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 ...

Single kinesin motor molecules were observed to buckle the microtubules along which they moved in a modified in vitro gliding assay. In this assay a central portion of the microtubule was clamped to the glass substrate via biotin-streptavidin bonds, while the plus end of the microtubule was free to interact ...

Toxoplasma gondii tachyzoites execute a complex and little understood combination of rapid movements to reach and penetrate human or other animals cells. In the present study, computer-assisted simulation was used to quantitatively analyze the motility of these parasites in three-dimensional space with spatial and temporal resolutions in the micrometer and ...

We have determined the relationship between microtubule length and translocation velocity from recordings of bovine brain microtubules translocating over a Paramecium 22S dynein substratum in an in vitro assay chamber. For comparison with untreated samples, the 22S dynein has been subjected to detergent and/or to pretreatments that induce phosphorylation of ...

Muscle contraction is driven by the cyclical interaction of myosin with actin, coupled to the breakdown of ATP. Studies of the interaction of filamentous myosin and of a double-headed proteolytic fragment, heavy meromyosin (HMM), with actin have demonstrated discrete mechanical events, arising from stochastic interaction of single myosin molecules with ...

We used the technique of electrorotation to apply steadily increasing external torque to tethered cells of the bacterium Escherichia coli while continuously recording the speed of cell rotation. We found that the bacterial flagellar motor generates constant torque when rotating forward at low speeds and constant but considerably higher torque ...

Force generation and relative sliding between the myosin and actin filaments in muscle are thought to be caused by tilting of the head region of the myosin crossbridges between the filaments. Structural and spectroscopic experiments have demonstrated segmental flexibility of myosin in muscle, but have not shown a direct linkage ...

Motor inhibition was studied in 3 versions of the stop-signal paradigm, with the stop signal requiring inhibition of any response (stop-all), a fixed alternative response (stop-change), or selective inhibition of only 1 of the responses (selective-stop). The lateralized readiness potential was used in Experiment 1 to distinguish between a selective, ...

We measured the dependence of the variance in the rotation rate of tethered cells of Escherichia coli on the mean rotation rate over a regime in which the motor generates constant torque. This dependence was compared with that of broken motors. In either case, motor torque was augmented with externally ...

The development of in vitro motility assays for motor proteins has been accompanied by a parallel development of advanced optical instrumentation capable of recording motion at the molecular level. Devices now exist that can record displacements to better than 0.1 nm at bandwidths in excess of 10 kHz, and that ...

A review is given of the structure and dynamics of the flagellar rotary motor. Force-generating elements in a motor driving a tethered bacterium (a cell fixed to the substratum by a single flagellum) exert forces of order 20 pN while moving at speeds of order 1 micron/s. Force-generating elements in ...

It was proposed by Huxley and Simmons (Nature 1971, 233:533-538) that force-generating cross-bridges are attached to actin in several stable positions. In this concept, isometric force is generated by the same mechanism as the quick tension recovery after an abrupt release of length; i.e., when crossbridges proceed from the first ...

Recent x-ray diffraction measurements of the axial periodicities of the actin and myosin filaments in contracting muscles show that they are stretched by small but significant amounts by the developed tension, so that at least one half, possibly more, of the observed compliance of a sarcomere must reside in the ...

With sliding actin-filament motility assays, filament velocity should be independent of changes in the level of actomyosin activation under unloaded conditions. Using a simple modification of the motility assay to measure relative changes in isometric force (activation), we determined that isometric force increased 200-fold with thiophosphorylation of the myosin regulatory ...

To elucidate the mechanism of force generation by actomyosin motor, a measuring system was constructed, in which an in vitro motility assay was combined with an optical trapping technique. An actin filament of several micron long was attached to a gelsolin-coated polystyrene bead, and was allowed to interact with a ...

Semmes-Weinstein monofilaments provide a repeatable instrument stimulus with a small standard deviation in contrast to other handheld test instruments, making them an optimum choice for objective sensory testing in a variety of clinics. Normal sensory detection thresholds for the entire body, and the stimulus force for each filament, were determined ...

Much of our current understanding of the molecular physiology of kinesin has come from in vitro motility assays: indeed, the discovery of kinesin relied upon such assays. By marrying in vitro assays with novel instruments capable of resolving movements on the molecular scale, it has proved possible to make measurements ...

