Shin'ichi Ishiwata

Microscopic analysis of polymerization dynamics with individual actin filaments.

The polymerization–depolymerization dynamics of actin is a key process in a variety of cellular functions. Many spectroscopic studies have been performed in solution, but studies on single actin filaments have just begun. Here, we show that the time course of polymerization of individual filaments consists of a polymerization phase and a subsequent steady-state phase. During the steady-state phase, a treadmilling process of elongation at the barbed end and shortening at the pointed end occurs, in which both components of the process proceed at approximately the same rate. The time correlation of length fluctuation of the filaments in the steady-state phase showed that the polymerization–depolymerization dynamics follow a diffusion (stochastic) process, which cannot be explained by simple association and dissociation of monomers at both ends of the filaments.

Dynamic study of F-actin by quasielastic scattering of laser light

Abstract A newly developing technique, quasielastic scattering of laser light, was applied to the dynamic study of F-actin interacting with other muscle proteins, heavy meromyosin, S-1 (i.e. a single head of myosin) and tropomyosin. The following three systems were studied: (1) the interaction between F-actin and heavy meromyosin or S-1 in the absence of ATP; (2) the interaction between F-actin and tropomyosin at various salt concentrations; and (3) the interaction between heavy meromyosin and the complex of F-actin and tropomyosin in the absence of ATP. F-actin was most flexible when the mole ratio of heavy meromyosin to F-actin monomer was 1:6. At this ratio, one heavy meromyosin molecule binds to about half a pitch of one strand of the actin filament. On the other hand, flexibility of F-actin was not changed on the addition of S-l. The addition of tropomyosin to F-actin solutions made F-actin stiffer. Tropomyosin appears to bind directly to F-actin up to a weight ratio of tropomyosin to F-actin of 1:6. This direct binding of tropomyosin affected the interaction between F-actin and heavy meromyosin. On the addition of heavy meromyosin to the complex solution of F-actin and tropomyosin, the complex was most flexible when the molar ratio of heavy meromyosin to F-actin was between 1:3 and 1:2. F-actin is not a rigid rod; rather, it appears to be a partially flexible chain. The flexibility of F-actin may have an important role in the molecular mechanism of muscular contraction.

Mechanochemical coupling of two substeps in a single myosin V motor.

Myosin V is a double-headed processive molecular motor that moves along an actin filament by taking 36-nm steps. Using optical trapping nanometry with high spatiotemporal resolution, we discovered that there are two possible pathways for the 36-nm steps, one with 12- and 24-nm substeps, in this order, and the other without substeps. Based on the analyses of effects of ATP, ADP and 2,3-butanedione 2-monoxime (a reagent shown here to slow ADP release from actomyosin V) on the dwell time and the occurrence frequency of the main and the intermediate states, we propose that the 12-nm substep occurs after ATP binding to the bound trailing head and the 24-nm substep results from a mechanical step following the isomerization of an actomyosin-ADP state on the bound leading head. When the isomerization precedes the 12-nm substep, the 36-nm step occurs without substeps.

