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A Ishijima

Publications and source records attributed to A Ishijima.

At least 19 recordsLinked to original sources
PubMed2001

Single molecule nanomanipulation of biomolecules.

The development of nanomanipulation techniques has given investigators the ability to manipulate single biomolecules and to record mechanical events of biomolecules at the single molecule level. The techniques were developed to elucidate the mechanism of molecular motors. We can directly monitor the unitary process of the mechanical work and the energy conversion processes by combining these techniques with the single molecule imaging techniques. Our results strongly suggest that the sliding movement of the actomyosin motor is driven by Brownian movement. Other groups have reported data that are more consistent with the lever arm model. These methods and imaging techniques enable us to monitor the behavior of biomolecules at work and will be applied to other molecular machines.

Actin Cytoskeleton↗
PubMed2001

Single molecule nanobioscience.

In recent years, the rapid development and progress of single-molecule detection techniques have opened up a new era of biological research. The advantage of single-molecule studies is that data are not obscured by the ensemble-averaged measurements inherent in classical biochemical experiments. These techniques are shedding light on the dynamic and mechanistic properties of molecular machines, both in vivo and in vitro. This review summarizes the single-molecule experiments that have been designed to investigate molecular motors, enzyme reactions, protein dynamics, DNA transcription and cell signaling.

Actins↗
PubMed2000

Single-motor mechanics and models of the myosin motor.

Recent progress in single-molecule detection techniques is remarkable. These techniques have allowed the accurate determination of myosin-head-induced displacements and how mechanical cycles are coupled to ATP hydrolysis, by measuring individual mechanical events and chemical events of actomyosin directly at the single-molecule level. Here we review our recent work in which we have made detailed measurements of myosin step size and mechanochemical coupling, and propose a model of the myosin motor.

Actins↗
PubMed1998

Simultaneous observation of individual ATPase and mechanical events by a single myosin molecule during interaction with actin.

We have developed a technique that allows mechanical and ligand-binding events in a single myosin molecule to be monitored simultaneously. We describe how steps in the ATPase reaction are temporally related to mechanical events at the single molecule level. The results show that the force generation does not always coincide with the release of bound nucleotide, presumably ADP. Instead the myosin head produces force several hundreds of milliseconds after bound nucleotide is released. This finding does not support the widely accepted view that force generation is directly coupled to the release of bound ligands. It suggests that myosin has a hysteresis or memory state, which stores chemical energy from ATP hydrolysis.

Actin Cytoskeleton↗
PubMed1998

Orientation dependence of displacements by a single one-headed myosin relative to the actin filament.

Displacements of single one-headed myosin molecules in a sparse myosin-rod cofilament were measured from bead displacements at various angles relative to an actin filament by dual optical trapping nanometry. The sparse myosin-rod cofilaments, 5-8 micron long, were synthesized by slowly mixing one-headed myosin prepared by papain digestion with myosin rods at molar ratios of 1:400 to 1:1500, so that one to four one-headed myosin molecules were on average scattered along the cofilament. The bead displacement was approximately 10 nm at low loads ( approximately 0.5 pN) and at angles of 5-10 degrees between the actin and myosin filaments (near physiologically correct orientation). The bead displacement decreased with an increase in the angle. The bead displacement at nearly 90 degrees was approximately 0 nm. When the angle was increased to approximately 150 degrees-170 degrees, the bead displacements increased to 5 nm. A native two-headed myosin showed similar size and orientation dependence of bead displacements as a one-headed myosin.

Actins↗
PubMed1996

Torsional rigidity of single actin filaments and actin-actin bond breaking force under torsion measured directly by in vitro micromanipulation.

Knowledge of the elastic properties of actin filaments is crucial for considering its role in muscle contraction, cellular motile events, and formation of cell shape. The stiffness of actin filaments in the directions of stretching and bending has been determined. In this study, we have directly determined the torsional rigidity and breaking force of single actin filaments by measuring the rotational Brownian motion and tensile strength using optical tweezers and microneedles, respectively. Rotational angular fluctuations of filaments supplied the torsional rigidity as (8.0 +/- 1.2) x 10(-26) Nm2. This value is similar to that deduced from the longitudinal rigidity, assuming the actin filament to be a homogeneous rod. The breaking force of the actin-actin bond was measured while twisting a filament through various angles using microneedles. The breaking force decreased greatly under twist, e.g., from 600-320 pN when filaments were turned through 90 degrees, independent of the rotational direction. Our results indicate that an actin filament exhibits comparable flexibility in the rotational and longitudinal directions, but breaks more easily under torsional load.

Actins↗
PubMed1996

Modification of the bi-directional sliding movement of actin filaments along native thick filaments isolated from a clam.

