西坂 崇之

Motor proteins are essential in life processes because they convert the free energy of ATP hydrolysis to mechanical work. However, the fundamental question on how they work when different amounts of free energy are released after ATP hydrolysis remains unanswered. To answer this question, it is essential to clarify how the stepping motion of a motor protein reflects the concentrations of ATP, ADP, and P-i in its individual actions at a single molecule level. The F, portion of ATP synthase, also called F-1-ATPase, is a rotary molecular motor in which the central gamma-subunit rotates against the alpha(3)beta(3) cylinder. The motor exhibits clear step motion at low ATP concentrations. The rotary action of this motor is processive and generates a high torque. These features are ideal for exploring the relationship between free energy input and mechanical work output, but there is a serious problem in that this motor is severely inhibited by ADP. In this study, we overcame this problem of ADP inhibition by introducing several mutations while retaining high enzymatic activity. Using a probe of attached beads, stepping rotation against viscous load was examined at a wide range of free energy values by changing the ADP concentration. The results showed that the apparent work of each individual step motion was not affected by the free energy of ATP hydrolysis, but the frequency of each individual step motion depended on the free energy. This is the first study that examined the stepping motion of a molecular motor at a single molecule level with simultaneous systematic control of Delta G(ATP). The results imply that microscopically defined work at a single molecule level cannot be directly compared with macroscopically defined free energy input.

F₁-ATPase is a rotary molecular motor in which unidirectional rotation of the central <symbol>γ</symbol>-subunit is powered by ATP hydrolysis in three catalytic sites arranged 120° apart around <symbol>γ</symbol>. To see how hydrolysis reactions produce mechanical rotation, we observed rotation of <symbol>γ</symbol> under the optical microscope, while watching which of the three sites bound and released a fluorescent ATP analog. The reaction scheme, including both the number of site occupancy and reactions that trigger substeps, is now established.<br>

F1-ATPase is a rotary molecular motor in which unidirectional rotation of the central γ subunit is powered by ATP hydrolysis in three catalytic sites arranged 120° apart around γ. To study how hydrolysis reactions produce mechanical rotation, we observed rotation under an optical microscope to see which of the three sites bound and released a fluorescent ATP analog. Assuming that the analog mimics authentic ATP, the following scheme emerges: (i) in the ATP-waiting state, one site, dictated by the orientation of γ, is empty, whereas the other two bind a nucleotide (ii) ATP binding to the empty site drives an ∼80° rotation of γ (iii) this triggers a reaction(s), hydrolysis and/or phosphate release, but not ADP release in the site that bound ATP one step earlier (iv) completion of this reaction induces further ∼40° rotation.

Position-dependent cycling of integrin interactions with both the cytoskeleton and extracellular matrix (ECM) is essential for cell spreading, migration, and wound healing. Whether there are regional changes in integrin concentration, ligand affinity or cytoskeleton crosslinking of liganded integrins has been unclear. Here, we directly demonstrate a position- dependent binding and release cycle of fibronectin-integrin-cytoskeleton interactions with preferential binding at the front of motile 3T3 fibroblasts and release at the endoplasm-ectoplasm boundary. Polystyrene beads coated with low concentrations of an integrin-binding fragment of fibronectin (fibronectin type III domains 7-10) were 3-4 times more likely to bind to integrins when placed within 0.5 microns vs. 0.5-3 microns from the leading edge. Integrins were not concentrated at the leading edge, nor did anti- integrin antibody-coated beads bind preferentially at the leading edge. However, diffusing liganded integrins attached to the cytoskeleton preferentially at the leading edge. Cytochalasin inhibited edge binding, which suggested that cytoskeleton binding to the integrins could alter the avidity for ligand beads. Further, at the ectoplasm-endoplasm boundary, the velocity of bead movement decreased, diffusive motion increased, and approximately one-third of the beads were released into the medium. We suggest that cytoskeleton linkage of liganded integrins stabilizes integrin- ECM bonds at the front whereas release of cytoskeleton-integrin links weakens integrin-ECM bonds at the back of lamellipodia.

Load dependence of the lifetime of the rigor bonds formed between a single myosin molecule (either heavy meromyosin, HMM, or myosin subfragment-1, S1) and actin filament was examined in the absence of nucleotide by pulling the barbed end of the actin filament with optical tweezers. For S1, the relationship between the lifetime (τ) and the externally imposed load (F) at absolute temperature T could be expressed as τ(F) = τ(0)·exp(-F·d/k(B)T) with τ(0) of 67 s and an apparent interaction distance d of 2.4 nm (k(B) is the Boltzmann constant). The relationship for HMM was expressed by the sum of two exponentials, with two sets of τ(0) and d being, respectively, 62 s and 2.7 nm, and 950 s and 1.4 nm. The fast component of HMM coincides with τ(F) for S1, suggesting that the fast component corresponds to single-headed binding and the slow component to double-headed binding. These large interaction distances, which may be a common characteristic of motor proteins, are attributed to the geometry for applying an external load. The pulling experiment has also allowed direct estimation of the number of myosin molecules interacting with an actin filament. Actin filaments tethered to a single HMM molecule underwent extensive rotational Brownian motion, indicating a low torsional stiffness for HMM. From these results, we discuss the characteristics of interaction between actin and myosin, with the focus on the manner of binding of myosin.

