Multiple- and single-molecule analysis of the actomyosin motor by nanometer-piconewton manipulation with a microneedle: unitary steps and forces

Abstract

We have developed a new technique for measurements of piconewton forces and nanometer displacements in the millisecond time range caused by actin-myosin interaction in vitro by manipulating single actin filaments with a glass microneedle. Here, we describe in full the details of this method. Using this method, the elementary events in energy transduction by the actomyosin motor, driven by ATP hydrolysis, were directly recorded from multiple and single molecules. We found that not only the velocity but also the force greatly depended on the orientations of 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 forces and displacements by myosin molecules correctly oriented in single synthetic myosin rod cofilaments. At a high myosin-to-rod ratio, large force fluctuations were observed when the actin filament interacted in the correct orientation with a cofilament. The noise analysis of the force fluctuations caused by a small number of heads showed that the 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 and the duty ratio were 12 +/- 2 s-1, and 22 +/- 4 s-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 head-1 for rapid length change or in rigor. At a very low myosin-to-rod ratio, distinct actomyosin attachment, force generation (the power stroke), and detachment events were directly detected. At high load, one power stroke generated a force spike with a peak value of 5-6 pN and a duration of 50 ms (k(-)-1), which were compatible with those of individual myosin heads deduced from the force fluctuations. As the load was reduced, the force of the power stroke decreased and the needle displacement increased. At near zero load, the mean size of single displacement 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 needle, was approximately 20 nm.

Kinetics of force generation by single kinesin molecules activated by laser photolysis of caged ATP

“…Recently many caged compounds, such as caged nucleotides (6), caged calcium (29), caged neurotransmitters (30), and caged fluorophores (31) have been synthesized and used to determine the rate of biomolecular chemical or mechanical reactions. In the previous studies, a high power pulsed laser or a xenon flash lamp was used for rapid photolysis of the caged compounds (6,7,17). Because the laser beam in this study was focused by an objective lens into a small region, a low-power UV laser (12 mW or 60 J per 5-ms pulse) was adequate for photolysis.…”

“…The trapping stiffness (0.07-0.25 pN⅐nm Ϫ1 ) was determined from thermal fluctuations of trapped beads using the equipartition law (10). Fluorescence images of fluorescently labeled microtubules and kinesin-coated beads, excited by a frequency-doubled Nd:YAG green laser (wavelength ϭ 532 nm; model 140-0534-100, Light Wave Electronics, Mountain View, CA), were recorded by a high-sensitivity silicone-intensified target camera (C2741, Hamamatsu Photonics, Hamamatsu City, Japan) and displayed on a television monitor (17). The transmitted bright-field image of the bead, illuminated by the green laser, was projected onto a quadrant photodiode (s944 -13, Hamamatsu Photonics) via an objective lens (Plan ϫ100, Nikon).…”

“…The absorbance of the solution at 405 nm was measured with a mercury arc lamp and a photodiode (HamamatsuPhotonics, S2545) and amplifier (Sentec OP701A) with a response time of 16 s. Data were recorded using MacLab software at a sampling rate of 50 kHz. The caged ATP was photolyzed by a 0.3-ms UV pulse (17). The absorbance increased during the UV pulse and then decreased exponentially with a rate constant k c of 290 s Ϫ1 at 25-27ЊC.…”

Protein Linear Molecular Motor‐Powered Nanodevices

“…Elaborate experiments derived from motility assays, [64,65] which involve optical traps, [4,7] microneedles, [8] or atomic force microscopy (AFM) [66] provided general engineering-relevant parameters. Apart from physical dimensions, [61] the velocity, the force generated, and ATP consumption are of prime interest.…”

“…The past four decades have witnessed extensive investigations into the molecular mechanisms that underpin the operation of these motors, which are central to biology. These fundamental investigations have been (i) experimental-aided by advances in molecular imaging, single molecule manipulation techniques, such as optical trapping, and development of motility assays, [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18] and (ii) theoretical-analyzing and attempting to explain the solely understanding their mechanisms per se. These devices are hybrids: consisting of naturally evolved protein motors, biochemically interfaced and housed in glass or polymeric microstructures, possibly with external (fluidic or EM) control of motility.…”