Basic properties of ATP-induced myosin head movement in hydrated myosin filaments, studied using the gas environmental chamber.

Although more than 50 years have passed since the monumental discovery of Huxley and Hanson that muscle contraction results from relative sliding between actin and myosin filaments, coupled with ATP hydrolysis, the mechanism underlying the filament sliding still remains to be a mystery. It is generally believed that the myofilament sliding is caused by cyclic attachment-detachment between myosin heads in myosin filaments and myosin-binding sites in actin filaments. Attempts to prove the myosin head movement using techniques of X-ray diffraction and chemical probes attached to myosin heads have failed to obtain clear results because of the asynchronous nature of myosin head movement. Using the gas environmental chamber (EC) attached to an electron microscope, we succeeded in recording myosin head movement in hydrated myosin filaments, coupled with ATP hydrolysis with the following results: (1)In the absence of actin filaments, myosin heads fluctuate around a definite neutral position, so that their time-averaged position remains unchanged; (2) On ATP application, myosin heads bind with ATP to be in the charged-up state, M-ADP-Pi, and perform a recovery stroke in the direction away from the myosin filament central bare zone and stay in the post-recovery stroke position; (3) In the actin-myosin filament mixture, myosin heads form rigor linkages with actin, and bind with applied ATP to be in the charged-up state, M-ADP-Pi, and perform a power stroke in the direction towards the myosin filament bare zone, while releasing ADP and Pi to stay in the post-power stroke position; (4) In both recovery and power strokes, myosin heads in the non charged-up state return to the neutral position. These results indicate that the charged-up myosin heads decide their direction of movement without being guided by actin filaments.

Effect of deuterium oxide on actomyosin motility in vitro.

Actin filament velocities in an in vitro motility assay system were measured both in heavy water (deuterium oxide, D(2)O) and water (H(2)O) to examine the effect of D(2)O on the actomyosin interaction. The dependence of the sliding velocity on pD of the D(2)O assay solution showed a broad pD optimum of around pD 8.5 which resembled the broad pH optimum (pH 8.5) of the H(2)O assay solution, but the maximum velocity (4.1+/-0.5 microm/s, n=11) at pD 8.5 in D(2)O was about 60% of that (7.1+/-1.1 microm/s, n=11) at pH 8.5 in H(2)O. The K(m) values of 95 and 80 microM and V(max) values of 3.2 and 5.1 microm/s for the D(2)O and H(2)O assay were obtained by fitting the ATP concentration dependence of the velocity (at pD and pH 7.5) to the Michaelis-Menten equation. The K(m) value of actin-activated Mg-ATPase activity of myosin subfragment 1 (S1) was decreased from 50 microM [actin] in H(2)O to 33 microM [actin] in D(2)O without any significant changes in V(max) (9.4 s(-1) in D(2)O and 9.3 s(-1) in H(2)O). The rate constants of ADP release from the acto-S1-ADP complex measured by the stopped flow method were 361+/-26 s(-1) (n=27) in D(2)O and 512+/-39 s(-1) (n=27) in H(2)O at 6 degrees C. These results suggest that the decrease in the in vitro actin-myosin sliding velocity in D(2)O results from a slowing of the release of ADP from the actomyosin-ADP complex and the increase in the affinity of actin for myosin in the presence of ATP in D(2)O.

Measurement of nucleotide exchange rate constants in single rabbit soleus myofibrils during shortening and lengthening using a fluorescent ATP analog.

