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Yoshiharu Ishii

Publications and source records attributed to Yoshiharu Ishii.

14 recordsLinked to original sources
PubMed2006

Cooperative actions between myosin heads bring effective functions.

A recent study with single molecule measurements has reported that muscle myosin, a molecular motor, stochastically generates multiple steps along an actin filament associated with the hydrolysis of a single ATP molecule [Kitamura, K., Tokunaga, M., Esaki, S., Iwane, A.H., Yanagida, T., 2005. Mechanism of muscle contraction based on stochastic properties of single actomyosin motors observed in vitro. Biophysics 1, 1-19]. We have built a model reproducing such a stochastic movement of a myosin molecule incorporated with ATPase reaction cycles and demonstrated that the thermal fluctuation was a key for the function of myosin molecules [Esaki, S., Ishii, Y., Yanagida, T., 2003. Model describing the biased Brownian movement of myosin. Proc. Jpn. Acad. 79 (Ser B), 9-14]. The size of the displacement generated during the hydrolysis of single ATP molecules was limited within a half pitch of an actin filament when a single myosin molecules work separately. However, in muscle the size of the displacement has been reported to be greater than 60 nm [Yanagida, T., Arata, T., Oosawa, F., 1985. Sliding distance of actin filament induced by a myosin crossbridge during one ATP hydrolysis cycle. Nature 316, 366-369; Higuchi et al., 1991]. The difference suggests cooperative action between myosin heads in muscle. Here we extended the model built for an isolated myosin head to a system in which myosin heads are aligned in muscle arrangement to understand the cooperativity between heads. The simulation showed that the rotation of the actin filament [Takezawa, Y., Sugimoto, Y., Wakabayashi, K., 1998. Extensibility of the actin and myosin filaments in various states of skeletal muscles as studied by X-ray diffraction. Adv. Exp. Med. Biol. 453, 309-317; Wakabayashi, K., Ueno, Y., Takezawa, Y., Sugimoto, Y., 2001. Muscle contraction mechanism: use of X-ray synchrotron radiation. Nat. Enc. Life Sci. 1-11] associated with the release of ATPase products and binding of ATP as well as interaction between myosin heads allowed the myosin filament to move greater than a half pitch of the actin filament while a single ATP molecule is hydrolyzed. Our model demonstrated that the movement is loosely coupled to the ATPase cycle as observed in muscle.

Animals↗
PubMed2006

Dynamic polymorphism of actin as activation mechanism for cell motility.

Actin filament dynamics are crucial in cell motility. Actin filaments, and their bundles, networks, and gels assemble and disassemble spontaneously according to thermodynamic rules. These dynamically changing structures of actin are harnessed for some of its functions in cells. The actin systems respond to external signals, forces, or environments by biasing the fluctuation of actin assembly structures. In this study, dynamic conformation of actin molecules was studied by monitoring conformational dynamics of actin molecules at the single molecule level in real time. Actin conformation spontaneously fluctuates between multiple conformational states. Regarding myosin motility, the dynamic equilibrium of actin conformation was interpreted as between states that activates and inhibits the motility. The binding of myosin to actin filaments activates myosin motility by shifting the conformational fluctuation of actin towards the state that activates the motility. Thus, the activation mechanism based on thermal fluctuation is suggested at molecular level as well as at cellular level.

Actins↗
PubMed2006

Brownian motion, fluctuation and life.

The measurements of dynamic behaviors of biomolecules in relation to their functions have been allowed using single molecule measurements. Thermal Brownian motion causes random step motion of motor proteins and structural fluctuation of protein molecules between multiple states. In hierarchic structure of life, the fluctuation is modulated. Random fluctuation is biased to directional motion and reactions as a result of interaction of proteins. The fluctuation of kinetic state of signaling proteins results in polarization and localization of cells. A recognition process in brain is also explained by the equation analogous to biochemical reaction at the molecular level. Thus dynamic processes originated from thermal motion may play an important role in activation processes in life.

Actins↗
PubMed2006

Dynamic polymorphism of Ras observed by single molecule FRET is the basis for molecular recognition.

