Polymerization of actin filaments coupled with adenosine triphosphate hydrolysis: Brownian dynamics and theoretical analysis

Polymerization dynamics of single actin filaments coupled with adenosine triphosphate (ATP) hydrolysis is investigated via both theoretical analysis and Brownian dynamics simulations. Brownian dynamics simulations have been applied recently to study the growth behaviors of long filaments as a function of the free actin monomer concentrations, C(T), which is found to be in agreement with the associated experiments. In the present study, both ATP cap length and length diffusivity are studied as a function of the free ATP-actin monomer concentrations, C(T). The exact analytical expressions are found to be in perfect consistency with Brownian dynamics simulations. Likewise, we find that the length diffusion coefficient is peaked near the critical concentration, C(T,cr). It is, therefore, expected that the dependence of length diffusivity on ATP-actin monomer concentrations is utilized to analyze the surprising experiments on the length fluctuations of individual actin filaments.     

The influence of the salt concentration on the aggregation behavior of viscoelastic detergents

The influence of excess NaCl on the properties of viscoelastic detergent solutions of Cetypyridiniumsalicylate (CPySal) has been studied by static and dynamic light scattering, electric birefringence and rheological measurements. It is obeserved that the rodlike micelles of length L which are present in these solutions and which are responsible for their elastic properties grow in length with the increase of the NaCl concentration. As long as the rods are shorter than their mean distance, their length can be determined from the rheological and electric birefringence measurements. For very small shear fields these solutions behave as Newtonian fluids. The viscosity of the solution increases strongly when the rods begin to overlap. Solutions with overlapping rods are elastic. It is postulated from the results that the cmc_II (the critical concentration above which rods are present) and the length L of the rods are partially determined by the intermicellar interaction energy. It is furthermore postulated that this intermicellar interaction energy has an influence on the polydispersity of the rods and seems to make the rods relatively monodisperse.     

Microtubules and actin filaments in teleost visual cone elongation and contraction.

Teleost retinal cones contract in light and elongate in darkness. This paper describes the disposition of microtubules and cytoplasmic filaments in cone cells of 2 species of fish (Haemulon sciurus and Lutjanus griseus). In Haemulon, the neck-like "myoid" region of the cone changes in length from 5 mu to 75 mu. Maximal observed rates of elongation and contraction are comparable to that of chromosome movement in mitosis (2-3 mu/min). Microtubules presumably participate in cone elongation, since numerous longitudinal microtubules are present in the myoid region, and colchicine blocks dark-induced elongation. Myoid shortening, on the other hand, appears to be an active contractile process. Disruption of microtubules in dark-adapted cones does not produce myoid shortening in the absence of light, and light-induced myoid shortening is blocked by cytochalasin-B. Cone cells possess longitudinally-oriented thin filaments which bind myosin subfragment-1 to form arrowhead complexes typical of muscle actin. Myoid thin filaments are clearly observed in negatively stained preparations of isolated cones which have been disrupted with detergent after attachment to grids. These myoid filaments are not, however, generally preserved by conventional fixation, though bundles of thin filaments are preserved in other regions of the cell. Thus, actin filaments are poorly retained by fixation in precisely the region of the cone cell where contraction occurs. Cone cells also possess longitudinally-oriented thick filaments 130-160 A in diameter. That these thick filaments may be myosin is suggested by the presence of side-arms with approximately 150 A periodicity. The linear organization of the contractile apparatus of the retinal cone cell makes this cell a promising model for morphological characterization of the disposition of actin and myosin filaments during contraction in a nonmuscle cell.     