The kinetics of displacement of a fluorescent nucleotide, 2'(3')-O-[N[2-[[Cy3]amido]ethyl]carbamoyl]-adenosine 5'-triphosphate (Cy3-EDA-ATP), bound to rabbit soleus muscle myofibrils were studied using flash photolysis of caged ATP. Use of myofibrils from this slow twitch muscle allowed better resolution of the kinetics of nucleotide exchange than previous studies with psoas muscle myofibrils (, Biophys. J. 73:2033-2042). Soleus myofibrils in the presence of Cy3-EDA-nucleotides (Cy3-EDA-ATP or Cy3-EDA-ADP) showed selective fluorescence staining of the A-band. The K(m) for Cy3-EDA-ATP and the K(d) for Cy3-EDA-ADP binding to the myofibril A-band were 1.9 microM and 3.8 microM, respectively, indicating stronger binding of nucleotide to soleus cross-bridges compared to psoas cross-bridges (2.6 microM and 50 microM, respectively). After flash photolysis of caged ATP, the A-band fluorescence of the myofibril in the Cy3-EDA-ATP solution under isometric conditions decayed exponentially with a rate constant of 0.045 +/- 0.007 s(-1) (n = 32) at 10 degrees C, which was about seven times slower than that for psoas myofibrils. When a myofibril was allowed to shorten with a constant velocity, the nucleotide displacement rate constant increased from 0.066 s(-1) (isometric) to 0.14 s(-1) at 20 degrees C with increasing shortening velocity up to 0.1 myofibril length/s (V(max), the shortening velocity under no load was approximately 0. 2 myofibril lengths/s). The rate constant was not significantly affected by an isovelocity stretch of up to 0.1 myofibril lengths/s. These results suggest that the cross-bridge kinetics are not significantly affected at higher strain during lengthening but depend on the lower strain during shortening. These data also indicate that the interaction distance between a cross-bridge and the actin filament is at least 16 nm for a single cycle of the ATPase.

Comparison of unitary displacements and forces between 2 cardiac myosin isoforms by the optical trap technique: molecular basis for cardiac adaptation.

To provide information on the mechanism of cardiac adaptation at the molecular level, we compared the unitary displacements and forces between the 2 rat cardiac myosin isoforms, V1 and V3. A fluorescently labeled actin filament, with a polystyrene bead attached, was caught by an optical trap and brought close to a glass surface sparsely coated with either of the 2 isoforms, so that the actin-myosin interaction took place in the presence of a low concentration of ATP (0.5 micromol/L). Discrete displacement events were recorded with a low trap stiffness (0.03 to 0.06 pN/nm). Frequency distribution of the amplitude of the displacements consisted of 2 gaussian curves with peaks at 9 to 10 and 18 to 20 nm for both V1 and V3, suggesting that 9 to 10 nm is the unitary displacement for both isoforms. The duration of the displacement events was longer for V3 than for V1. On the other hand, discrete force transients were recorded with a high trap stiffness (2.1 pN/nm), and their amplitude showed a broad distribution with mean values between 1 and 2 pN for V1 and V3. The durations of the force transients were also longer for V3 than for V1. These results indicate that both the unitary displacements and forces are similar in amplitude but different in duration between the 2 cardiac myosin isoforms, being consistent with the reports that the tension cost is higher in muscles consisting mainly of V1 than those consisting mainly of V3.

Measurement of ATP turnover during shortening and lengthening of rabbit psoas myofibrils using a fluorescent ATP analog.

In order to study ATP turnover during shortening and lengthening of rabbit psoas myofibrils, we have used fluorescence microscopy in which the displacement of a fluorescent nucleotide analog, 2'(3')-O-[N-[2-[[Cy3] amido] ethyl] carbamoyl]-adenosine 5' triphosphate (Cy3-EDA-ATP) bound to cross-bridge on flash photolysis of caged ATP was measured [Chaen et al. (1997) Biophys. J. 73, 2033-2042]. In the previous paper, we reported that when a myofibril was imposed to shorten with a constant velocity by a piezo-electric actuator, the nucleotide displacement rate constant initially increased to 0.7 s-1 with increasing shortening velocity and then declined with a further increase in shortening velocity. The rate constant during lengthening measured in the present experiment was found to be not significantly affected. These results suggest that the cross-bridge kinetics show a asymmetrical dependence on the mechanical strain in the cross-bridges, namely, the rate constants are not significantly affected at higher strain during lengthening but depend on the lower strain during shortening.

Evanescent excitation microscopy. Its application to the study of single molecular process kinetics of actomyosin motor.

Evanescent field was generated on the stage of an inverted microscope upon an incidence of 532 nm Nd-YAG laser beam on interface between aqueous solution and fused silica glass. Thick filaments isolated from Mytilus edulis were adsorbed to the glass surface and nanomolar concentration of adenosine triphosphate (ATP) labeled with rhodamine was allowed to interact with thick filaments. The fluorescence from the surface was observed by triple-view microscopy at video rate. There were many fluorescent spots at the interface, which we identified as individual fluorescent ATP molecules. We found that the fluorescence from those spots was polarized. Fluorescence intensity of individual spots fluctuated considerably. We interpret the latter observation as a result of change in the orientation of emission dipole of the fluorescent ATP analog.

