MULTIPLE-MOLECULE 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 force s and nanometer displacements in the millisecond time range caused by actin-myosin interaction in vitro by manipulating single actin filamen ts with a glass microneedle. Here, we describe in full the details of this method. Using this method, the elementary events in energy transd uction by the actomyosin motor, driven by ATP hydrolysis, were directl y recorded from multiple and single molecules. We found that not only the velocity but also the force greatly depended on the orientations o f 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 fo rces and displacements by myosin molecules correctly oriented in singl e synthetic myosin rod cofilaments. At a high myosin-to-rod ratio, lar ge force fluctuations were observed when the actin filament interacted in the correct orientation with a cofilament. The noise analysis of t he force fluctuations caused by a small number of heads showed that th e 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 a nd the duty ratio were 12 +/- 2s(-1), and 22 +/- 4s(-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) h ead(-1) for rapid length change or in rigor. At a very low myosin-to-r od ratio, distinct actomyosin attachment, force generation (the power stroke), and detachment events were directly detected. At high load, o ne power stroke generated a force spike with a peak value of 5-6 pN an d a duration of 50 ms (k_(-1)), which were compatible with those of in dividual myosin heads deduced from the force fluctuations. As the load was reduced, the force of the power stroke decreased and the needle d isplacement increased, At near zero load, the mean size of single disp lacement 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 ne edle, was approximately 20 nm.