The properties of bi-directional sliding of F-actin prepared from rabbit skeletal muscle moving along clam thick filaments have been characterized in the presence of agents known to modify unloaded shortening velocity in muscle to determine if the sliding characteristics of actin are similar in the two directions of movement. Actin filaments moved at a fast velocity towards the central bare zone (11.1 +/- 0.2 microns s-1) and at a slower velocity away from the bare zone (3.9 +/- 0.3 microns s-1). Movement of filaments at the slow sliding velocity is thought to be sustained by a change in orientation of the myosin head. The Michaelis Menten constant (Km values) of approximately 0.3 mM in the presence of MgATP concentrations of 0.01-2.0 mM at an ionic strength of 43.5 mM were reduced to approximately 0.1 mM at low ionic strength (18.5 mM) although the Km values at the fast and slow sliding velocities at each ionic strength were similar. In the presence of constant concentrations of MgATP, increasing the MgADP concentrations from 0.5 to 2mM, decreased the bi-directional sliding velocity of actin. The data were well fitted with an equation described by Michaelis Menten kinetics yielding mean absolute Km and Ki values of 0.41 +/- 0.01 and 0.44 +/- 0.05 mM for the fast velocity and 0.29 +/- 0.07 and 0.45 +/- 0.02 mM for the slow velocity of sliding, respectively. The Km and Ki values were not significantly different from each other at either the fast or slow sliding velocities. The actin filament sliding velocity appeared to be controlled through the thick filament as actin was devoid of regulatory proteins and the presence of Ca2+ modified the MgATP dependent movement of actin. The pCa value for half maximal sliding velocity was 7.0 for both fast and slow velocities. The Km and Ki values and the Ca2+ sensitivity of the actin movement at the fast and slow sliding velocity are similar suggesting that no major biochemical changes have occurred in the myosin head as a result of a change in orientation.

Actins↗
PubMed1996

Multiple- and single-molecule analysis of the actomyosin motor by nanometer-piconewton manipulation with a microneedle: unitary steps and forces.

We have developed a new technique for measurements of piconewton forces and nanometer displacements in the millisecond time range caused by actin-myosin interaction in vitro by manipulating single actin filaments with a glass microneedle. Here, we describe in full the details of this method. Using this method, the elementary events in energy transduction by the actomyosin motor, driven by ATP hydrolysis, were directly recorded from multiple and single molecules. We found that not only the velocity but also the force greatly depended on the orientations of myosin relative to the actin filament axis. Therefore, to avoid the effects of random orientation of myosin and association of myosin with an artificial substrate in the surface motility assay, we measured forces and displacements by myosin molecules correctly oriented in single synthetic myosin rod cofilaments. At a high myosin-to-rod ratio, large force fluctuations were observed when the actin filament interacted in the correct orientation with a cofilament. The noise analysis of the force fluctuations caused by a small number of heads showed that the myosin head generated a force of 5.9 +/- 0.8 pN at peak and 2.1 +/- 0.4 pN on average over the whole ATPase cycle. The rate constants for transitions into (k+) and out of (k-) the force generation state and the duty ratio were 12 +/- 2 s-1, and 22 +/- 4 s-1, and 0.36 +/- 0.07, respectively. The stiffness was 0.14 pN nm-1 head-1 for slow length change (100 Hz), which would be approximately 0.28 pN nm-1 head-1 for rapid length change or in rigor. At a very low myosin-to-rod ratio, distinct actomyosin attachment, force generation (the power stroke), and detachment events were directly detected. At high load, one power stroke generated a force spike with a peak value of 5-6 pN and a duration of 50 ms (k(-)-1), which were compatible with those of individual myosin heads deduced from the force fluctuations. As the load was reduced, the force of the power stroke decreased and the needle displacement increased. At near zero load, the mean size of single displacement spikes, i.e., the unitary steps caused by correctly oriented myosin, which were corrected for the stiffness of the needle-to-myosin linkage and the randomizing effect by the thermal vibration of the needle, was approximately 20 nm.

Actomyosin↗
PubMed1995

Neonatal alloimmune thrombocytopenia involving HPA-5b (Br(a)): a rare Japanese case.

Neonatal alloimmune thrombocytopenia (NAIT) is caused by platelet antigen incompatibility between the mother and fetus. The frequency of NAIT varies among ethnic groups. In Caucasians, HPA-5b (Br(a)) is the antigen that is second most frequently implicated. In Japan, NAIT due to anti-HPA-5b antibody is quite rare. The present case is the second case of Br(a)-NAIT in Japanese and the first case serologically confirmed by monoclonal antibody-specific immobilization of platelet antigens.

Antigens, Human Platelet↗
PubMed1994

Direct measurement of stiffness of single actin filaments with and without tropomyosin by in vitro nanomanipulation.

In order to explain the molecular mechanism of muscle contraction, it is crucial to know the distribution of the sarcomere compliance of active muscle. Here, we directly measure the stiffness of single actin filaments with and without tropomyosin, using a recently developed technique for nanomanipulation of single actin filaments with microneedles. The results show that the stiffness for 1-micron-long actin filaments with and without tropomyosin is 65.3 +/- 6.3 and 43.7 +/- 4.6 pN/nm, respectively. When the distribution of crossbridge forces along the actin filament is taken into account, the elongation of a 1-micron-long thin filament during development of isometric contraction is calculated to be approximately 0.23%. The time constant of force in response to a sudden length change is < 0.2 ms, indicating that the viscoelasticity is negligible in the millisecond time range. These results suggest that approximately 50% of the sarcomere compliance of active muscle is due to extensibility of the thin filaments.