ATP-induced axial movement of myosin heads in living thick filaments recorded with a gas environmental chamber attached to the electron microscope.

Using a gas environmental (hydration) chamber, in which biological specimens can be kept in wet state, we succeeded in recording images of 'living' muscle thick filaments with gold position markers attached to the myosin heads. The position of individual myosin heads did not change appreciably with time in the absence of ATP, indicating stability of the myosin head mean position. On application of ATP, the position of individual myosin heads was found to move by approximately 20 nm along the filament axis, while no appreciable movement of the filaments was detected. The ATP-induced myosin head movement was not observed in filaments in which ATPase activity of the myosin heads was eliminated. Application of ADP produced no appreciable myosin head movement. These results show that the ATP-induced myosin head movement takes place in the absence of the thin filaments. Since ATP reacts rapidly with the myosin head (M) to form the complex (M.ADP.Pi) having average lifetime of > 10 s, the observed myosin head movement may be mostly associated with reaction, M + ATP-->M.ADP.Pi. This work will open a new research field to study dynamic structural changes of individual biomolecules which are kept in 'living' state in an electron microscope.

Distinct kinetic properties of cardiac myosin isoforms revealed by in vitro studies.

To clarify the physiological significance of myosin isoform redistribution in cardiac adaptation process, we compared the kinetic property of the two cardiac myosin isoforms using in vitro motility assay techniques. Cardiac myosin isoforms V1 and V3 were obtained from ventricular muscle of young rats and hypothyroid rats respectively. On each of these myosin isoforms fixed on a glass coverslip, fluorescently labeled actin filaments were made to slide in the presence of ATP. To measure the force generated by actomyosin interaction, a small latex bead was attached to the barbed end of an actin filament and the bead was captured by the laser optical trap installed in a microscope. The force was determined from the distance between the bead and the trap positions under either auxotonic or isometric conditions. The time-averaged force generated by multiple cross-bridges did not differ significantly between the two isoforms. On the other hand, the unitary force measurement revealed the same level of amplitude but a longer duration for V3 isoform. The same level of time-averaged force is in agreement with not only our previous finding but the results of maximum force measurement in muscle preparations. The difference in kinetic characteristics of the two isoforms could account for the difference in economy of force development and the basis for cardiac adaptation mechanism.

Measurement of nucleotide release kinetics in single skeletal muscle myofibrils during isometric and isovelocity contractions using fluorescence microscopy.

Rabbit psoas muscle myofibrils, in the presence of the fluorescent nucleotide analog 2'(3')-O-[N-[2-[[Cy3]amido]ethyl]carbamoyl]-adenosine 5' triphosphate (Cy3-EDA-ATP), showed selective fluorescence staining of the A-band with a reduced fluorescence at the M-line. Addition of Cy3-EDA-ATP to a myofibril in the presence of Ca2+ caused auxotonic shortening against a compliant glass microneedle. These results indicate that Cy3-EDA-ATP is a substrate for myosin in the myofibril system. The kinetics of nucleotide release from a single myofibril, held isometrically between two needles, were measured by the displacement of prebound Cy3-EDA-ATP on flash photolysis of caged ATP. The A-band fluorescence of the myofibril decayed exponentially with a rate constant of 0.3 s(-1) at 8 degrees C, an order of magnitude faster than that for isolated thick filaments in the absence of actin. When a myofibril was imposed to shorten with a constant velocity by a piezoelectric actuator, the nucleotide displacement rate constant initially increased to 0.7 s(-1) with increasing shortening velocity and then declined with a further increase in shortening velocity. These results demonstrate that the displacement rates of Cy3-EDA-nucleotides bound to the cross-bridges in the contracting myofibril reflect a process that shows strain dependence.

Dynamic electron microscopy of ATP-induced myosin head movement in living muscle thick filaments.

