Hiroto Tanaka

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.

X-ray diffraction evidence for the extensibility of actin and myosin filaments during muscle contraction.

To clarify the extensibility of thin actin and thick myosin filaments in muscle, we examined the spacings of actin and myosin filament-based reflections in x-ray diffraction patterns at high resolution during isometric contraction of frog skeletal muscles and steady lengthening of the active muscles using synchrotron radiation as an intense x-ray source and a storage phosphor plate as a high sensitivity, high resolution area detector. Spacing of the actin meridional reflection at approximately 1/2.7 nm-1, which corresponds to the axial rise per actin subunit in the thin filament, increased about 0.25% during isometric contraction of muscles at full overlap length of thick and thin filaments. The changes in muscles stretched to approximately half overlap of the filaments, when they were scaled linearly up to the full isometric tension, gave an increase of approximately 0.3%. Conversely, the spacing decreased by approximately 0.1% upon activation of muscles at nonoverlap length. Slow stretching of a contracting muscle increased tension and increased this spacing over the isometric contraction value. Scaled up to a 100% tension increase, this corresponds to a approximately 0.26% additional change, consistent with that of the initial isometric contraction. Taken together, the extensibility of the actin filament amounts to 3-4 nm of elongation when a muscle switches from relaxation to maximum isometric contraction. Axial spacings of the layer-line reflections at approximately 1/5.1 nm-1 and approximately 1/5.9 nm-1 corresponding to the pitches of the right- and left-handed genetic helices of the actin filament, showed similar changes to that of the meridional reflection during isometric contraction of muscles at full overlap. The spacing changes of these reflections, which also depend on the mechanical load on the muscle, indicate that elongation is accompanied by slight changes of the actin helical structure possibly because of the axial force exerted by the actomyosin cross-bridges. Additional small spacing changes of the myosin meridional reflections during length changes applied to contracting muscles represented an increase of approximately 0.26% (scaled up to a 100% tension increase) in the myosin periodicity, suggesting that such spacing changes correspond to a tension-related extension of the myosin filaments. Elongation of the myosin filament backbone amounts to approximately 2.1 nm per half sarcomere. The results indicate that a large part (approximately 70%) of the sarcomere compliance of an active muscle is caused by the extensibility of the actin and myosin filaments; 42% of the compliance resides in the actin filaments, and 27% of it is in the myosin filaments.

The motor domain determines the large step of myosin-V

Class-V myosin proceeds along actin filaments with large (∼36 nm) steps. Myosin-V has two heads, each of which consists of a motor domain and a long (23 nm) neck domain. In accordance with the widely accepted lever-arm model, it was suggested that myosin-V steps to successive (36 nm) target zones along the actin helical repeat by tilting its long neck (lever-arm). To test this hypothesis, we measured the mechanical properties of single molecules of myosin-V truncation mutants with neck domains only one-sixth of the native length. Our results show that the processivity and step distance along actin are both similar to those of full-length myosin-V. Thus, the long neck domain is not essential for either the large steps or processivity of myosin-V. These results challenge the lever-arm model. We propose that the motor domain and/or the actomyosin interface enable myosin-V to produce large processive steps during translocation along actin.

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.

Single molecule analysis of the actomyosin motor.

Progress in imaging techniques and nano-manipulation of single molecules has been remarkable. These techniques have allowed the accurate determination of myosin-head-induced displacements and of how the mechanical cycles of the actomyosin motor are coupled to ATP hydrolysis. This has been achieved by measuring mechanical and chemical events of actomyosin directly at the single molecule level. Recent studies have made detailed measurements of myosin step size and mechanochemical coupling. The results of these studies suggest a new model for the mechanism of motion underlying actomyosin motors, which differs from the currently accepted lever-arm swinging model.

