F-actin Solutions Research Articles

Evolutionary analysis of actinins(ACTN) gene family for positive selective sites by finding the dn/ds (ω) value

Identifying positively selected amino acid sites is an important approach for making a conclusion about the function of proteins; an amino acid site that is undergoing positive selection is likely to play a key role in the function of the protein. Here, we used codon-based substitution models and maximum-likelihood (ML) methods to identify positively selected sites that are likely to influence the cohesiveness and mechanics of the cytoskeleton by cross-linking actinins filaments and other cytoskeleton components to create a scaffold that imparts stability and forms a bridge between the cytoskeleton and signaling pathways. We use MEGA 7 and PAML for polygenetic Analysis and positive selective Sites. Alpha-actinins from skeletal muscle as a protein factor promoting the super precipitation of actomyosin and inducing the gelation of F-actin solutions. The protein is located in Z-bands of sarcomeres in skeletal and cardiac muscles as well as near the fascia adherents of intercalated discs in cardiac muscle. In smooth muscle, it exists in cytoplasmic dense bodies and membrane-associated dense plaques. We used PAMLX to find out specific sites in these DNA sequences. These results show the location and identification of positively selected sites in the ACTININS gene family. ACTN1, ACTN2, ACTN3 and ACTN4 had (248G,250L), (234I,236N ,238P, 239K, 252F, 260E, 261Q, 314Q, 590N, 607L, 869S), (NO) and (248G, 250L) positive selective sites respectively. The aims of our work are the prediction of these sites to find the specific location of these sites in 3D structure and to replace it with suitable amino acids for the cure of obesity, muscles, cancer, and other chronic diseases

Micro-mechanical response and power-law exponents from the longitudinal fluctuations of F-actin solutions.

We investigate the local fluctuations of filamentous actin (F-actin), with a focus on the skeletal thin filament, using single-particle optical trapping interferometry. This experimental technique allows us to detect the Brownian motion of a tracer bead immersed in a complex fluid with nanometric resolution at the microsecond time-scale. The mean square displacement, loss modulus, and velocity autocorrelation function (VAF) of the trapped microprobes in the fluid follow power-law behaviors, whose exponents can be determined in the short-time/high-frequency regime over several decades. We obtain 7/8 subdiffusive power-law exponents for polystyrene depleted microtracers at low optical trapping forces. Microrheologically, the elastic modulus of these suspensions is observed to be constant up to the limit of high frequencies, confirming that the origin of this subdiffusive exponent is the local longitudinal fluctuations of the polymers. Deviations from this value are measured and discussed in relation to the characteristic length scales of these F-actin networks and probes' properties, and also in connection with the different power-law exponents detected in the VAFs. Finally, we observed that the thin filament, composed of tropomyosin (Tm) and troponin (Tn) coupled to F-actin in the presence of Ca2+, shows exponent values less dispersed than that of F-actin alone, which we interpret as a micro-measurement of the filament stabilization.

Stress relaxation in F-actin solutions by severing.

Networks of filamentous actin (F-actin) are important for the mechanics of most animal cells. These cytoskeletal networks are highly dynamic, with a variety of actin-associated proteins that control cross-linking, polymerization and force generation in the cytoskeleton. Inspired by recent rheological experiments on reconstituted solutions of dynamic actin filaments, we report a theoretical model that describes stress relaxation behavior of these solutions in the presence of severing proteins. We show that depending on the kinetic rates of assembly, disassembly, and severing, one can observe both length-dependent and length-independent relaxation behavior.

Cysteine-rich protein 2 accelerates actin filament cluster formation.

