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The mechanical properties of the mammalian cell regulate many cellular functions and are largely dictated by the cytoskeleton, a composite network of protein filaments, including actin, microtubules, and intermediate filaments. Interactions between these distinct filaments give rise to emergent mechanical properties that are difficult to generate synthetically, and recent studies have made great strides in advancing our understanding of the mechanical interplay between actin and microtubule filaments. While intermediate filaments play critical roles in the stress response of cells, their effect on the rheological properties of the composite cytoskeleton remains poorly understood. Here, we use optical tweezers microrheology to measure the linear viscoelastic properties and nonlinear stress response of composites of actin and vimentin with varying molar ratios of actin to vimentin. We reveal a surprising, nearly opposite effect of actin-vimentin network mechanics compared to single-component networks in the linear versus nonlinear regimes. Namely, the linear elastic plateau modulus and zero-shear viscosity are markedly reduced in composites compared to single-component networks of actin or vimentin, whereas the initial response force and stiffness are maximized in composites versus single-component networks in the nonlinear regime. While these emergent trends are indicative of distinct interactions between actin and vimentin, nonlinear stiffening and longtime stress response appear to both be dictated primarily by actin, at odds with previous bulk rheology studies. We demonstrate that these complex, scale-dependent effects arise from the varied contributions of network density, filament stiffness, non-specific interactions, and poroelasticity to the mechanical response at different spatiotemporal scales. Cells may harness this complex behavior to facilitate distinct stress responses at different scales and in response to different stimuli to allow for their hallmark multifunctionality.
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Actin plays a vital role in maintaining the stability and rigidity of biological cells while allowing for cell motility and shape change. The semiflexible nature of actin filaments-along with the myriad actin-binding proteins (ABPs) that serve to crosslink, bundle, and stabilize filaments-are central to this multifunctionality. The effect of ABPs on the structural and mechanical properties of actin networks has been the topic of fervent investigation over the past few decades. Yet, the combined impact of filament stabilization, stiffening and crosslinking via ABPs on the mechanical response of actin networks has yet to be explored. Here, we perform optical tweezers microrheology measurements to characterize the nonlinear force response and relaxation dynamics of actin networks in the presence of varying concentrations of α-actinin, which transiently crosslinks actin filaments, and phalloidin, which stabilizes filamentous actin and increases its persistence length. We show that crosslinking and stabilization can act both synergistically and antagonistically to tune the network resistance to nonlinear straining. For example, phalloidin stabilization leads to enhanced elastic response and reduced dissipation at large strains and timescales, while the initial microscale force response is reduced compared to networks without phalloidin. Moreover, we find that stabilization switches this initial response from that of stress stiffening to softening despite the increased filament stiffness that phalloidin confers. Finally, we show that both crosslinking and stabilization are necessary to elicit these emergent features, while the effect of stabilization on networks without crosslinkers is much more subdued. We suggest that these intriguing mechanical properties arise from the competition and cooperation between filament connectivity, bundling, and rigidification, shedding light on how ABPs with distinct roles can act in concert to mediate diverse mechanical properties of the cytoskeleton and bio-inspired polymeric materials.
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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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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.
Assuntos
Citoesqueleto de Actina/química , DNA/química , Fenômenos Mecânicos , Microscopia de Força Atômica/métodos , Microesferas , Simulação de Dinâmica Molecular , Pinças Ópticas , Reologia/métodos , Substâncias Viscoelásticas/químicaRESUMO
Localized deformation is ubiquitous in many natural and engineering materials as they approach failure, and a significant effort has been made to understand localization processes with simple continuum models. Real materials are much more commonly heterogeneous but it is unclear exactly how heterogeneity affects outcomes. In this work we study the response of an idealized heterogenous elastic sheet on a soft foundation as it is uniaxially compressed. The patterned surface layers are created by selective ultraviolet/ozone treatment of the top surface of a polydimethylsiloxane (PDMS) sample using a TEM grid as a mask. By controlling the exposure time of UV/O3, samples ranging from continuous thin films to sets of isolated small plates were created. We find that patterned regions noticeably localize while bulk regions appear as uniform wrinkles, and that local and global strains depend on the pattern pitch, exposure levels and the treatment protocol. Remarkably, various responses can be modeled using well-understood theory that ignores pattern details aside from the small distance between the adjacent boundaries and the local value of strain.
