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The movements of single actin filaments along a myosin-fixed glass surface were observed under a conventional fluorescence microscope. Although random at a low concentration, moving directions of filaments were aligned by the presence of over 1.0 mg/mL of unlabeled filaments. We found that actin filaments when at the intermediate concentrations ranging from 0.1 to 1.0 mg/mL, formed winding belt-like patterns and moved in a two-directional manner along the belts. These patterns were spread over a millimeter range and found to have bulged on the glass in a three-dimensional manner. Filaments did not get closer than about 37.5 nm to each other within each belt-pattern. The average width and the curvature radius of the pattern did not apparently change even when the range of actin concentrations was between 0.05 and 1.0 mg/mL or the sliding velocity between 1.2 and 3.2 μm/sec. However, when the length of filaments was shortened by ultrasonic treatments or the addition of gelsolin molecules, the curvature radius became small from 100 to 60 μm. These results indicate that this belt-forming nature of actin filaments may be due to some inter-filament interactions.

Polymerization induces hydrolysis of ATP bound to actin, followed by γ-phosphate release, which helps advance the disassembly of actin filaments into ADP-G-actin. Mechanical understanding of this correlation between actin assembly and ATP hydrolysis has been an object of intensive studies in biochemistry and structural biology for many decades. Although actin polymerization and depolymerization occur only at either the barbed or pointed ends and the kinetic and equilibrium properties are substantially different from each other, characterizing their properties is difficult to do by bulk assays, as these assays report the average of all actin filaments in solution and are therefore not able to discern the properties of individual actin filaments. Biochemical studies of actin polymerization and hydrolysis were hampered by these inherent properties of actin filaments. Total internal reflection fluorescence (TIRF) microscopy overcame this problem by observing single actin filaments. With TIRF, we now know not only that each end has distinct properties, but also that the rate of γ-phosphate release is much faster from the terminals than from the interior of actin filaments. The rate of γ-phosphate release from actin filament ends is even more accelerated when latrunculin A is bound. These findings highlight the importance of resolving structural differences between actin molecules in the interior of the filament and those at either filament end. This review provides a history of observing actin filaments under light microscopy, an overview of dynamic properties of ATP hydrolysis at the end of actin filament, and structural views of γ-phosphate release.

We examined the movement of an actin filament sliding on a mixture of normal and genetically modified myosin molecules that were attached to a glass surface. For this purpose, we used a Dictyostelium G680V mutant myosin II whose release rates of Pi and ADP were highly suppressed relative to normal myosin, leading to a significantly extended life-time of the strongly bound state with actin and virtually no motility. When the mixing ratio of G680V mutant myosin II to skeletal muscle HMM (heavy myosin) was 0.01%, the actin filaments moved intermittently. When they moved, their sliding velocities were about two-fold faster than the velocity of skeletal HMM alone. Furthermore, sliding movements were also faster when the actin filaments were allowed to slide on skeletal muscle HMM-coated glass surfaces in the motility buffer solution containing G680V HMM. In this case no intermittent movement was observed. When the actin filaments used were copolymerized with a fusion protein consisting of Dictyostelium actin and Dictyostelium G680V myosin II motor domain, similar faster sliding movements were observed on skeletal muscle HMM-coated surfaces. The filament sliding velocities were about two-fold greater than the velocities of normal actin filaments. We found that the velocity of actin filaments sliding on skeletal muscle myosin molecules increased in the presence of a non-motile G680V mutant myosin motor.

We observed a three-dimensional up-and-down movement of an actin filament sliding on heavy mero-myosin (HMM) molecules in an in vitro motility assay. The up-and-down movement occurred along the direction perpendicular to the planar glass plane on which the filament demonstrated a sliding movement. The height length of the up-and-down movement was measured by monitoring the extent of diminishing fluorescent emission from the marker attached to the filament in the evanescent field of attenuation. The height lengths whose distribution exhibits a local maximum were found around the two values, 150 nm and 90 nm, separately. This undulating three-dimensional movement of an actin filament suggests that the interactions between myosin (HMM) molecules and the actin filament may temporally be modulated during its sliding movement.

Fourier's law of heat transfer addressing temperature differences is intrinsically selective in favoring the mitigation of the differences proceeding as fast as possible. We present an experimental demonstration of such selective behavior of material origin. When an actin filament equipped with nano-scale heat acceptors was placed under heat pulsation, it demonstrated a unidirectional movement without the presence of myosin or ATP. The prime factor for the unidirectional movement was the temperature differences between the locally heated portions on the actin filament and the cooler material bodies in the surroundings. The unidirectional movement could be enhanced in the process of mitigating the temperature differences as fast as possible.

An ATP-activated actin filament sliding on myosin molecules exhibited mechanical distortions or fluctuations both longitudinally and transversally along the filament. Although actin filaments exhibited a uniform sliding movement longitudinally as the ATP concentration increased, the longitudinal fluctuations were found to vary their magnitude with the concentration. The magnitude of longitudinal fluctuations reached its maximum at approximately 100 microM of the ATP concentration. The local enhancement of the longitudinal fluctuations as responding to changes in the ATP concentration is associated with a critical phenomenon bridging the two different kinds of mechanical distortions, either contractile or protractile ones, occurring within a sliding actin filament.