Filamentous actin (F-actin) forms many types of structures and dynamically regulates cell morphology and movement, and plays a mechanosensory role for extracellular stimuli. In this study, we determined that the smooth muscle-related transcription factor, cysteine-rich protein 2 (CRP2), regulates the supramolecular networks of F-actin. The structures of CRP2 and F-actin in solution were analyzed by small-angle X-ray solution scattering (SAXS). The general shape of CRP2 was partially unfolded and relatively ellipsoidal in structure, and the apparent cross sectional radius of gyration (Rc) was about 15.8 Å. The predicted shape, derived by ab initio modeling, consisted of roughly four tandem clusters: LIM domains were likely at both ends with the middle clusters being an unfolded linker region. From the SAXS analysis, the Rc of F-actin was about 26.7 Å, and it was independent of CRP2 addition. On the other hand, in the low angle region of the CRP2-bound F-actin scattering, the intensities showed upward curvature with the addition of CRP2, which indicates increasing branching of F-actin following CRP2 binding. From biochemical analysis, the actin filaments were augmented and clustered by the addition of CRP2. This F-actin clustering activity of CRP2 was cooperative with α-actinin. Thus, binding of CRP2 to F-actin accelerates actin polymerization and F-actin cluster formation.

Measuring Protein Binding to F-actin by Co-sedimentation.

Filamentous actin (F-actin) organization within cells is regulated by a large number of actin-binding proteins that control actin nucleation, growth, cross-linking and/or disassembly. This protocol describes a technique – the actin co-sedimentation, or pelleting, assay – to determine whether a protein or protein domain binds F-actin and to measure the affinity of the interaction (i.e., the dissociation equilibrium constant). In this technique, a protein of interest is first incubated with F-actin in solution. Then, differential centrifugation is used to sediment the actin filaments, and the pelleted material is analyzed by SDS-PAGE. If the protein of interest binds F-actin, it will co-sediment with the actin filaments. The products of the binding reaction (i.e., F-actin and the protein of interest) can be quantified to determine the affinity of the interaction. The actin pelleting assay is a straightforward technique for determining if a protein of interest binds F-actin and for assessing how changes to that protein, such as ligand binding, affect its interaction with F-actin.

Measuring Protein Binding to F-actin by Co-sedimentation

Filamentous actin (F-actin) organization within cells is regulated by a large number of actin-binding proteins that control actin nucleation, growth, cross-linking and/or disassembly. This protocol describes a technique – the actin co-sedimentation, or pelleting, assay – to determine whether a protein or protein domain binds F-actin and to measure the affinity of the interaction (i.e., the dissociation equilibrium constant). In this technique, a protein of interest is first incubated with F-actin in solution. Then, differential centrifugation is used to sediment the actin filaments, and the pelleted material is analyzed by SDS-PAGE. If the protein of interest binds F-actin, it will co-sediment with the actin filaments. The products of the binding reaction (i.e., F-actin and the protein of interest) can be quantified to determine the affinity of the interaction. The actin pelleting assay is a straightforward technique for determining if a protein of interest binds F-actin and for assessing how changes to that protein, such as ligand binding, affect its interaction with F-actin.

Direct observation of orientation distributions of actin filaments in a solution undergoing shear banding.

Shear banding is frequently observed in complex fluids. However, the configuration of macromolecules in solutions undergoing shear banding has not yet been directly observed. In this study, by using the fact that F-actin solutions exhibit shear banding and actin filaments are visualized by fluorescent labels, we directly observed the intrinsic states of an actin solution undergoing shear banding. By combining the 3D imaging of labeled actin filaments and particle image velocimetry (PIV), we obtained orientation distributions of actin filaments in both high and low shear rate regions, whose quantitative differences are indicated. In addition, by using the orientation distributions and applying stress expression for rod-like polymers, we estimated stress tensors in both high and low shear rate regions. This evaluation indicates that different orientation distributions of filamentous macromolecules can exhibit a common shear stress.

Two-point particle tracking microrheology of nematic complex fluids.

Many biological and technological complex fluids exhibit tight microstructural alignment that confers them nematic mechanical properties. Among these we count liquid crystals and biopolymer networks, which are often available in microscopic amounts. However, current microrheological methods cannot measure the directional viscoelastic coefficients that appear in the constitutive relation of nematic complex fluids. This article presents directional two-point particle-tracking microrheology (D2PTM) - a novel microrheology technique to determine these coefficients. We establish the theoretical foundation for D2PTM by analyzing the motion of a probing microscopic particle embedded in a nematic complex fluid, and the mutual hydrodynamic interactions between pairs of distant particles. From this analysis, we generalize the formulation of two-point particle tracking microrheology for nematic complex fluids, and demonstrate that the new formulation provides sufficient information to fully characterize the anisotropic viscoelastic coefficients of such materials. We test D2PTM by simulating the Brownian motion of particles in nematic viscoelastic fluids with prescribed directional frequency-dependent shear moduli, showing that D2PTM accurately recovers the prescribed shear moduli. Furthermore, we experimentally validate D2PTM by applying it to a lyotropic nematic liquid crystal, and demonstrate that this new microrheology method provides results in agreement with dynamic light scattering measurements. Lastly, we illustrate the experimental application of the new technique to characterize nematic F-actin solutions. These experiments constitute the first microrheological measurement of the directional viscoelastic coefficients of an anisotropic soft material.