Structural transition of actin filament in a cell-sized water droplet with a phospholipid membrane

Actin filament, F-actin, is a semiflexible polymer with a negative charge, and is one of the main constituents of cell membranes. To clarify the effect of cross talk between a phospholipid membrane and actin filaments in cells, we conducted microscopic observations on the structural changes in actin filaments in a cell-sized (several tens of micrometers in diameter) water droplet coated with a phospholipid membrane such as phosphatidylserine (PS; negatively charged head group) or phosphatidylethanolamine (PE; neutral head group) as a simple model of a living cell membrane. With PS, actin filaments are distributed uniformly in the water phase without adsorption onto the membrane surface between 2 and 6 mM Mg2+, while between 6 and 12 mM Mg2+, actin filaments are adsorbed onto the inner membrane surface. With PE, the actin filaments are uniformly adsorbed onto the inner membrane surface between 2 and 12 mM Mg2+. With both PS and PE membranes, at Mg2+ concentrations higher than 12 mM, thick bundles are formed in the bulk water droplet accompanied by the dissolution of actin filaments from the membrane surface. The attraction between actin filaments and membrane is attributable to an increase in the translational entropy of counterions accompanied by the adsorption of actin filaments onto the membrane surface. These results suggest that a microscopic water droplet coated with phospholipid can serve as an easy-to-handle model of cell membranes.     

Diffusion dynamics of motor-driven transport: gradient production and self-organization of surfaces

The interaction between cytoskeletal filaments (e.g., actin filaments) and molecular motors (e.g., myosin) is the basis for many aspects of cell motility and organization of the cell interior. In the in vitro motility assay (IVMA), cytoskeletal filaments are observed while being propelled by molecular motors adsorbed to artificial surfaces (e.g., in studies of motor function). Here we integrate ideas that cytoskeletal filaments may be used as nanoscale templates in nanopatterning with a novel approach for the production of surface gradients of biomolecules and nanoscale topographical features. The production of such gradients is challenging but of increasing interest (e.g., in cell biology). First, we show that myosin-induced actin filament sliding in the IVMA can be approximately described as persistent random motion with a diffusion coefficient (D) given by a relationship analogous to the Einstein equation (D = kT/gamma). In this relationship, the thermal energy (kT) and the drag coefficient (gamma) are substituted by a parameter related to the free-energy transduction by actomyosin and the actomyosin dissociation rate constant, respectively. We then demonstrate how the persistent random motion of actin filaments can be exploited in conceptually novel methods for the production of actin filament density gradients of predictable shapes. Because of regularly spaced binding sites (e.g., lysines and cysteines) the actin filaments act as suitable nanoscale scaffolds for other biomolecules (tested for fibronectin) or nanoparticles. This forms the basis for secondary chemical and topographical gradients with implications for cell biological studies and biosensing.     

The effect of pyrene labelling on the thermal stability of actin filaments

The ability of actin to form filaments is fundamental to its biological function and often characterised by various methods in vitro. One of the most frequently used methods capitalises on the observation that the fluorescence emission of a pyrene label on the Cys-374 residue of actin is enhanced by a factor of ∼20 during polymerisation. This method inherently involves the chemical modification of actin monomers with pyrene. It was reported earlier that the pyrene labelling of actin monomers has only small effect on the polymerisation and depolymerisation rates of actin, indicating that the method is suitable to characterise the effect of actin-binding proteins or peptides on the polymerisation kinetics.In our present work we tested the effect of the pyrene labelling on the thermal denaturation of actin filaments by using the method of differential scanning calorimetry (DSC). By recording the heat denaturation profiles of unlabelled and pyrene labelled actin filaments we observed that pyrene labelling shifted the melting point (T_m) of actin filaments from 66 to 68 °C. A similar effect was detected in the presence of equimolar concentration of phalloidin where the T_m shifted from 79 to 82 °C. We concluded that the observed pyrene labelling induced differences of the thermal denaturation of actin filaments were small. The DSC results, therefore, confirmed that the methods based on the measurements of pyrene intensity during actin polymerisation are suitable to characterise the polymerisation kinetics of actin under in vitro conditions.     

