distinct topology-driven conformations. Our collective results reveal that topology-dependent conformational changes, unique to crowded environments, enable DNA to overcome the classically expected mobility reduction that high-viscosity crowded environments impose. Such coupled universal dynamics suggest a mechanism for DNA to maintain sufficient mobility required for wide-ranging biological processes despite severe cellular crowding.
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ADN Bacteriano/metabolismo , Benzoxazoles/química , ADN Bacteriano/química , Dextranos/química , Difusión , Escherichia coli/genética , Microscopía Fluorescente , Conformación de Ácido Nucleico , Compuestos de Quinolinio/químicaRESUMEN
We drive optically trapped microspheres through entangled F-actin at constant speeds and distances well beyond the linear regime, and measure the microscale force response of the entangled filaments during and following strain. Our results reveal a unique crossover to appreciable nonlinearity at a strain rate of [small gamma, Greek, dot above]c ≈ 3 s(-1) which corresponds remarkably well with the theoretical rate of relaxation of entanglement length deformations 1/τent. Above [small gamma, Greek, dot above]c, we observe stress stiffening which occurs over very short time scales comparable to the predicted timescale over which mesh size deformations relax. Stress softening then takes over, yielding to an effectively viscous regime over a timescale comparable to the entanglement length relaxation time, τent. The viscous regime displays shear thinning but with a less pronounced viscosity scaling with strain rate compared to flexible polymers. The relaxation of induced force on filaments following strain shows that the relative relaxation proceeds more quickly for increasing strain rates; and for rates greater than [small gamma, Greek, dot above]c, the relaxation displays a complex power-law dependence on time. Our collective results reveal that molecular-level nonlinear viscoelasticity is driven by non-classical dynamics of individual entanglement segments that are unique to semiflexible polymers.