The sliding velocity of actin filaments was found to increase in the presence of ATP analogues. At 0.5 mM ATP, the presence of 2.0 mM of AMP-PNP enhanced the filament velocity from 3.2 up to 4.5 microm/s. However, 2 mM ADP decreased the velocity down to 1.1 microm/s. The results suggest that the complex conformations of myosin cross-bridges interacting with an actin filament in the presence of ATP analogues makes the entire filament move faster.

Oligomerization of amino acids proceeded on or inside lipid vesicles as a model of prebiotic cells in a simulated hydrothermal environment. When the suspension of lipid vesiclestaking up monomeric glycine underwent a sudden temperature dropby traversing from a hot (180 °C) to a cold (0 °C) region repeatedly while circulating through a closedreaction circuit, oligopeptides up to heptaglycine were formed even in the absence of condensing agents.

An actin filament sliding on myosin moleculesdemonstrates both longitudinal distortions and transversal fluctuationswith the linear dimension far exceeding the diameter of an actinmonomer. Local swaying of a single actin filament was identified byreading speckled fluorescent markers attached on the filament. Theaccuracy of reading each speckled marker was about 10.4 nm (r.m.s.).Longitudinal distortions of an actin filament at a low ATP concentrationof 20 μM were as much as 0.5 μm for the average filament lengthof 5.4 μm. The magnitude of transversal fluctuations was as much as60 nm, that was independent of the filament length. Both longitudinaldistortions and transversal fluctuations are suggested to play a pivotalrole for facilitating a smooth sliding movement of an actin filament.

An actin filament contacting myosin molecules increased the fluctuation intensity of the filamental displacement as the ATP concentration increased. In particular, fluctuations in the filamental displacement in the planar plane in which the sliding movement takes place were isotropic at a low ATP concentration, and became anisotropic as the concentration increased. The build-up of the sliding movement of an actin filament was associated with the transformation from isotropic to anisotropic fluctuations of the filamental displacement.

An actin filament exhibits distortions longitudinally when it slides upon myosin molecules. We observed that the actin filament demonstrated contractile distortions at low ATP concentrations and protractile distortions at high concentrations. Temporal development of such distortions was identified, by tracing each of several speckled fluorescent markers attached to the actin filament. Close association of the sliding movement to the moving distortions of an actin filament suggests the presence of a unitary mechanism regulating the apparently two different modes of dynamic movement.

An actin filament sliding on myosin molecules in the presence of an extremely low concentration of ATP exhibited a staggered movement. Longitudinally sliding movement of the filament was frequently interrupted by its non-sliding, fluctuating movements both in the longitudinal and transversal directions. Intermittent sliding movements of an actin filament indicate establishment of a coordination of ATP-mediated active sites distributed along the filament.

Myosin molecules contacting an actin filament in the presence of ATP were found to regulate the filamental fluctuations due to ATP hydrolysis in a communicative manner along the filament. As an evidence of the occurrence of the communication, ATP-activated fluctuating displacements of the filament in the direction perpendicular to its longitudinal axis were identified to propagate at a finite velocity not less than about 0.2 micron/s unidirectionally along the filament.

Actin filaments, when partially decorated with troponin T-I complexes, can slide faster on myosin heads than those with no decoration. Purified troponin T-I complexes bind to actins, and inhibit the actin activated myosin adenosine 5′-triphosphatase activity completely when the molar ratio of troponin T-I complex to actin is increased to 1 to 1. Those actin filaments decorated with troponin T-I complexes up to 20 to 50% of their molar ratio exhibit enhancement of the velocity of sliding on myosins up to 20% compared to those without such decoration. As the molar ratio of decoration further increases, the sliding velocity decreases. These results are consistent with the observation that even if some of actin monomers do not participate in the ATPase activity directly, they can interact with myosin heads and take part in the sliding movement.

A comparison was made between fluctuations in a length of sarcomere from a myofibril during relaxed and rigor conditions. The fluctuations of the length associated with rigor states was due entirely to thermal fluctuations in the ambient. In contrast, the fluctuations associated with relaxed states were accompanied by extremely slow and macroscopically modulated thermal fluctuations. A slow, macroscopic modulation of the thermal fluctuations of the length of sarcomere was found to be unique to myofibrils in their relaxed conditions.

We found that a solution of actin filaments can form a periodic texture in the process of drying on a flat glass surface in the air; the periodic texture was composed of smooth meandering bundles of actin filaments. We also found that a branched salt crystal grows in the space between the meandering bundles of actin filaments. The distance between the adjacent striae (striation period) in the resulting dried two-dimensional pattern of striation decreased from about 50 to 2 μm, as the ambient temperature was increased from 4 to 40°C at 1 mg/ml actin, and showed an increasing tendency from a few to several tens μm with the increase in the initial concentration of actin filaments from 0.6 to 2.0 mg/ml at room temperature. As the speed of drying is increased at a certain temperature, the striation period was also found to decrease. We propose that the formation of the two-dimensional striation pattern of bundles of actin filaments is the result of condensation of proteins due to dehydration, and suggest that the solvent flow from the center to the periphery of the sample causes the meandering of actin filaments.