F1-ATPase: A Rotary Motor Made of a Single Molecule

Nucleotide-driven motors, including F1, share common structural motifs near the nucleotide-binding site (18xVale, R.D. J. Cell Biol. 1996; 135: 291–302Crossref | PubMedSee all References, 14xNoji, H, Amano, T, and Yoshida, M. J. Bioenerg. Biomemb. 1996; 28: 451–457Crossref | PubMedSee all References), suggesting that these motors might employ common principles in some aspects of their mechanisms. As a general principle, we propose that the distinction between bending and binding is important.Bending (conformational change) of a motor protein alone could produce motion and force relative to its rail, the latter serving merely as a base that securely holds the “sole” of the “foot” of the motor. Myosin is considered to bend its leg forward when attached to actin, producing the “unitary step” (Figure 3Figure 3, pink myosin on the left; Goldman 1998xGoldman, Y.E. Cell. 1998; 93: 1–4Abstract | Full Text | Full Text PDF | PubMed | Scopus (83)See all ReferencesGoldman 1998). The machinery for bending could all be in myosin, because isolated myosin changes its conformation depending on the bound nucleotide (Gulick and Rayment 1997xGulick, A.M and Rayment, I. Bioessay. 1997; 19: 561–569Crossref | PubMedSee all ReferencesGulick and Rayment 1997). The free-energy changes associated with myosin ATPase, however, indicate that myosin alone would be unable to produce a large amount of work. Moreover, when myosin interacts with actin, as much as half of the free energy of ATP hydrolysis is used for unbinding of myosin from actin. Subsequent rebinding thus liberates energy. If myosin is to work at high efficiency, it should convert the energy gained during rebinding to mechanical output, by cooperation with actin.A model by A. F. Huxley 1957xHuxley, A.F. Progr. Biophys. Biophys. Chem. 1957; 7: 255–318PubMedSee all ReferencesHuxley 1957 is on the other extreme: that binding alone produces motion and force. Myosin fluctuates thermally, and when it fluctuates in the correct direction, it binds to actin resulting in displacement and pull. In binding-alone models, thermal diffusion brings the motor and rail close to the binding configuration, and binding energy is used to stabilize that configuration. Work has to be done in the diffusion process, and can be done as shown below. Diffusive displacement of a particle of diameter d over a distance L takes a time of the order of (L2/2) · (3πηd/kBT), which is ∼1 μs for L = d = 10 nm at room temperature (thermal energy kBT ≈ 4 pN · nm) in water (the viscosity η ≈ 10−3 N · s · m−2). If this displacement is to produce work W (against a load), the time for displacement is multiplied by ∼exp(W/kBT), which is 2 × 104 for W = 10 kBT ≈ 40 pN · nm and 5 × 108 for W = 20 kBT ≈ 80 pN · nm. Thus, work below 10 kBT can be done if the frequency of motor operation is below ∼102/s. Binding models also require a mechanism that ensures correct choice of a binding site, or proper directional biasing of diffusion. The mechanism is not specified in the Huxley model.An elegant interplay between bending and binding has been proposed for kinesin and its cousin ncd (Hirose et al. 1996xHirose, K, Lockhart, A, Cross, R.A, and Amos, L.A. Proc. Natl. Acad. Sci. USA. 1996; 93: 9539–9544Crossref | PubMed | Scopus (124)See all ReferencesHirose et al. 1996). When one foot of kinesin (or ncd) is bound to a microtubule (rail), the other foot is unbound and undergoes thermal motion. They have shown that the unbound foot of kinesin, which walks toward the plus end of a microtubule, swings toward the plus end presumably by bending of the bound leg (Figure 3Figure 3, pink kinesin), and the unbound foot of minus-directed ncd swings toward the minus end. The bending biases the Brownian search of the unbound foot for the next binding site, for the plus direction for kinesin, and minus for ncd. The 8 nm step of kinesin (yet unresolved for ncd), and associated force, are produced when the foot lands on the binding site. A substep(s) and partial force may be produced by the bending, but the major mechanical output of this motor likely comes from the binding of the motor to its rail.The three-foot F1 (Figure 3Figure 3) could in principle operate by binding alone, stepping among the three stable configurations with the correct direction being dictated by the bound nucleotide. (“Binding” for the case of F1 should be interpreted as a transition to the most stable configuration between βs and γ, and might involve repulsive rather than attractive interactions.) The large mechanical output of ∼20 kBT per step, however, cannot be achieved by a purely diffusive process because it would be too infrequent to account for the observed rate of rotation. Probably, the effective potential between βs and γ is downhill toward the next stable configuration, thus assisting the diffusion against an external load. The work per step would be determined by the total height of the potential slope, which is not dependent on the rotational speed. Bending of the three βs alone is unlikely to rotate γ by 120° because of the obstruction by intervening α subunits.Of course the distinction between bending and binding becomes less obvious as one inquires more deeply into the mechanism. What we wish to stress here is that molecular motors must work through close cooperation of the two partners. The rail, in particular, is not a simple support, and binds and unbinds its nucleotide-hydrolyzing partner, supplying binding energy and controlling hydrolysis. The two aspects, bending and binding, should be useful in analyzing the mechanism of cooperation. The F1 motor in which the two partners never detach from each other provides a wonderful opportunity to explore the details of the cooperation experimentally.

Effect of calcium ions on the flexibility of reconstituted thin filaments of muscle studied by quasielastic scattering of laser light

Abstract Quasielastic scattering of laser light was used to examine the effect of calcium ions on the flexibility of a reconstituted thin filament, namely, an F-actin/tropomyosin/troponin complex of striated muscle. The results showed that the flexibility of a reconstituted thin filament changed reversibly at about 1 μ m of free calcium ions (i.e. the physiological concentration). Below 1 μ m -Ca 2+ a tropomyosin/troponin system suppressed the bending motion of F-actin. This suppression was removed by calcium ions above 1 μ m , and the F-actin flexibility became almost the same as that of an F-actin/tropomyosin complex. A quantitative study showed that calcium ions affected the reconstituted thin filament in two ways at the free calcium ion concentrations of about 1 μ m and 20 μ m , corresponding, respectively, to the two different calcium binding constants of troponin. An over-all conformational change of a reconstituted thin filament is brought about by calcium ions. It is suggested that the effect of calcium ions on troponin can be transferred to F-actin by altering the binding between tropomyosin and several monomer units in F-actin. The possible role of the dynamic nature of F-actin in muscle contraction is discussed.