Actin filament guidance on a chip: toward high-throughput assays and lab-on-a-chip applications

Biological molecular motors that are constrained so that function is effectively limited to predefined nanosized tracks may be used as molecular shuttles in nanotechnological applications. For these applications and in high-throughput functional assays (e.g., drug screening), it is important that the motors propel their cytoskeletal filaments unidirectionally along the tracks with a minimal number of escape events. We here analyze the requirements for achieving this for actin filaments that are propelled by myosin II motor fragments (heavy meromyosin; HMM). First, we tested the guidance of HMM-propelled actin filaments along chemically defined borders. Here, trimethylchlorosilane (TMCS)-derivatized areas with high-quality HMM function were surrounded by SiO(2) domains where HMM did not bind actin. Guidance along the TMCS-SiO(2) border was almost 100% for filament approach angles between 0 and 20 degrees but only about 10% at approach angles near 90 degrees . A model (Clemmens, J.; Hess, H.; Lipscomb, R.; Hanein, Y.; Bohringer, K. F.; Matzke, C. M.; Bachand, G. D.; Bunker, B. C.; Vogel, V. Langmuir 2003, 19, 10967-10974) accounted for essential aspects of the data and also correctly predicted a more efficient guidance of actin filaments than previously shown for kinesin-propelled microtubules. Despite the efficient guidance at low approach angles, nanosized (<700 nm wide) TMCS tracks surrounded by SiO(2) were not effective in guiding actin filaments. Neither was there complete guidance along nanosized tracks that were surrounded by topographical barriers (walls and roof partially covering the track) unless there was also chemically based selectivity between the tracks and surroundings. In the latter case, with dually defined tracks, there was close to 100% guidance. A combined experimental and theoretical analysis, using tracks of the latter type, suggested that a track width of less than about 200-300 nm is sufficient at a high HMM surface density to achieve unidirectional sliding of actin filaments. In accord with these results, we demonstrate the long-term trapping of actin filaments on a closed-loop track (width < 250 nm). The results are discussed in relation to lab-on-a-chip applications and nanotechnology-assisted assays of actomyosin function.     

Formation of polymer microrods in shear flow by emulsification--solvent attrition mechanism

Rodlike polymer particles could have interesting properties and could find many practical applications; however, few methods for the production of such particles are available. We report a systematic study of a droplet shearing process for the formation of polymer rods with micrometer or submicrometer diameter and a length of up to tens of micrometers. The process is based on emulsification of a polymer solution under shear, combined with solvent attrition in the surrounding organic medium. The droplets deform and elongate into cylinders, which solidify when the solvent transfers to the dispersion medium. Stopped flow experiments allow distinguishing all stages of the mechanism. The results are interpreted on the basis of the theory of droplet elongation and breakup under shear. The effects of the viscosity ratio and shear stress are matched against the theoretical expectations. The method is simple, efficient, and scalable, and we demonstrate how it can be controlled and modified. The experimental parameters that allow varying the rod size and aspect ratio include shear rate, medium viscosity, and polymer concentration. Examples of the specific properties of the polymer rods, including self-organization, alignment in external fields and in fluid flows, and stabilization of bubbles, droplets, and capsules, are presented.     

Selective chemical imaging of static actin in live cells

We have characterized rationally designed and optimized analogues of the actin-stabilizing natural products jasplakinolide and chondramide C. Efficient actin staining was achieved in fixed permeabilized and non-permeabilized cells using different combinations of dye and linker length, thus highlighting the degree of molecular flexibility of the natural product scaffold. Investigations into synthetically accessible, non-toxic analogues have led to the characterization of a powerful cell-permeable probe to selectively image static, long-lived actin filaments against dynamic F-actin and monomeric G-actin populations in live cells, with negligible disruption of rapid actin dynamics.     