Cell adhesion. The minimal cadherin-catenin complex binds to actin filaments under force.

Linkage between the adherens junction (AJ) and the actin cytoskeleton is required for tissue development and homeostasis. In vivo findings indicated that the AJ proteins E-cadherin, β-catenin, and the filamentous (F)-actin binding protein αE-catenin form a minimal cadherin-catenin complex that binds directly to F-actin. Biochemical studies challenged this model because the purified cadherin-catenin complex does not bind F-actin in solution. Here, we reconciled this difference. Using an optical trap-based assay, we showed that the minimal cadherin-catenin complex formed stable bonds with an actin filament under force. Bond dissociation kinetics can be explained by a catch-bond model in which force shifts the bond from a weakly to a strongly bound state. These results may explain how the cadherin-catenin complex transduces mechanical forces at cell-cell junctions.

Direct visualization of flow-induced conformational transitions of single actin filaments in entangled solutions.

While semi-flexible polymers and fibres are an important class of material due to their rich mechanical properties, it remains unclear how these properties relate to the microscopic conformation of the polymers. Actin filaments constitute an ideal model polymer system due to their micron-sized length and relatively high stiffness that allow imaging at the single filament level. Here we study the effect of entanglements on the conformational dynamics of actin filaments in shear flow. We directly measure the full three-dimensional conformation of single actin filaments, using confocal microscopy in combination with a counter-rotating cone-plate shear cell. We show that initially entangled filaments form disentangled orientationally ordered hairpins, confined in the flow-vorticity plane. In addition, shear flow causes stretching and shear alignment of the hairpin tails, while the filament length distribution remains unchanged. These observations explain the strain-softening and shear-thinning behaviour of entangled F-actin solutions, which aids the understanding of the flow behaviour of complex fluids containing semi-flexible polymers.

What is “hypermobile” water?: detected in alkali halide, adenosine phosphate, and F-actin solutions by high-resolution microwave dielectric spectroscopy

Abstract Experimental observation by high-resolution microwave dielectric spectroscopy of hydration properties of alkali halide ions, adenosine phosphate ions, and F-actin revealed the existence of hypermobile water (HMW) molecules around those solutes. To understand the molecular process of HMW, two theoretical approaches are reviewed here. One is based on a statistical mechanical approach to analyze the rotational freedom of water molecules around a charged particle. Another approach reports direct calculation of dielectric relaxation process of water molecules around an ion. Experimentally observed HMW molecules are theoretically explained with the significance of multi-correlations among an ion and water molecules.

3P190 F-アクチン溶液のシアバンディング(細胞生物的課題,ポスター,第52回日本生物物理学会年会(2014年度))

3P190 F-アクチン溶液のシアバンディング(細胞生物的課題,ポスター,第52回日本生物物理学会年会(2014年度))

Shear Banding in an F-Actin Solution

We report herein the first evidence that an F-actin solution shows shear banding, which is characterized by the spontaneous separation of homogeneous shear flow into two macroscopic domains of different definite shear rates. The constant shear stress observed in the F-actin solution is explained by the banded flow with volume fractions that obey the lever rule. Nonhomogenous reversible flows were observed in the F-actin solution with respect to upward and downward changes in the shear rate. This is the first time shear banding has been observed in a simple biomacromolecule. The biological implications and dynamic aspects of shear flow velocity characteristic patterns are discussed.

1PT003 中性子散乱によるF-アクチン周辺水分子のダイナミクスの研究(日本生物物理学会第50回年会(2012年度))

1PT003 中性子散乱によるF-アクチン周辺水分子のダイナミクスの研究(日本生物物理学会第50回年会(2012年度))

Flow birefringence property of desmin filaments.