Although muscle contraction is known to result from movement of the myosin heads on the thick filaments while attached to the thin filaments, the myosin head movement coupled with ATP hydrolysis still remains to be investigated. Using a gas environmental (hydration) chamber, in which biological specimens can be kept in wet state, we succeeded in recording images of living muscle thick filaments with gold position markers attached to the myosin heads. The position of individual myosin heads did not change appreciably with time in the absence of ATP, indicating stability of the myosin head mean position. On application of ATP, the position of individual myosin heads was found to move by approximately 20 nm along the filament axis, whereas no appreciable movement of the filaments was detected. The ATP-induced myosin head movement was not observed in filaments in which ATPase activity of the myosin heads was eliminated. Application of ADP produced no appreciable myosin head movement. These results show that the ATP-induced myosin head movement takes place in the absence of the thin filaments. Because ATP reacts rapidly with the myosin head (M) to form the complex (M. ADP.Pi) with an average lifetime of >10 s, the observed myosin head movement may be mostly associated with reaction, M + ATP --> M.ADP. Pi. This work will open a new research field to study dynamic structural changes of individual biomolecules, which are kept in a living state in an electron microscope.

Lower activation energy for sliding of F-actin on a less thermostable isoform of carp myosin.

We have examined the temperature-dependence of sliding velocity of fluorescent F-actin on myosins isolated from 10 degrees C- and 30 degrees C-acclimated carp. Activation energies for the sliding of F-actin were 63 and 111 kJ/mol for the 10 degrees C- and 30 degrees C-acclimated carp myosins, respectively. Arrhenius plots of the sliding velocity from 10 degrees C- and 30 degrees C-acclimated carp myosin were shown to intersect at high temperature (about 30 degrees C). The thermostability estimated by measuring the Ca(2-)-ATPase activity was less for myosin from 10 degrees C- than 30 degrees C-acclimated carp. We suggest that a less thermostable structure in cold-acclimated carp myosin results in a reduced activation energy for the contractile process, which allows the F-actin to slide fast even at low temperatures.

Different cardiac myosin isoforms exhibit equal force-generating ability in vitro.

We measured forces generated by myosin molecules and a single actin filament using an optical trap system. The force per unit length of actin filament did not differ significantly between cardiac myosin isoforms. V1 and V3. This indicates that the ability to generate force is equal between V1 and V3, despite their difference in the unloaded sliding velocity past actin.

The mode of ATP-dependent microtubule-kinesin sliding in the auxotonic condition.

Kinesin is a motor protein that converts chemical energy derived from ATP hydrolysis into mechanical work to transport cellular components along microtubules. We studied the properties of ATP-dependent microtubule-kinesin sliding with two different in vitro assay systems. In one assay system, a kinesin-coated glass microneedle (elastic coefficient, 1-2.5 pN microns -1) was made to slide along an axoneme. Using this system, we obtained the relationship between the force (= load) on the microneedle and the velocity of microneedle-kinesin sliding in the auxotonic condition, in which the load on the microtubule-kinesin contacts increased as sliding progressed. The force-velocity curve was upwardly convex (maximum velocity Vmax, 0.58 +/- 0.15 microns s-1; maximum isometric force P0, 5.0 +/- 1.6 pN) and was similar to that of in vitro actin-myosin sliding in the auxotonic condition, suggesting that the two motor protein systems have fundamental kinetic properties in common. In the other assay system, an axoneme attached to a glass microneedle (elastic coefficient, 4-5 pN microns -1) was made to slide on a kinesin-coated glass surface (Vmax, 0.68 +/- 0.17 microns s-1; P0, 46.1 +/- 18.6 pN). The change in shape of the axoneme indicated an enormous flexibility of randomly oriented kinesin molecules.

Force-velocity relations of rat cardiac myosin isozymes sliding on algal cell actin cables in vitro.

The difference in kinetic properties between two myosin isozymes (V1 and V3) in rat ventricular myocardium was studied by determining the steady-state force-velocity (P-V) relations in the ATP-dependent movement of V1 and V3-coated polystyrene beads on actin cables of giant algal cells mounted on a centrifuge microscope. The maximum unloaded velocity of bead movement was larger for V1 than for V3. The velocity of bead movement decreased with increasing external load applied by the centrifuge microscope, and eventually reached zero when the load was equal to the maximum isometric force (P0) generated by the myosin heads. The maximum isometric force P0 was less than 10 pN, and did not differ significantly between V1 and V3. The P-V curves consisted of a hyperbolic part in the low force range and a non-hyperbolic part in the high force range. The critical force above which the curve deviated from the hyperbola was much smaller for V1 than for V3. An analysis using a model with an extremely small number of myosin heads involved in the bead movement suggested a marked difference in kinetic properties between V1 and V3.