Subdiffraction‐Resolution Fluorescence Microscopy of Myosin–Actin Motility

Subdiffraction‐resolution imaging by subsequent localization of single photoswitchable molecules can achieve a spatial resolution in the range of ～20 nm with moderate excitation intensities, but have so far been too slow for imaging faster dynamics in biology. Herein, we introduce a novel approach for video‐like subdiffraction microscopy based on rapid and reversible photoswitching of commercially available organic carbocyanine fluorophores. With the present concept, we demonstrate in vitro studies on the motility of fluorophore‐labeled actin filaments along myosin II. Actin filaments were densely labeled with carbocyanine fluorophores, and the gliding velocity adjusted by the concentration of ATP. At imaging frame rates of ～100 Hz, only 100 consecutive frames are sufficient to generate a single high‐resolution image of moving actin filaments with a lateral resolution of ～30 nm. A video‐like sequence is generated from individual reconstructed images by additionally applying a sliding window algorithm. We measured velocities of individual actin filaments of up to ～0.18 μm s^?1, observed strong bending and disruption of filaments as well as locally immobile fragments.     

The effect of toxins on the thermal stability of actin filaments by differential scanning calorimetry

In our present study we performed the detailed characterisation of jasplakinolide and phalloidin on the thermal stability of actin filaments. The heat absorption curves were analysed by using the model established by Sanchez-Ruiz et al. [1]. The analysis provided the activation energies attributed to the heat denaturation of actin filaments in the absence and in the presence of toxins. The results indicated that there are kinetic differences between the toxin-mediated stabilization of the Ca²⁺-and Mg²⁺-actin filaments. The effect of toxins appeared to be cation dependent.     

Single-filament dynamics and long-range ordering of semiflexible biopolymers under flow and confinement

We report the collective and single-filament dynamics of long semiflexible actin filaments flowing in an evaporating droplet adhering on glass and accumulating along the physical barrier constituted by the droplet triple line. The observation of fluorescent reporter filaments embedded in the entangled network enables us to relate the final collective organization of the accumulated filaments to the individual filament dynamics. Three areas corresponding to distinct filament organizations are observed in the region of the initial triple line pinning, after complete evaporation of the droplet. A nematic liquid-crystal-like alignment of the filaments is observed at the edge of the droplet because of the dynamic filament alignment, whereas a less-ordered packing is generated because of the bending and folding of most of the filaments. The latter unconventional dynamics is analyzed in terms of the amplification of undulation modes typical of semiflexible polymers. The receding regime of the droplet triple line leads finally to a remaining film of actin filaments showing random organization.     

The effects of formins on the conformation of subdomain 1 in actin filaments

In this study we investigated the effects of formins on the conformation of actin filaments by using the method of fluorescence quenching. Actin was labelled with IAEDANS at Cys³⁷⁴ and the quencher was acrylamide. The results showed that formin binding induced structural changes in the subdomain 1 of actin protomers which were reflected by greater quenching constants (K_SV). Simultaneously the fraction of the fluorophore population accessible for the quencher (α) decreased. These observations suggest that the conformational distribution characteristic for the actin protomers became broader after the binding of formins, for which the structural framework was provided by a more flexible protein matrix in the microenvironment of the label. The effects of formins depended on the formin:actin molar ratio, and also on the ionic strength of the medium. These observations are in agreement with previous results and underline the importance of the intramolecular conformational changes induced by formins in the structure of actin filaments.     

Thermal characterisation of actin filaments prepared from adp-actin monomers

The thermodynamic properties of the ADP- and ATP-actin filaments were compared by the method of differential scanning calorimetry. The lower melting point for the ADP-F-actin filament (58.4 vs. 64.5°C for ATP-F-actin) indicated that compared to the ATP-actin filaments its structure was less resistant to heat denaturation. The detailed thermodynamic characterisation of the proteins was carried out by the analysis of the calorimetric enthalpy, the entropy and the free enthalpy changes. All of the determined parameters gave lower values to the ADP-actin filaments than to the ATP-actin filaments. The calculated values of the activation energy also demonstrated that compared to the ADP-F-actin the ATP-F-actin was thermodynamically more resistant to the denaturing effect of heat. Based on all of this information we have concluded that the actin filament prepared from ADP containing magnesium saturated actin monomers at pH 8.0 is thermodynamically less stable than the ones obtained from ATP-actin monomers.     