Changes in the x-ray diffraction patterns produced by 100-microseconds-length steps imposed during tetanic stimulation were recorded from single intact fibers of frog tibialis anterior muscle. For shortening steps, a staircase length change was applied, with a 20-ms interval between steps. For stretches, each 20-ms cycle started with a stretch, followed ...

The mechanical properties of cytoplasm are considered to be of underlying importance in the mechanism of cell movement and are to a large extent determined by an actin-containing cytoskeleton. Several laboratories have begun to accumulate data on the mechanical or rheologic properties of protein systems derived from the actin cytoskeleton. ...

To probe the mechanism by which the motor protein kinesin moves along microtubules, we have developed a highly sensitive technique for measuring the force exerted by a single motor molecule. In this technique, one end of a microtubule is attached to the tip of a flexible glass fiber of calibrated ...

The flagellar axoneme of the mutant pf18 lacking the central pair does not beat, but undergoes a nanometer-scale, high-frequency oscillation (hyper-oscillation) in the presence of ATP [Yagi et al., 1994: Cell Motil. Cytoskeleton 29:177-185]. The present study demonstrates that the amplitude of the hyper-oscillation increases significantly in the simultaneous presence ...

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 ...

The second part of my tutorial stresses the systematic importance of two parameters of discrimination training: (a) the magnitude of the physical difference between the positive and the negative stimulus (disparity) and (b) the magnitude of the difference between the positive stimulus, in particular, and the background stimulation (salience). It ...

To address large discrepancies reported in the literature, the viscoelastic properties of gels formed by purified actin filaments have been measured by five different techniques and five different instruments using actin preparations purified separately in four different laboratories. These measurements consistently showed that the elastic shear modulus of 2 mg/ml ...

Myosin subfragment-1 (S-1) has been shown to induce single actin filaments into bundles (Ando & Scales (1985) J. Biol. Chem. 260, 2321-2327 ; Ando (1987) J. Mol. Biol. 195, 351-358). We examined the influence of the alkali light chains of myosin on the actin bundling. Only S-1 isoenzyme possessing alkali ...

How motor proteins induce mechanical movement at the molecular level has been a focus of biophysicists for a long time. While the whole picture is yet to be completely revealed, recent developments in looking at nanometer-scale movement with millisecond-time resolution driven by single motors have revealed important new details about ...

In order to determine the relative motions of an actin filament and a myosin molecule upon hydrolysis of one ATP, an in vitro motility assay, in which individual actin filaments slide over heavy meromyosin molecules bound to a substrate, was combined with an optical trapping technique. An actin filament, attached ...

The elementary events in energy transduction by the actomyosin motor, driven by ATP hydrolysis, were directly recorded from multiple and single molecules using a recently developed technique for nano-manipulation of single actin filaments by a microneedle. In order to avoid the effects of random orientation of myosin and association of ...

A new in vitro assay using a feedback enhanced laser trap system allows direct measurement of force and displacement that results from the interaction of a single myosin molecule with a single suspended actin filament. Discrete stepwise movements averaging 11 nm were seen under conditions of low load, and single ...

Movement of single myosin filaments, synthesized by copolymerization of intact myosin and fluorescently labeled light meromyosin, were observed along a single actin filament suspended in solution by a dual laser trap in a fluorescence microscope. The sliding velocity of the myosin filaments was 11.0 +/- 0.2 micron/s at 27 degrees ...

Plants respond to mechanical stress by adaptive changes in growth. Although this phenomenon is well established, the mechanism of the perception of mechanical forces by plant cells is not yet known. We provide evidence that the cortical microtubules subadjacent to the growth-controlling outer epidermal cell wall of maize coleoptiles respond ...

Unloaded contraction of a single sarcomere was studied on fiber fragments (1-micron thick, 4- to 6-microns wide, and less than 50 microns long) from glycerinated scallop striated muscle. The fragment was activated from rigor by a rapid solution exchange method. Sarcomere length (SL) was measured in the intensity profile caused ...

Cells of the bacterium Escherichia coli were tethered and spun in a high-frequency rotating electric field at a series of discrete field strengths. This was done first at low field strengths, then at field strengths generating speeds high enough to disrupt motor function, and finally at low field strengths. Comparison ...