Loading direction regulates the affinity of ADP for kinesin.

Kinesin is an ATP-driven molecular motor that moves processively along a microtubule. Processivity has been explained as a mechanism that involves alternating single- and double-headed binding of kinesin to microtubules coupled to the ATPase cycle of the motor. The internal load imposed between the two bound heads has been proposed to be a key factor regulating the ATPase cycle in each head. Here we show that external load imposed along the direction of motility on a single kinesin molecule enhances the binding affinity of ADP for kinesin, whereas an external load imposed against the direction of motility decreases it. This coupling between loading direction and enzymatic activity is in accord with the idea that the internal load plays a key role in the unidirectional and cooperative movement of processive motors.

Myosin V is a left-handed spiral motor on the right-handed actin helix

Myosin V is a two-headed, actin-based molecular motor implicated in organelle transport. Previously, a single myosin V molecule has been shown to move processively along an actin filament in discrete ∼36 nm steps. However, 36nm is the helical repeat length of actin, and the geometry of the previous experiments may have forced the heads to bind to, or halt at, sites on one side of actin that are separated by 36 nm. To observe unconstrained motion, we suspended an actin filament in solution and attached a single myosin V molecule carrying a bead duplex. The duplex moved as a left-handed spiral around the filament, disregarding the right-handed actin helix. Our results indicate a stepwise walking mechanism in which myosin V positions and orients the unbound head such that the head will land at the 11th or 13th actin subunit on the opposing strand of the actin double helix.

Kinesin–microtubule binding depends on both nucleotide state and loading direction

Kinesin is a motor protein that transports organelles along a microtubule toward its plus end by using the energy of ATP hydrolysis. To clarify the nucleotide-dependent binding mode, we measured the unbinding force for one-headed kinesin heterodimers in addition to conventional two-headed kinesin homodimers under several nucleotide states. We found that both a weak and a strong binding state exist in each head of kinesin corresponding to a small and a large unbinding force, respectively; that is, weak for the ADP state and strong for the nucleotide-free and adenosine 5′-[β,γ-imido]triphosphate states. Model analysis showed that (i) the two binding modes in each head could be explained by a difference in the binding energy and (ii) the directional instability of binding, i.e., dependence of unbinding force on loading direction, could be explained by a difference in the characteristic distance for the kinesin–microtubule interaction during plus- and minus-end-directed loading. Both these factors must play an important role in the molecular mechanism of kinesin motility.

Characterization of single actomyosin rigor bonds: load dependence of lifetime and mechanical properties.

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 (tau) and the externally imposed load (F) at absolute temperature T could be expressed as tau(F) = tau(0).exp(-F.d/k(B)T) with tau(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 tau(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 tau(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.

Submillisecond rotational dynamics of spin-labeled myosin heads in myofibrils.

The rotational motion of crossbridges, formed when myosin heads bind to actin, is an essential element of most molecular models of muscle contraction. To obtain direct information about this molecular motion, we have performed saturation transfer EPR experiments in which spin labels were selectively and rigidly attached to myosin heads in purified myosin and in glycerinated myofibrils. In synthetic myosin filaments, in the absence of actin, the spectra indicated rapid rotational motion of heads characterized by an effective correlation time of 10 microseconds. By contrast, little or no submillisecond rotational motion was observed when isolated myosin heads (subfragment-1) were attached to glass beads or to F-actin, indicating that the bond between the myosin head and actin is quite rigid on this time scale. A similar immobilization of heads was observed in spin-labeled myofibrils in rigor. Therefore, we conclude that virtually all of the myosin heads in a rigor myofibril are immobilized, apparently owing to attachment of heads to actin. Addition of ATP to myofibrils, either in the presence or absence of 0.1 mM Ca2+, produced spectra similar to those observed for myosin filaments in the absence of actin, indicating rapid submillisecond rotational motion. These results indicate that either (a) most of the myosin heads are detached at any instant in relaxed or activated myofibrils or (b) attached heads bearing the products of ATP hydrolysis rotate as rapidly as detached heads.