Effect of phalloidin on filaments polymerized from heart muscle ADP-actin monomers

The effect of phalloidin on filaments polymerized from ADP-actin monomers of the heart muscle was investigated with differential scanning calorimetry. Heart muscle contains α-skeletal and α-cardiac actin isoforms. In the absence of phalloidin the melting temperature was 55°C for the α-cardiac actin isoform and 58°C for the α-skeletal one when the filaments were generated from ADP-actin monomers. After the binding of phalloidin the melting temperature was isoform independent (85.5°C). We concluded that phalloidin stabilized the actin filaments of α-skeletal and α-cardiac actin isoforms to the same extent when they were polymerized from ADP-actin monomers.     

F-Actin reassembly during focal adhesion impacts single cell mechanics and nanoscale membrane structure

Focal adhesions play an important role in cell spreading,migration,and overall mechanical integrity.The relationship of cell structural and mechanical properties was investigated in the context of focal adhesion processes.Combined atomic force microscopy(AFM) and laser scanning confocal microscopy(LSCM) was utilized to measure single cell mechanics,in correlation with cellular morphology and membrane structures at a nanometer scale.Characteristic stages of focal adhesion were verified via confocal fluorescent studies,which confirmed three representative F-actin assemblies,actin dot,filaments network,and long and aligned fibrous bundles at cytoskeleton.Force-deformation profiles of living cells were measured at the single cell level,and displayed as a function of height deformation,relative height deformation and relative volume deformation.As focal adhesion progresses,single cell compression profiles indicate that both membrane and cytoskeleton stiffen,while spreading increases especially from focal complex to focal adhesion.Correspondingly,AFM imaging reveals morphological geometries of spherical cap,spreading with polygon boundaries,and elongated or polarized spreading.Membrane features are dominated by protrusions of 41-207 nm tall,short rods with 1-6 μm in length and 10.2-80.0 nm in height,and long fibrous features of 31-246 nm tall,respectively.The protrusion is attributed to local membrane folding,and the rod and fibrous features are consistent with bilayer decorating over the F-actin assemblies.Taken collectively,the reassembly of F-actin during focal adhesion formation is most likely responsible for the changes in cellular mechanics,spreading morphology,and membrane structural features.     

Structure and dynamics of actin filament solutions in the presence of latrunculin A

We study the polymerization kinetics and linear rheology of actin filaments in the absence and in the presence of latrunculin A. Filamentous actin is a semiflexible polymer, and latrunculin A is an organic, actin-binding molecule. Using diffusing wave spectroscopy (DWS), we monitor the thermally excited motion of monodisperse polystyrene microspheres in semidilute solutions of actin filaments. From these measurements, we extract the microspheres mean-square displacement, which is related to the viscoelastic nature of the actin solutions. These optical measurements, along with mechanical measurements, suggest that despite its depolymerizing effect, latrunculin A promotes the strengthening of actin networks. DWS shows that while the scaling nature of the viscoelastic properties of actin filaments is essentially unmodified in the presence of latrunculin A at small time scales, the elasticity of actin solutions becomes enhanced for increasing latrunculin concentrations at large time scales. Complementary electron-microscopy measurements help uncover the structural origin of this enhanced elasticity at small time scales. © 1998 John Wiley & Sons, Inc. J Polym Sci B: Polym Phys 36: 3007–3015, 1998     

Morphological and biochemical abnormalities in hearts of cardiac mutant salamanders (Ambystoma mexicanum).

