Motility of single one-headed kinesin molecules along microtubules.

The motility of single one-headed kinesin molecules (K351 and K340), which were truncated fragments of Drosophila two-headed kinesin, has been tested using total internal reflection fluorescence microscopy. One-headed kinesin fragments moved continuously along the microtubules. The maximum distance traveled until the fragments dissociated from the microtubules for both K351 and K340 was approximately 600 nm. This value is considerably larger than the space resolution of the measurement system (SD approximately 30 nm). Although the movements of the fragments fluctuated in forward and backward directions, statistical analysis showed that the average movements for both K340 and K351 were toward the plus end of the microtubules, i.e., forward direction. When BDTC (a 1.3-S subunit of Propionibacterium shermanii transcarboxylase, which binds weakly to a microtubule), was fused to the tail (C-terminus) of K351, its movement was enhanced, smooth, and unidirectional, similar to that of the two-headed kinesin fragment, K411. However, the travel distance and velocity of K351BDTC molecules were approximately 3-fold smaller than that of K411. These observations suggest that a single kinesin head has basal motility, but coordination between the two heads is necessary for stabilizing the basal motility for the normal level of kinesin processivity.

Single-motor mechanics and models of the myosin motor.

Recent progress in single-molecule detection techniques is remarkable. These techniques have allowed the accurate determination of myosin-head-induced displacements and how mechanical cycles are coupled to ATP hydrolysis, by measuring individual mechanical events and chemical events of actomyosin directly at the single-molecule level. Here we review our recent work in which we have made detailed measurements of myosin step size and mechanochemical coupling, and propose a model of the myosin motor.

Direct inhibition of microtubule-based kinesin motility by local anesthetics.

Local anesthetics are known to inhibit neuronal fast anterograde axoplasmic transport (FAAT) in a reversible and dose-dependent manner, but the precise mechanism has not been determined. FAAT is powered by kinesin superfamily proteins, which transport membranous organelles, vesicles, or protein complexes along microtubules. We investigated the direct effect of local anesthetics on kinesin, using both in vitro motility and single-molecule motility assays. In the modified in vitro motility assay, local anesthetics immediately and reversibly stopped the kinesin-based microtubule movement in an all-or-none fashion without lowering kinesin ATPase activity. QX-314, a permanently charged derivative of lidocaine, exerted an effect similar to that of lidocaine, suggesting that the effect of anesthetics is due to the charged form of the anesthetics. In the single-molecule motility assay, the local anesthetic tetracaine inhibited the motility of individual kinesin molecules in a dose-dependent manner. The concentrations of the anesthetics that inhibited the motility of kinesin correlated well with those blocking FAAT. We conclude that the charged form of local anesthetics directly and reversibly inhibits kinesin motility in a dose-dependent manner, and it is the major cause of the inhibition of FAAT by local anesthetics.

Mechanical and chemical properties of cysteine-modified kinesin molecules.

To probe the structural changes within kinesin molecules, we made the mutants of motor domains of two-headed kinesin (4-411 aa) in which either all the five cysteines or all except Cys45 were mutated. A residual cysteine (Cys45) of the kinesin mutant was labeled with an environment-sensitive fluorescent probe, acrylodan. ATPase activity, mechanical properties, and fluorescence intensity of the mutants were measured. Upon acrylodan-labeled kinesin binding to microtubules in the presence of 1 mM AMPPNP, the peak intensity was enhanced by 3.4-fold, indicating the structural change of the kinesin head by the binding. Substitution of cysteines decreased both the maximum microtubule-activated ATPase and the sliding velocity to the same extent. However, the maximum force and the step size were not affected; the force produced by a single molecule was 6-6.5 pN, and a step size due to the hydrolysis of one ATP molecule by kinesin molecules was about 10 nm for all kinesins. This step size was close to a unitary step size of 8 nm. Thus, the mechanical events of kinesin are tightly coupled with the chemical events.

A single myosin head moves along an actin filament with regular steps of 5.3 nanometres.

Actomyosin, a complex of actin filaments and myosin motor proteins, is responsible for force generation during muscle contraction. To resolve the individual mechanical events of force generation by actomyosin, we have developed a new instrument with which we can capture and directly manipulate individual myosin subfragment-1 molecules using a scanning probe. Single subfragment-1 molecules can be visualized by using a fluorescent label. The data that we obtain using this technique are consistent with myosin moving along an actin filament with single mechanical steps of approximately 5.3 nanometres; groups of two to five rapid steps in succession often produce displacements of 11 to 30 nanometres. This multiple stepping is produced by a single myosin head during just one biochemical cycle of ATP hydrolysis.

Movements of truncated kinesin fragments with a short or an artificial flexible neck.

To investigate the role of the neck domain of kinesin, we used optical trapping nanometry to perform high-resolution measurements of the movements and forces produced by recombinant kinesin fragments in which the neck domains were shortened or replaced by an artificial random coil. Truncated kinesin fragments (K351) that contain a motor domain consisting of approximately 340 aa and a short neck domain consisting of approximately 11 aa showed fast movement (800 nm/s) and 8-nm steps. Such behavior was similar to that of recombinant fragments containing the full-length neck domain (K411) and to that of native kinesin. Kinesin fragments lacking the short neck domain (K340), however, showed very slow movement (<50 nm/s), as previously reported. Joining an artificial 11-aa sequence that was expected to form a flexible random chain to the motor domain (K340-chain) produced normal fast ( approximately 700 nm/s) and stepwise movement. The results suggest that the neck domain does not act as a rigid lever arm to magnify the structural change at the catalytic domain as has been believed for myosin, but it does act as a flexible joint to guarantee the mobility of the motor domain.