We have investigated the flow birefringence property and assembly process of desmin, a muscle specific intermediate protein. Solution of non-polar desmin filaments showed birefringence when aligned in the sheared flow. The amount of birefringence of desmin filaments was considerably lower when compared with that of F-actin solution. Assembly of desmin from soluble state was followed by the birefringence measurements. At any desmin concentrations examined, the degree of flow birefringence increased rapidly just after the addition of the assembly buffer and reached a saturated level within 30 min. The time to reach half-maximal values of flow birefringence slightly but definitely depended on the initial soluble desmin concentrations. The plotting of the initial velocity of the assembly against the soluble desmin concentrations showed a slope of 1.4. This result suggested that the assembly process detected by flow birefringence measurements followed second-order kinetics, and the process corresponded to the second step of the three stage model for type III intermediate filament assembly proposed by Herrmann and his colleagues; the annealing of unit length filaments into filaments.

Fragmentation Is Crucial for the Steady-State Dynamics of Actin Filaments

Despite the recognition that actin filaments are important for numerous cellular processes, and decades of investigation, the dynamics of in vitro actin filaments are still not completely understood. Here, we follow the time evolution of the length distribution of labeled actin reporter filaments in an unlabeled F-actin solution via fluorescence microscopy. Whereas treadmilling and diffusive length fluctuations cannot account for the observed dynamics, our results suggest that at low salt conditions, spontaneous fragmentation is crucial.

Scale-dependent diffusion of spheres in solutions of flexible and rigid polymers: mean square displacement and autocorrelation function for FCS and DLS measurements

We present a theoretical description of diffusion of a sphere in polymer solution. The depletion layer around the sphere affects its motion and leads to scale-dependent diffusion. We propose the model of walking confined diffusion. Although it is different from anomalous diffusion, we show that the experimental data generated by this process may have features characteristic of anomalous diffusion. We give analytical formulas for the autocorrelation functions describing this type of motion in: i) dynamic light scattering experiments and ii) fluorescence correlation spectroscopy experiments. We compare our results to existing experimental data for polyethylene oxide, fd-virus, and F-actin solutions.

Microrheology of solutions embedded with thread-like supramolecular structures

A family of methods uses colloidal particles as a mechanical probe for deforming the medium in conjunction with a procedure to trace the movement of the particles to get rheological information in a very wide frequency range. All of them are under the heading of microrheology. In the last decade, they have been developed up to the point of being a useful tool for understanding the structure and the dynamics of solutions with embedded thread-like supramolecular structures. This is the case of wormlike micellar solutions, which is the main interest of the paper. Here the impact of microrheology has been essential, providing structural information not easily obtained by other methods. Microrheology has also made an important contribution to the understanding of other threadlike system, as in the case of F-actin or fd virus solutions; they will also be discussed.

Two-dimensional Periodic Pattern of Dried F-actin Solution

Two-dimensional Periodic Pattern of Dried F-actin Solution

Hyper-mobility of water around actin filaments revealed using pulse-field gradient spin-echo 1H NMR and fluorescence spectroscopy

This paper reports that water molecules around F-actin, a polymerized form of actin, are more mobile than those around G-actin or in bulk water. A measurement using pulse-field gradient spin-echo 1H NMR showed that the self-diffusion coefficient of water in aqueous F-actin solution increased with actin concentration by ∼5%, whereas that in G-actin solution was close to that of pure water. This indicates that an F-actin/water interaction is responsible for the high self-diffusion of water. The local viscosity around actin was also investigated by fluorescence measurements of Cy3, a fluorescent dye, conjugated to Cys 374 of actin. The steady-state fluorescence anisotropy of Cy3 attached to F-actin was 0.270, which was lower than that for G-actin, 0.334. Taking into account the fluorescence lifetimes of the Cy3 bound to actin, their rotational correlation times were estimated to be 3.8 and 9.1 ns for F- and G-actin, respectively. This indicates that Cy3 bound to F-actin rotates more freely than that bound to G-actin, and therefore the local water viscosity is lower around F-actin than around G-actin.