We examined the capacity of tropomyosin molecules regulating the sliding movement of actin filaments on myosin molecules in the presence of ATP molecules to be hydrolyzed. For this objective, we prepared tropomyosin molecules modified to be a little bit stiffer compared to the intact ones by applying a fixed cross-linker between a pair of twisted tropomyosin monomers. The cross-linked tropomyosin molecules, when complexed with actin filaments, were found to inhibit the sliding movement of the filaments on myosin molecules even in the absence of calcium-regulated troponin molecules. It is then suggested that the mechanical flexibility of tropomyosin molecules may be instrumental to actualizing the proper functional regulation of the sliding movement of actin filaments.

An actin filament with heat acceptors attached to its Cys374 residue in each actin monomer could move unidirectionally even under heat pulsation alone, while in the total absence of both ATP and myosin. The prime driver for the movement was temperature gradients operating between locally heated portions on an actin filament and its cooler surroundings. In this report, we investigated how the mitigation of the temperature gradients induces a unidirectional movement of an actin filament. We then observed the transversal fluctuations of the filament in response to heat pulsation and their transition into longitudinally unidirectional movement. The transition was significantly accelerated when Cys374 and Lys336 were simultaneously excited within an actin monomer. These results suggest that the mitigation of the temperature gradients within each actin monomer first went through the energy transformation to transversal fluctuations of the filament, and then followed by the transformation further down to longitudinal movements of the filament. The faster mitigation of temperature gradients within actin monomer helps build up the transition from the transversal to longitudinal movements of the filament by coordinating the interaction between the neighboring monomers.

Troponin extracted from rabbit skeletal muscle directly binds to an actin filament in a molar ratio of 1:1 even in the absence of tropomyosin. An actin filament decorated with troponin did not exhibit significant difference from pure actin filaments in the maximum rate of actomyosin ATP hydrolysis and the sliding velocity of the filament examined by means of an in vitro motility assay. However, the relative number of troponin-bound actin filaments moving in the absence of calcium ions decreased to half that in their presence. The amount of HMM bound to the filaments was less than 4% of actin monomers in the presence of TNs. In addition, actin filaments could not move when Tn molecules were bound in the molar ratio of about 1:1 although they sufficiently bind to myosin heads. These results indicate that troponin can transform an actin monomer within a filament into an Off-state without sterically blocking of the myosin-binding sites with tropomyosin molecules.

We examined both longitudinal and transversal fluctuations of displacements of an actin filament sliding upon Chara myosin molecules. Although the magnitude of transversal fluctuations remained rather independent of ATP concentration, the longitudinal ones were found to increase their magnitude as the concentration increased. In addition, the longitudinal fluctuations gradually increased as the sliding velocity of the filament increased.

The moving velocities of vesicles in the cytoplasmic streaming of a slime mold were measured, in which all of the vesicles passing through a designated window were counted. Vesicles in the streaming are distributed in their moving velocities and the distribution itself varies with time. The mean velocity of vesicles and its standard deviation were found to exhibit a linear relationship, suggesting a possibility that vesicles in the cytoplasm would also be involved in force generation.

Troponin-tropomyosin complex from skeletal muscles was observed to regulate sliding movement of actin filaments on myosin molecules in a manner independent of their ATPase activity. When actin molecules were crosslinked with DSS (disuccinimidyl suberate), the myosin ATPase activity in the presence of the modified actin filaments complexed with both troponin and tropomyosin was only 10% less than that in the case of unmodified actin, and the ATPase activation was independent of calcium ions. In contrast, the sliding velocity of the modified actin filaments on myosin molecules decreased to zero below pCa 6.5. The present results indicate that troponin-tropomyosin complex regulates contractile movement of actomyosin systems through direct alternation of a mechanochemical property of the thin filaments, not through a decrease in the ATPase activity of the myosin molecules.

Single filaments of actin were observed to fluctuate in the direction perpendicular to their longitudinal axes when they hydrolyzed ATP in the presence of myosin. The transversal fluctuations of actin filaments were identified by reading the transversal displacements of the filaments under a fluorescence microscope. The transversal fluctuations in the absence of ATP decreased their intensity as the number of myosin molecules contacting directly with actin filaments increased. In the presence of ATP, on the other hand, the amplitude of the transversal fluctuations increased in proportion to the ATP concentration up to a certain level, while the sliding velocity of the filaments did not increase significantly over the same range of ATP concentration. The present observation suggests that the chemical energy released from ATP along actin filament binding to myosin molecules is first and primarily converted into kinetic energy of fluctuations in the form of the displacement movements of the filaments in the direction perpendicular to their longitudinal axes.

We measured both the ATPase activity of actomyosin complex and the sliding velocity of actin filaments on myosin heads under hydrophobic conditions in the presence of ethanol. Both the ATPase activity and the sliding velocity decrease with the increase of ethanol concentration, if the ionic strength is not too high. As ionic strength increases, there appears an optimum concentration of ethanol that can enhance both the ATPase activity and the sliding velocity.