Smooth muscle myosin phosphorylated at single head shows sustained mechanical activity

Smooth muscle contraction is regulated by the phosphorylation of myosin. It is well known that tonic smooth muscles can maintain force with low energy consumption (latch state); however, the molecular mechanism underlying this phenomenon is unresolved. Here we show that single-head phosphorylated smooth myosin (SHPMII) exhibits fast (∼24 s–1) and slow prolonged (∼1 s–1) actin interactions, whereas double-head phosphorylated myosin (DHPMII) predominantly exhibits the fast (∼29 s–1) interaction, suggesting that the phosphorylated head of SHPMII is mechanically as active as that of DHPMII. Both the fast and the slow actin interactions of SHPMII support the positive net mechanical displacement of actin. The actin translocating velocity of SHPMII was much slower than that of DHPMII, which is consistent with the slow actin interaction of SHPMII. We propose that the “latch state” can be explained by the motor characteristics of SHPMII that is present during the sustained phase of contraction.

A case of very large intrahepatic bile duct adenoma followed for 7 years

A 70-year-old man was referred to our hospital due to abnormal liver function. A tumor of 92 mm × 61 mm was detected on ultrasound screening of the left liver lobe. Although the tumor was suspected to be intrahepatic bile duct carcinoma, he had chronic heart disease and was unable to undergo surgery. Therefore, he was followed without further testing. No increase in tumor serum markers or tumor size was observed for the subsequent 7 years. We continued to suspect intrahepatic bile duct carcinoma, and we decided to perform a tumor biopsy. Tumor biopsy findings indicated intrahepatic bile duct adenoma (BDA), which is a rare benign epithelial liver tumor typically ranging from 1 mm to 20 mm. We herein report a case of very large BDA followed for 7 years.

A high-dimensional chaotic discrete-time neuron model and bursting phenomena

Abstract Bursting is one of the most important activities in a single neuron, but it is not observed in known discrete-time models, which have one-dimensional dynamics. In this Letter, we propose a high-dimensional discrete-time model of a single neuron by generalization of Aiharas. We analyze its dynamics and bifurcations, and observe bursting phenomena.

Molecular motors and single-molecule enzymology.

Publisher Summary This chapter provides information on the molecular motors and single-molecule enzymology. Molecular motors are the molecular machines that perform mechanical work using the energy released from the hydrolysis of ATP. Mechanical measurements in muscle fibers are carried out extensively, whereas only a few biochemical measurements are reported. In contrast to muscle fibers, the purified protein solutions have been used for biochemical measurements at the expense of the mechanical measurements. Molecular motors—such as myosin and kinesin—are linear motors that slide along protein tracks actin and microtubule, respectively. To elucidate how the myosin molecule works, it is necessary to measure the mechanical events generated by a single myosin and ATP molecule. In the in vitro measurements of biological function, it is critical to keep the proteins intact during the measurements. In the in vitro motility assay, the immobilization of molecular motors on the glass surface is required. The immobilization of the proteins is also required for the imaging of single molecules.

Velocity-Dependent Actomyosin ATPase Cycle Revealed by In Vitro Motility Assay with Kinetic Analysis

The actomyosin interaction plays a key role in a number of cellular functions. Single-molecule measurement techniques have been developed to study the mechanism of the actomyosin contractile system. However, the behavior of isolated single molecules does not always reflect that of molecules in a complex system such as a muscle fiber. Here, we developed a simple method for studying the kinetic parameters of the actomyosin interaction using small numbers of molecules. This approach does not require the specialized equipment needed for single-molecule measurements, and permits us to observe behavior that is more similar to that of a complex system. Using an in vitro motility assay, we examined the duration of continuous sliding of actin filaments on a sparsely distributed heavy meromyosin-coated surface. To estimate the association rate constant of the actomyosin motile system, we compared the distribution of experimentally obtained duration times with a computationally simulated distribution. We found that the association rate constant depends on the sliding velocity of the actin filaments. This technique may be used to reveal new aspects of the kinetics of various motor proteins in complex systems.