We have developed temperature-pulse microscopy in which the temperature of a microscopic sample is raised reversibly in a square-wave fashion with rise and fall times of several ms, and locally in a region of approximately 10 μm in diameter with a temperature gradient up to 2°C/μm. Temperature distribution was imaged pixel by pixel by image processing of the fluorescence intensity of rhodamine phalloidin attached to (single) actin filaments. With short pulses, actomyosin motors could be activated above physiological temperatures (higher than 60°C at the peak) before thermally induced protein damage began to occur. When a sliding actin filament was heated to 40-45°C, the sliding velocity reached 30 μm/s at 25 mM KCl and 50 μm/s at 50 mM KCl, the highest velocities reported for skeletal myosin in usual in vitro assay systems. Both the sliding velocity and force increased by an order of magnitude when heated from 18°C to 40-45°C. Temperature- pulse microscopy is expected to be useful for studies of biomolecules and cells requiring temporal and/or spatial thermal modulation.

We sumrnarize the mechanical and functional properties of a motormolecule of muscle studied with a new type of an in vitro motility assay system and a multi-imaging microscope system. First, supercoiling of an actin filament is shown, suggesting that the filament moves forward like a right-handed screw. Second, we describe the unbinding force and the lifetime of a rigor bond between a single myosin molecule and an actin filament measured by using optical tweezers. From these results, the mechanism of sliding and force generation of the motor molecule is discussed.

To elucidate the mechanism of force generation by actomyosin motor, a measuring system was constructed, in which an in vitro motility assay was combined with an optical trapping technique. An actin filament of several μm long was attached to a gelsolin-coated polystyrene bead, and was allowed to interact with a small number (~1/1-μm actin filament) of rabbit skeletal heavy meromyosin (an active subfragment of myosin) molecules bound to a nitrocellulose-coated coverglass. The bead position was determined at 33-ms intervals. We measured the force generation event at relatively low (100-400 nM) ATP concentration so that the occurrence of individual force generation events could be detected with our time resolution. The actin-bound bead held in the optical trap moved in a stepwise manner in the direction of the actin filament only in the presence of ATP. At the trap strength of 0.3 pN/nm, the maximum size of the step was 11 nm, and the maximum force associated with the movement was 3.3 pN.

THE unbinding and rebinding of motor proteins and their substrate filaments are the main components of sliding movement1. We have measured the unbinding force between an actin filament and a single motor molecule of muscle, myosin, in the absence of ATP, by pulling the filament with optical tweezers2. The unbinding force could be measured repeatedly on the same molecule, and was independent of the number of measurements and the direction of the imposed loads within a range of ±90°. The average unbinding force was 9.2 ± 4.4 pN, only a few times larger than the sliding force3"5 but an order of magnitude smaller than other intermolecular forces6,7. From its kinetics8 we suggest that unbinding occurs sequentially at the molecular interface, which is an inherent property of motor molecules. © 1995 Nature Publishing Group.

In order to determine the relative motions of an actin filament and a myosin molecule upon hydrolysis of one ATP, an in vitro motility assay, in which individual actin filaments slide over heavy meromyosin molecules bound to a substrate, was combined with an optical trapping technique. An actin filament, attached to a gelsolin-coated bead, was captured with an optical trap. The surface-bound heavy meromyosin molecules pulled the filament against the trapping force, which resulted in back and forth motions of the actin-bound bead. The number of heavy meromyosin molecules interacting with an actin filament (at most 1/ μm filament) and the ATP concentration (≤0.5 μM) were chosen so as to facilitate detection of each "pull." Calculation of the centroid of the bead image revealed abrupt displacements of the actin filament. The frequency of such displacements was between 0.05 and 0.1 per 1 s per 1μm actin filament, being consistent with calculated values based on the reported bimolecular binding constants of ATP and the actomyosin rigor complex. The distribution of the displacements peaked around 7 nm at a trapping force of 0.016 pN/nm, but it became broader, and some displacements were as large as 30 nm, when the trapping force was reduced to 0.0063 pN/nm, suggesting that the force generation due to the structural change of a myosin head may be insufficient to explain such displacements. © 1994 BY The Journal of Biochemistry.

MUSCLE contraction occurs by mutual sliding between thick (myosin) and thin (actin) filaments1,2. But the physical and chemical properties of the sliding force are not clear even the precise direction of sliding force generated at each cross-bridge is not known. We report here the use of a recently developed in vitro motile assay system3-5 to show supercoiling of an actin filament in which the front part of the filament was fixed to a glass surface through cross-linked heavy-meromyosin and the rear part was able to slide on a track of heavy-meromyosin. A left-handed single turn of superhelix formed just before supercoiling, suggesting that the sliding force has a right-handed torque component that induces the right-handed rotation of an actin filament around its long axis. The presence of the torque component in the sliding force will explain several properties of the contractile system of muscle. © 1993 Nature Publishing Group.

A new microscope technique, termed ''W'' (double view video) microscopy, enables simultaneous observation of two different images of an object through a single video camera or by eye. The image pair may, for example, be transmission and fluorescence, fluorescence at different wavelengths, or mutually perpendicular components of polarized fluorescence. Any video microscope can be converted into a dual imager by simple insertion of a small optical device. The continuous appearance of the dual image assures the best time resolution in existing and future video microscopes. As an application, orientations of actin protomers in individual, moving actin filaments have been imaged at the video rate. Asymmetric calcium influxes into a cell exposed to an intense electric pulse have also been visualized.