The dynamic range of the microrheometry based on the analysis of the enforced motion of colloidal force probes by video microscopy has been extended to 40 Hz. For that purpose a novel rapid image processing system has been developed enabling the real-time data acquisition and analysis thus improving the time resolution of particle tracking to 6 ms. A second advancement consists of the development of a correction procedure accounting for two effects: First, for the smearing out of the diffraction image of the beads due to the finite data acquisition time and, second, for systematic phase shifts of magnetic bead deflection with respect to the force due to the finite response time of the superparamagnetic beads in the direction of the gradient of the magnetic field. The new method has been applied to re-study the dynamic scaling law of the frequency dependence of the viscoelastic impedance of entangled actin solutions in the frequency regime determined by the conformational dynamics and entropic tension of single filaments. The frequency dependence of the storage and loss modulus obeys the scaling laws G′(ω)∝G″(ω)∝ωα with α=0.83(8) which is only slightly higher than the theoretical prediction (α=0.75).
We present a study on filamentous actin solutions containing heavy meromyosin subfragments of myosin II motor molecules. We focus on the viscoelastic phase behavior and internal dynamics of such networks during ATP depletion. Upon simultaneously using micro-rheology and fluorescence microscopy as complementary experimental tools, we find a sol-gel transition accompanied by a sudden onset of directed filament motion. We interpret the sol-gel transition in terms of myosin II enzymology, and suggest a "zipping" mechanism to explain the filament motion in the vicinity of the sol-gel transition.PACS numbers: 87.16. Ka, 87.15.La, 87.15.Nn, 87.16.Nn Eucaryotic cells show an amazing versatility in their mechanical properties. Not only can they sustain stresses ranging from some tenths to hundreds of Pascals, but they can equally well perform such complex processes as cytokinesis and cell locomotion. A vital role for these and other cellular functions is played by the cytoskeleton, the structural framework of the cell composed of a network of protein filaments. A major component is filamentous actin (F-Actin), whose physical properties are by now well characterized [1]. The length distribution, spatial arrangement and connectivity of these filaments is controlled by a great variety of regulatory proteins.An important family of these regulatory proteins are cross-linkers, which can be further classified as passive or active. The function of passive cross-linkers, e.g. α-actinin, is mainly determined by their molecular structure and the on-off kinetics of their binding sites to actin. Upon changing the association-dissociation equilibrium and hence the degree of cross-linking by varying the temperature the network can be driven from a sol into a gel state [2]. Depending on both the concentration and the affinity of these cross-linkers for F-actin there is a tendency to either form random networks or bundles [3]. Motor proteins of the myosin family can also act as active cross-linkers. When both of its functional head groups are bound to two different filaments they can use the energy of adenosine-triphosphate (ATP) hydrolysis to exert relative forces and motion between them. However, such an event is very unlikely under physiological conditions and ATP saturation, because then myosin II spends only a short fraction of its chemomechanical cycle attached to the filament (duty ratio: r 0.02 [4]). Active relative transport yet becomes possible due to the concerted action of several motors if in vitro myosin II proteins assemble into multimeric minifilaments [5].In this letter we study actin networks containing the heavy meromyosin (HMM) subfragment lacking the light meromyosin domain responsible for myosin II assembly. Our focus is on the viscoelastic phase behavior and internal dynamics of such networks during ATP depletion. We use an experimental setup combining microrheology with fluorescent microscopy of labeled filaments. This allows us to identify a sol-gel transition accompanied by a sudden onset of directed filament...
This paper deals with correlations between the viscoelastic impedance of entangled actin networks and the slow conformational dynamics and diffusive motions of single filaments. The single filament dynamics is visualized and analysed by analysing the Brownian motion of attached colloidal beads, which enables independent measurements of characteristic viscoelastic response times such as the entanglement and reptation times. We further studied the frequency-dependent viscoelastic impedance of active actin-heavy-meromyosin II networks by magnetic-tweezers microrheometry to gain insight into the effect of such highly dynamic and force-generating crosslinkers (exhibiting bond lifetimes of less than 1 s) on the rheological properties. We show that at high frequencies (higher than 1 Hz) the viscoelastic loss modulus is slightly increased relative to the entangled network (associated with an increase in the energy dissipated during mechanical excitations), while at low frequencies the plateau of the impedance spectrum becomes more pronounced as a consequence of the cross-linking of the network and the suppression of the terminal regime. Our data provide evidence that the myosin motor protein may play a role as softener of the actin cortex, enabling the adaptive reduction of the yield stress of cells and thus facilitating cellular deformations.
This paper deals with correlations between the viscoelastic impedance of entangled actin networks and the slow conformational dynamics and diffusive motions of single filaments. The single filament dynamics is visualized and analysed by analysing the Brownian motion of attached colloidal beads, which enables independent measurements of characteristic viscoelastic response times such as the entanglement and reptation times. We further studied the frequency-dependent viscoelastic impedance of active actin-heavy-meromyosin II networks by magnetic-tweezers microrheometry to gain insight into the effect of such highly dynamic and force-generating crosslinkers (exhibiting bond lifetimes of less than 1 s) on the rheological properties. We show that at high frequencies (higher than 1 Hz) the viscoelastic loss modulus is slightly increased relative to the entangled network (associated with an increase in the energy dissipated during mechanical excitations), while at low frequencies the plateau of the impedance spectrum becomes more pronounced as a consequence of the cross-linking of the network and the suppression of the terminal regime. Our data provide evidence that the myosin motor protein may play a role as softener of the actin cortex, enabling the adaptive reduction of the yield stress of cells and thus facilitating cellular deformations.
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