Single molecule imaging of fluorophores and enzymatic reactions achieved by objective-type total internal reflection fluorescence microscopy.

Imaging of single fluorescent molecules has been achieved in a relatively simple manner using objective-type total internal reflection fluorescence microscopy (TIRFM). Switching from epi-fluorescence microscopy to objective-type TIRFM was achieved by translation of a single mirror in the system. Clear images of single molecules of an orange fluorescent dye, Cy3, were obtained with a fluorescence-to-background ratio of 12, using a conventional high aperture objective (PlanApo, 100 x, NA 1.4) with ordinary coverslips and immersion oil. This method allowed visualization of single molecules under scanning probe microscopes. Taking advantage of the technique of single molecule imaging, individual ATP turnovers have been visualized with a fluorescent ATP analogue, Cy3-ATP, using a simple experimental strategy. Clear on/off signals were obtained that correspond to the association and dissociation of single Cy3-ATP/ADP molecules with a single myosin head molecule. This method will allow a variety of single-molecular assays of biomolecular functions to be performed using fluorescently labeled substrates, ligands, messengers, and biologically active molecules. Thus, the present technique provides a simple yet powerful and universal tool for researchers to probe the events of single molecules.

Single molecular assay of individual ATP turnover by a myosin-GFP fusion protein expressed in vitro.

Fusion proteins of a truncated mutant of myosin subfragment-1 (S1dC) and green fluorescent protein (GFP) were expressed in vitro by T7 RNA polymerase and rabbit reticulocyte lysate. Single S1dC-GFP fusion proteins were clearly seen and their individual ATP turnovers were directly monitored using low background total internal reflection fluorescence microscopy (LBTIRFM), recently developed by our laboratory. LBTIRFM using GFP as a fluorescent tag allowed us to assay functions of single protein molecules expressed in vitro. Thus, the results suggested that this method may be particularly useful to analyze functions of proteins that cannot be produced in an active form and/or in large quantities in conventional heterologous expression systems.

Myosin subfragment-1 is fully equipped with factors essential for motor function.

The sliding velocity of actin filaments propelled by chicken skeletal myosin subfragment-1 (S1) was measured when the tail end of S1 was specifically bound to the glass surface. To achieve the specific binding, a regulatory light chain was replaced by a recombinant fusion protein of biotin-dependent transcarboxylase (BDTC) and chicken gizzard smooth muscle regulatory light chain (cgmRLC). The BDTC-cgmRLC of S1 was then attached to the glass surface using a biotin-avidin system. The velocity of actin filaments caused by S1 bound to the surface in this manner was 6.8 +/- 0.6 microm/sec at 29 degrees C, which was 3.5-fold greater than that (1.9 +/- 0.3 microm/sec) when bound directly to the surface as in previous studies, but similar to that caused by native chicken skeletal myosin (6.5 +/- 0.6 microm/sec). The actin-activated Mg-ATPase activity was similar to that of S1 before the RLC of S1 was exchanged for BDTC-cgmRLC. The results indicate that S1 can produce a normal fast movement of actin filaments as well as hydrolyse ATP and generate force.

Dual-colour microscopy of single fluorophores bound to myosin interacting with fluorescently labelled actin using anti-Stokes fluorescence.

We have refined prismless total internal reflection fluorescence microscopy with extremely low background to visualize single fluorophores attached to protein molecules interacting with a filamentous biopolymer labelled with different colour fluorophores. By using Stokes and anti-Stokes fluorescence, two different colour fluorescences from two different colour fluorophores excited with a single wavelength laser can be observed simultaneously. This microscopy was applied to visualize motor proteins, actin and myosin molecules. Single myosin molecules labelled with a tetramethylrhodamine-5-iodoacetamide interacting with a BODIPY FL-labelled actin filament, a filamentous polymer of actin molecules, were observed clearly and simultaneously in aqueous solution. Individual hydrolysis reactions of Cy3-labelled ATP by single myosin molecules and sliding of a BODIPY FL-labelled actin filament along the myosin molecules could also be observed simultaneously. Thus, this technique is useful for observing single molecular processes of proteins interacting with a biological macromolecule such as an actin filament and a DNA.

Two-dimensional crystals of the Kdp-ATPase of Escherichia coli.

A variant form of the Kdp-ATPase of Escherichia coli was overproduced to a level approaching 37% of the protein in the inner membrane of this organism. Membranes from overproducing cells were prepared with an inside-out orientation. Incubation of the membranes on ice for 1-2 weeks in the presence of sodium vanadate resulted in the formation of two-dimensional crystals of the Kdp-ATPase. The calculated projection map of the p1 crystal form showed three prominent density peaks at a resolution of 22 A. This technique is a useful and simple method to obtain low-resolution structures of membrane proteins.