Ras regulates signal transduction pathway function by dynamically interacting with various effectors. To understand the basis for Ras function, its conformational dynamics were measured in the absence and presence of effectors using single molecule fluorescence resonance energy transfer (FRET) between probes located on the Switch II region and GTP. The time trajectories of FRET efficiency from GTP-bound Ras showed that this conformation spontaneously varies among multiple states. Among them, a low FRET state was identified as an inactive state. The transition involving the inactive conformational state occurred in the time range of seconds. In contrast, fluctuation occurring most probably between multiple active high FRET conformational states lasted approximately 30 ms but converged to a specific conformational state upon binding to an effector. Thus, Ras conformation spontaneously fluctuates to readily interact with various effectors.

Fluorescence Resonance Energy Transfer↗
PubMed2006

Dynamic polymorphism of single actin molecules in the actin filament.

Actin filament dynamics are critical in cell motility. The structure of actin filament changes spontaneously and can also be regulated by actin-binding proteins, allowing actin to readily function in response to external stimuli. The interaction with the motor protein myosin changes the dynamic nature of actin filaments. However, the molecular bases for the dynamic processes of actin filaments are not well understood. Here, we observed the dynamics of rabbit skeletal-muscle actin conformation by monitoring individual molecules in the actin filaments using single-molecule fluorescence resonance energy transfer (FRET) imaging with total internal reflection fluorescence microscopy (TIRFM). The time trajectories of FRET show that actin switches between low- and high-FRET efficiency states on a timescale of seconds. If actin filaments are chemically cross-linked, a state that inhibits myosin motility, the equilibrium shifts to the low-FRET conformation, whereas when the actin filament is interacting with myosin, the high-FRET conformation is favored. This dynamic equilibrium suggests that actin can switch between active and inactive conformations in response to external signals.

Actin Cytoskeleton↗
PubMed2006

Single supramolecule spectroscopy of natural and alkaline-treated chlorosomes from green sulfur photosynthetic bacteria.

Fluorescence emission properties of intact and alkaline-treated chlorosomes containing bacteriochlorophyll(BChl)-c, d, and e, which were isolated from four species of green sulfur photosynthetic bacteria, were successfully studied at the single-unit level using a total internal reflection fluorescence microscope. Single intact chlorosomes containing BChl-c from Chlorobium (Chl.) tepidum exhibited heterogeneous emission bands of BChl-c self-aggregates. In contrast, fluorescence spectra of chlorosomal BChl self-aggregates in single intact chlorosomes from the other three Chlorobium species were less heterogeneous than those from Chi. tepidum. Removal of energy-accepting BChl-a/protein complexes called baseplates from the intact chlorosomes by treatments with alkaline media hardly changed spectral shapes of BChl aggregates and their peak distributions at the single-chlorosome level. The similarity of spectral properties at the single-unit level between intact and alkaline-treated chlorosomes of four Chlorobium species clearly indicated that the removal of base-plates from intact chlorosomes by the alkaline-treatment did not affect BChl self-aggregates inside single chlorosomes.

Alkalies↗
PubMed2005

Entropy rectifies the Brownian steps of kinesin.

Kinesin is a stepping motor that successively produces forward and backward 8-nm steps along microtubules. Under physiological conditions, the steps powering kinesin's motility are biased in one direction and drive various biological motile processes. The physical mechanism underlying the unidirectional bias of the kinesin steps is not fully understood. Here we explored the mechanical kinetics and thermodynamics of forward and backward kinesin steps by analyzing their temperature and load dependence. Results show that the frequency asymmetry between forward and backward steps is produced by entropy. Furthermore, the magnitude of the entropic asymmetry is 6 k(B)T, more than three times greater than expected from a current model, in which a mechanical conformational change within the kinesin molecular structure directly biases the kinesin steps forward. We propose that the stepping direction of kinesin is preferably caused by an entropy asymmetry resulting from the compatibility between the kinesin and microtubule interaction based on their polar structures.

Animals↗
PubMed2004

Single-molecule imaging of the dynamic interactions between macromolecules.