Actins↗
PubMed1994

Single-molecule analysis of the actomyosin motor using nano-manipulation.

The elementary events in energy transduction by the actomyosin motor, driven by ATP hydrolysis, were directly recorded from multiple and single molecules using a recently developed technique for nano-manipulation of single actin filaments by a microneedle. In order to avoid the effects of random orientation of myosin and association of myosin with an artificial substrate in the surface motility assay, we used single myosin-rod cofilaments with various ratios. Distinct actomyosin attachment, force generation (the power stroke) and detachment events were detected at a very low myosin: rod ratio. At high load, one power stroke generated 5-6 pN peak force and 2.3 pN force averaged over the cycle, which were compatible with those deduced from noise analysis of force fluctuations caused by multiple molecules. As the load was reduced, the length of the power stroke increased. At near zero load, the length of a power stroke was approximately 17 nm. The results suggested that an ATPase cycle produces one power stroke at high load and many ones at low load.

Actins↗
PubMed1993

Force-generating domain of myosin motor.

To understand the underlying mechanism of force generation by myosin motor, it is crucial to know which part of the molecule is essential for the process. Recent structure determination of myosin motor domain at atomic resolution has revealed that the domain comprises two smaller domains, the "ATPase domain" consisting of only an N-terminal segment of the heavy chain and the "neck domain" consisting of a long alpha-helix of the heavy chain and two light chains. This atomic structure begs the question of whether both domains are required for force generation. To answer it, we genetically truncated the head to generate a recombinant fragment composed of the "ATPase domain" alone. The truncated head drove sliding movement of actin filaments and generated force in a novel in vitro assay system, which allows us to hold a specific site of the head on a glass surface. These results indicate that the compact ATPase domain functions as a force-generating machinery of the myosin motor.

Actins↗
PubMed1993

Charge-reversion mutagenesis of Dictyostelium actin to map the surface recognized by myosin during ATP-driven sliding motion.

Amino acid residues D24/D25, E99/E100, E360/E361, and D363/E364 in subdomain 1 of Dictyostelium actin were replaced with histidine residues by site-directed mutagenesis. Mutant actins were expressed in Dictyostelium cells and purified to homogeneity. The sliding movement of mutant actin filaments on heavy meromyosin attached to a glass surface was measured to assess the effect of the mutation on the motility of actin. For two C-terminal mutants, force generated by a single actin filament and myosin was also measured. These measurements indicated that both D24/D25 and E99/E100 are involved in ATP-driven sliding, whereas E360/E361/D363/E364 are not essential for ATP-driven sliding and force generation.

Actins↗
PubMed1993

Coupling between ATPase and force-generating attachment-detachment cycles of actomyosin in vitro.

We have developed a high resolution force measurement system in vitro by manipulating a single actin filament attached to a microneedle. The system could resolve forces less than a piconewton, and has time resolution in the submillisecond range. We have used this system to detect force fluctuations produced by individual molecular interactions. We observed large force fluctuations during isometric force generations. Noise analysis of the force fluctuations showed that the force was produced by stochastic and independent attachment-detachment cycles between actin and myosin heads, and one force-generating attachment-detachment cycle corresponded to each ATPase cycle. But, the force fluctuations almost completely disappeared during sliding at the velocities of 20 to 70% of the maximum one at zero load. The analysis indicated that myosin heads produced an almost constant force for most (probably > 70%) of the ATPase cycle time, i.e., the duty ratio > 0.7. Since the myosin step size was given as (velocity) x (the duty ratio) x (the ATPase cycle time, 30 ms), it was calculated to be 40 to 110 nm, corresponding to velocities of 20 to 70% of the maximum one (9 microns/s), respectively. These values are much greater than the displacement by a single attachment-detachment cycle of actomyosin (10-20 nm), indicating that multiple force-generating attachment-detachment cycles correspond to each ATPase cycle during sliding at velocities of > 20% of the maximum one. In conclusion, the coupling between the ATPase and the force-generating attachment-detachment cycles of actomyosin is not rigidly determined in a one-to-one fashion but is variable depending on the load.

Actins↗
PubMed1993

Nano-manipulation of actomyosin molecular motors in vitro: a new working principle.

Techniques have been recently developed that allow the direct observation of single actin filaments and their manipulation, using glass microneedles, in the nanometer range. Further development of these techniques has made possible the detection of subpiconewton-level forces of individual myosin heads. This in vitro motility model is sensitive in the submillisecond range and has allowed us to determine the force generation of an actomyosin motor directly at the molecular level. The results have led to a new conceptual framework for chemo-mechanical energy transduction in the molecular motor.

Actins↗
PubMed1992

Super helix formation of actin filaments in an in vitro motile system.

Muscle contraction results from relative sliding of actin and myosin filaments. However, the possibility that actin filaments twist or rotate during sliding has not yet been experimentally investigated. We found that a super helix of an actin filament is formed in an in vitro motile system. This fact suggests that an actin filament twists and rotates due to a torque component of a sliding force generated at cross-bridges.

Actin Cytoskeleton↗