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This study sought to confirm and investigate further recently published information regarding the occurrence of Neospora caninum in cattle in Ethiopia and investigate infection in dogs, the canine definitive host, in this region. Faecal samples from 383 dogs in Hawassa, Ethiopia were examined by microscopy for Neospora-like oocysts, and positive samples then analysed by a molecular approach (DNA isolation, PCR and sequencing at the ITS1 gene). Brain tissue samples from four late term aborted foetuses, one congenitally defective calf (hind leg arthrogryposis) and placental tissue from cattle in the same area were also examined by the same molecular approach. All foetal, calf and placental tissue were associated with Neospora seropositive dams. A high prevalence of Neospora-like oocysts (11.5 µm±1.5 µm diameter) was observed in faecal samples from dogs (37 positive samples; 9.7% prevalence), and in 17 of these the identification was confirmed by PCR, giving a prevalence of confirmed infection of 4.4%. N. caninum DNA was also detected in all foetal and calf brain tissue samples. Sequencing revealed only minor differences among all PCR products, whether from oocysts or from brain tissue samples. These data provide molecular evidence of the presence of N. caninum infection in both dog and cattle in this region of Ethiopia. Moreover these findings highlight the role of dogs in maintaining and spreading the infection horizontally in the study area. The high frequency of N. caninum infection in household dogs as well as farm dogs is worthy of further investigation.
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Encéfalo/parasitologia , Doenças dos Bovinos/parasitologia , Coccidiose/veterinária , Doenças do Cão/parasitologia , Fezes/parasitologia , Neospora/isolamento & purificação , Oocistos/parasitologia , Animais , Bovinos , Doenças dos Bovinos/epidemiologia , Coccidiose/epidemiologia , Coccidiose/parasitologia , DNA de Protozoário/genética , Doenças do Cão/epidemiologia , Cães , Etiópia , Feminino , Masculino , Neospora/genética , Reação em Cadeia da Polimerase/veterináriaRESUMO
Polymer glasses have numerous advantageous mechanical properties in comparison to other materials. One of the most useful is the high degree of toughness that can be achieved due to significant yield occurring in the material. Remarkably, the onset of plasticity in polymeric materials is very poorly quantified, despite its importance as the ultimate limit of purely elastic behavior. Here, we report the results of a novel experiment which is extremely sensitive to the onset of yield and discuss its impact on measurement and elastic theory. In particular, we use an elastic instability to locally bend and impart a local tensile stress in a thin, glassy polystyrene film, and directly measure the resulting residual stress caused by the bending. We show that plastic failure is initiated at extremely low strains, of the order 10(-3) for polystyrene. Not only is this critical strain found to be small in comparison to bulk measurement, we show that it is influenced by thin film confinement--leading to an increase in the critical strain for plastic failure as film thickness approaches zero.
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The lethal toxicity of major components of the essential oils of Ocimium kilimandscharicum and O. kenyense and of selected blends of these against Sitophilus zeamais and Rhyzopertha dominica were compared with those of the full blends of the essential oils. The compounds were assayed in amounts and proportions present in the minimum 100% lethal dose of the oils. Whereas a major component of O. kilimandscharicum was found to be largely responsible for the toxic action of its essential oil against R. dominica, the results with the other treatments indicated that the toxic action of the essential oils were due to the combined effects of different components, either with or without significant individual toxic action of their own against the insects. The significance of the results and their implication in screening and using plants and their phytochemicals for pest and microbial control are highlighted.