The force-velocity relationship of the ATP-dependent actin-myosin sliding causing cytoplasmic streaming in algal cells, studied using a centrifuge microscope.

When uncoated polystyrene beads suspended in Mg-ATP solution were introduced into the internodal cell of an alga Chara corallina, the beads moved along the actin cables with directions and velocities (30-62 microns s-1) similar to those of native cytoplasmic streaming. Bead movement was inhibited both in the absence of ATP and in the presence of CA2+, as with native cytoplasmic streaming. These results indicate that bead movement is caused by cytoplasmic myosin molecules attached to the head surface interacting with actin cables. The steady-state force-velocity relationship of the actin-myosin sliding that produces cytoplasmic streaming was determined by applying constant centrifugal forces to the beads moving on the actin cables. The force-velocity curve in the positive load region was nearly straight, and the implications of this shape are discussed in connection with the kinetic properties of the actin-myosin interaction in cytoplasmic streaming. It is suggested that the time for which a cytoplasmic myosin head is detached from actin in one cycle of actin-myosin interaction is very short. The Ca(2+)-induced actin-myosin linkages, responsible for the Ca(2+)-induced stoppage of cytoplasmic streaming, were shown to be much stronger than the rigor actin-myosin linkages.

Effect of lateral forces on the movement of myosin-coated beads on actin cables studied using a centrifuge microscope.

We developed an in vitro motility assay system, in which myosin-coated polystyrene beads were made to slide on actin filament arrays (actin cables) in giant algal cells and subjected to centrifugal forces, which were parallel to the direction of bead movement to serve as external loads on actin-myosin sliding (Oiwa et al. (1990) Proc Natl Acad Sci USA 87: 7893-7897), and succeeded in determining the steady-state force-velocity relation of ATP-dependent actin-myosin sliding. To give further information about the properties of actin-myosin sliding, we have applied centrifugal forces, in parallel with the plane of actin-myosin sliding but at right angles with the direction of bead movement, and have found that such "lateral" centrifugal forces reduced the velocity of bead movement. In addition, we have also found that the velocity of bead movement is reduced more markedly with lateral forces applied from the left side of the bead ("left" lateral forces) than those applied from the right side of the bead ("right" lateral forces). These results are discussed in connection with the direction of sliding force generated by the myosin heads on the bead which interact with the right-handed double helix of actin monomers constituting actin filaments.

Relation between magnetically-applied force and velocity in beads coated with rabbit myosin, sliding on actin cables in Nitellopsis cells.

We have succeeded in controlling the sliding movement of myosin-coated magnetizable beads on actin cables in Nitellopsis cells by the inhomogeneous magnetic field adjacent to a small, strong permanent magnet. The relation between magnetic force acting on the bead and the bead velocity was, in many respects, similar to that obtained from the same system by the use of centrifugal force (Oiwa et al., 1990). In particular, force favouring the motion (negative load) had little effect on the velocity until it was sufficient to pull the bead off the actin, whereas a relatively small positive load caused a reduction in velocity to a plateau value. Although the present method does not allow a good control of force direction, it demonstrates the promise of magnetic force in studying in vitro motility.

Dependence of the work done by ATP-induced actin-myosin sliding on the initial baseline force: its implications for kinetic properties of myosin heads in muscle contraction.

The properties of the ATP-dependent actin-myosin sliding responsible for muscle contraction was studied using an in vitro force-movement assay system, in which a myosin-coated glass microneedle was made to slide on actin filament arrays (actin cables) in the giant algal cell with iontophoretic application of ATP. With a constant amount of ATP application, the amount of work done by the actin-myosin sliding increased with increasing baseline force from zero to 0.4-0.6 Po, and then decreased with further increasing baseline force, thus giving a bell-shaped work versus baseline force relation. The result that the maximum actin-myosin sliding velocity did not change appreciably with increasing baseline force up to 0.4-0.6 Po implies, together with the limited number of myosin heads involved, that (1) the rate of power output of actin-myosin sliding is determined primarily by the amount of external load rather than the velocity of actin-myosin sliding, and (2) the bell shaped work versus baseline force relation (and also the hyperbolic force-velocity relation) results from the kinetic properties of individual myosin head rather than the change in the number of myosin heads involved.