The effect of homozygosity for recessive gene c in Ambystoma mexicanum is the absence of a heartbeat even though initially heart development appears normal. Mutant embryos (c/c) are first distinguishable from their normal siblings (+/+;+/c) at stage 34 (7 days after fertilization) when the normals develop contracting hearts. The mutant hearts at this stage, upon gross examination, appear structurally normal but fail to beat. Nevertheless, the mutants survive through stage 41, which is about 20 days beyond the heartbeat stage, and they exhibit normal swimming movements, indicating that gene c does not affect skeletal muscle. Electron microscopic studies of normal hearts show some myofibrils to be present at stage 34; by stage 41, the normal myocardial cells have become highly differentiated muscle cells. Although some mutant heart cells contain a few thin 60 A and thick 150 A filaments, organized myofibrils are absent. Instead, amorphous proteinaceous collections are prominent. Heavy meromyosin (HMM) binding experiments were performed on mutant hearts to determine whether the myocardial cells contain actin. Mutant myocardial cells that are glycerinated but not treated with HMM contain intact amorphous bodies. After incubation in HMM, the amorphous collections are no longer present and large numbers of decorated actin filaments appear. The.results suggest that the amorphous proteinaceous collections contain actin in a nonfilamentous form, and the addition of HMM induces this actin to polymerize into filaments. SDS-polyacrylamide gel electrophoresis of mutant heart tissue supports this conclusion by showing a prominent 43,000 dalton band suggestive of actin. The electrophoresis experiments also demonstrate a significant reduction of myosin heavy chain (200,000 daltons) in mutant hearts when compared to normal, and this latter observation is confirmed by radioimmunoassay experiments. Muscle tropomyosin (34,000 daltons), prominent in normal hearts, is virtually nonexistent in mutants. Thus, it appears that this single gene mutation affects the accumulation and organization of several different muscle proteins, including actin, myosin, and tropomyosin.     

Guidance of actin filament elongation on filament-binding tracks

Biomolecular motors, which convert chemical energy into mechanical work in intracellular processes, have high potential in bionanotechnology in vitro as molecular shuttles or nanoscale actuators. In this context, guided elongation of actin filaments in vitro could be used to lay tracks for myosin motor-based shuttles or to direct nanoscale actuators based on actin filament end-tracking motors. To guide the direction of filament polymerization on surfaces, microcontact printing was used to create tracks of chemically modified myosin, which binds to, but cannot exert force on, filaments. These filament-binding tracks captured nascent filaments from solution and guided the direction of their subsequent elongation. The effect of track width and protein surface density on filament alignment and elongation rate was quantified. These results indicate that microcontact printing is a useful method for guiding actin filament polymerization in vitro for biomolecular motor-based applications.     

Selective attachment of F-actin with controlled length for developing an intelligent nanodevice

Development of the nanodevice that myosin-coated beads "walk" on actin filaments (F-actin) tracks for in vitro nanotransportation was hindered due to the difficulty of assembling large-area well-orientated F-actin tracks on the surface. In this work, we present a selective attachment of F-actin with controlled length on a patterned surface by employing biotinylated capped protein gelsolin as intermediate anchoring bridge. A patterned streptavidin layer was formed via coupling with a biotin layer that photo-actively attached to an amine-functionalized glass surface. The patterned film was found stable and homogenous compared to that obtained by microcontact printing method, according to the profiling with fluorescence microscopy. By a secondary blocking process, non-specific binding of F-actin to the patterned surface through electrostatic adsorption can be resisted. The length variation of F-actin as a function of gelsolin concentration was also investigated, implying that F-actin is appropriately of 2.5 μm in average length once F-actin/gelsolin molar ratio is 4:1. Finally, the selective attachment of F-actin was well characterized with quantifying the number of attached F-actin per unit area in the patterned areas over that in blocked areas. The density of F-actin was estimated at c.a. 2 μm(2) per actin filament molecule so that the distance between one another actin filament is estimated as c.a. 1.41-1.97 μm. The unique properties of F-actin, e.g. well flexibility or electrical conductivity, make it feasible to lay them down and form unidirectional aligned tracks by fluidic flow or electrical field. This may open a possibility for the long-distant movement of myosin-coated beads, offering a novel discipline for the development of micro-biochip in vitro.