Techniques have been recently developed that allow the direct observation of single actin filaments and their manipulation, using glass microneedles, in the nanometer range. Further development of these techniques has made possible the detection of subpiconewton-level forces of individual myosin heads. This in vitro motility model is sensitive in the ...

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 ...

The photocycle of bacteriorhodopsin (BR) was studied in the 0.3 microsecond to 10 s time interval after excitation, using a wide range of actinic light intensities (10 ns half-duration, 0.06-60 mJ/cm2), at neutral and alkaline pH values. The relative weights of the rapidly and the slowly decaying components of the ...

Most models of mitotic congression and segregation assume that only poleward pulling forces occur at kinetochores. However, there are reports for several different cell types that both mono-oriented and bi-oriented chromosomes oscillate toward and away from the pole throughout mitosis. We used new methods of high resolution video microscopy and ...

Kinesin is a microtubule-based motor protein that uses energy released from Mg-ATP hydrolysis to generate force for the movement of intracellular membranes towards the fast-growing (plus) ends of microtubule tracks in cells. Kinesin-driven microtubule movement can be visualized and quantified using light microscope motility assays but our understanding of how ...

It is generally thought that to generate active force in muscle, myosin heads (cross-bridges) that are attached to actin undergo large-scale conformational changes. However, evidence for conformational changes of the attached cross-bridges associated with force generation has been ambiguous. In this study, we took advantage of the recent observation that ...

The influence of microtubule-associated protein 2 (MAP2) on the dynamics of microtubule assembly and disassembly from axonemal fragments was characterized in vitro in solutions of pure tubulin and varying concentrations of MAP2. A mechanistic description of interactions between MAP2 and individual microtubules was developed from analysis of recorded images obtained ...

Differentiation of the filamentous cyanobacteria Calothrix sp strains PCC 7601 and PCC 7504 is regulated by light spectral quality. Vegetative filaments differentiate motile, gas-vacuolated hormogonia after transfer to fresh medium and incubation under red light. Hormogonia are transient and give rise to vegetative filaments, or to heterocystous filaments if fixed ...

Length steps (complete in 0.2 ms; amplitude < 2% of the fibre length) were applied during the tetanus plateau of intact frog muscle fibres (1.7-3.2 degrees C). The effects of varied tonicity on the early changes in tension in response to the length steps were studied. The solutions were made ...

The force response of Ca(2+)-activated, permeabilized segments of rabbit psoas muscle fibers to stretches and releases was studied. These length changes were imposed (i) during isometric steady state contraction, (ii) as a restretch at the end of a ramp-shaped prerelease, and (iii) during isotonic steady state shortening. The speed of ...

The possibility that weakly binding bridges are attached to actin in the absence of Ca2+ under physiological conditions was investigated by studying the force response of unstimulated intact muscle fibres of the frog to fast ramp stretches. The force response during the stretching period is divided into two phases: phase ...

We have developed a high resolution force measurement system in vitro by manipulating a single actin filament attached to a microneedle. The system could resolve forces less than a piconewton, and has time resolution in the submillisecond range. We have used this system to detect force fluctuations produced by individual ...

The higher force observed in fixed-end tetani relative to sarcomere-isometric tetani is commonly attributed to sarcomere length inhomogeneity; sarcomeres in the end regions of the fiber shorten extensively at the expense of the central sarcomeres. By shortening, these sarcomeres supposedly attain higher force production capacity and can thus account for ...

To study the kinetic properties of the ATP-dependent actin-myosin sliding responsible for muscle contraction, we developed an in vitro force-movement assay system, in which centrifugal forces were applied to myosin-coated polystyrene beads sliding along actin cables of giant algal cells in the presence of ATP. Under constant centrifugal forces directed ...

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 ...

We have visualized, under an optical microscope, the orientations of actin monomers in individual actin filaments undergoing Brownian motion in solution, actively sliding past myosin molecules, or immobile on a surface. For the visualization, two strategies have been adopted. One is to exploit the fluorescence polarization of a fluorescent probe ...

Rheological measurements of the frequency-dependent complex elastic module G*(omega) of entangled F-actin solutions in the frequency range 10(-5)-1 Hz were carried out in three dynamic regimens: 1.) A terminal relaxation from gel-like to liquid-like behaviour measured at frequencies omega < or = tau d-1, 2.) a rubber-type plateau and 3.) ...