In recent years, the development of single-molecule detection techniques has allowed the dynamic properties of biomolecules, which are normally obscured in conventional ensemble measurements, to be measured. One of these single-molecule detection techniques allows the measurement of dissociation and association events of individual molecules to be measured. This technique is based on the unique premise that the mobility between molecules that are bound and the mobility between those that are free in solution are different. The binding of ATP at the beginning and its dissociation at the end of the hydrolysis reaction were detected at the single-molecule level in real time. In this study, we extended this technique to image the dynamic interactions between large biomolecules (protein/protein and protein/polysaccharide). The binding and dissociation of fluorescently labeled macromolecules to partner molecules fixed on a glass surface were visualized by total internal reflection fluorescence microscopy. The dynamic interactions between the proteins in two energy conversion systems, that is, signaling proteins and enzyme molecules moving on dextran, have been measured. In these systems, the dynamic interactions were sensitive to the factors determining the chemical reactions. Thus, the dynamic interactions monitored in the single-molecule measurements provided useful information to further the understanding of the underlying mechanisms of energy conversion systems.

Adenosine Triphosphate↗
PubMed2004

Mechano-chemical coupling of molecular motors revealed by single molecule measurements.

Single molecule measurements have allowed series of kinetic events of biomolecules to be monitored without interruption. The stepwise movement of molecular motors was measured and analyzed in relation to the hydrolysis reaction of ATP. In the case of kinesin, forward and backward steps occurred stochastically at the same chemical state. The directional movement was explained by the asymmetric potential created by the interaction between kinesin and microtubules. Similarly thermal Brownian movement of myosin during the hydrolysis of single ATP molecules was biased through an asymmetric potential, resulting in directional movement. Thus, single molecule measurements have provided new approaches to analyze the function of molecular motors which often consist of several different events.

Actin Cytoskeleton↗
PubMed2003

Stochastic processes in nano-biomachines revealed by single molecule detection.

Proteins and their assemblies are in the size of nanometers and are exposed to thermal disturbances. Many molecular processes in these nano-biomachines are stochastic, reflecting the fact that the input energy level is comparable to that of thermal energy. These stochastic properties have been revealed by recently developed single molecule detection techniques. The movement of molecular motors, myosin, and kinesin, has been suggested to be thermally driven. Random thermal movement is biased using the energy of the ATP hydrolysis. Thus, the molecular motors may harness thermal energy. This unique mechanism may be important in understanding the operation of the biosystems.

Adenosine Triphosphate↗
PubMed2003

Single molecule processes on the stepwise movement of ATP-driven molecular motors.

Movement is a fundamental characteristic of all living things. This biogenic function that is attributed to the molecular motors such as kinesin, dynein and myosin. Molecular motors generate forces by using chemical energy derived from the hydrolysis reaction of ATP molecules. Despite a large number of studies on this topic, the chemomechanical energy transduction mechanism is still unsolved. In this study, we have investigated the chemomechanical coupling of the ATPase cycle to the mechanical events of the molecular motor kinesin using single molecule detection (SMD) techniques. The SMD techniques allowed to detection of the movement of single kinesin molecules along a microtubule and showed that kinesin steps mainly in the forward direction, but occasionally in the backward. The stepping direction is determined by a certain load-dependent process, on which the stochastic behavior is well characterized by Feynman's thermal ratchet model. The driving force of the stepwise movement is essentially Brownian motion, but it is biased in the forward direction by using the free energy released from the hydrolysis of ATP.

Adenosine Triphosphate↗
PubMed2002

Spectral heterogeneity in single light-harvesting chlorosomes from green sulfur photosynthetic bacterium chlorobium tepidum.

The fluorescence emission properties of single chlorosomes from the green sulfur photosynthetic bacterium Chlorobium (Chl.) tepidum are studied for the first time, using a total internal reflection fluorescence microscope. The fluorescence peak positions of bacteriochlorophyll (BChl)-c self-aggregates in a single chlorosome of Chl. tepidum were widely distributed in the wavelength region between 750 and 768 nm, and the standard deviation (s.d. = 4.1 nm, n = 51) was larger than that of single chlorosomes of Chloroflexus (Cfl.) (s.d. = 1.9 nm, n = 50). The spectral heterogeneity among single chlorosomes from Chl. tepidum was in sharp contrast to those from Cfl. aurantiacus. The difference of chlorosomal spectral properties between Chl. tepidum and Cfl. aurantiacus at the single-unit level would be ascribed to the homolog composition of BChl-c--chlorosomes of Chl. tepidum have BChl substituted with various alkyl groups at both the 8- and 12-positions, whereas light-harvesting BChl-c molecules in Cfl. chlorosomes have the same substituents at the 8- (ethyl group) and 12- (methyl group) positions.

Chlorobi↗