In order to explain the molecular mechanism of muscle contraction, it is crucial to know the distribution of the sarcomere compliance of active muscle. Here, we directly measure the stiffness of single actin filaments with and without tropomyosin, using a recently developed technique for nanomanipulation of single actin filaments with microneedles.The results show that the stiffness for 1-,Im-long actin filaments with and without tropomyosin is 65.3 ± 6.3 and 43.7 ± 4.6 pN/nm, respectively. When the distribution of crossbridge forces along the actin ifiament is taken into account, the elongation of a l-,um-long thin rilament during development of isometric contraction is calculated to be -0.23%. The time constant of force in response to a sudden length change is <0.2 ms, indicating that the viscoelasticity is negligible in the millisecond time range. These results suggest that '50% of the sarcomere compliance of active muscle is due to extensibility of the thin riaments.In striated muscles, contraction is due to cyclic interactions of myosin crossbridges with thin filaments in the sarcomere (1, 2). The mechanical properties ofthe interaction have often been studied by analyzing tension transients of contracting muscle fibers. In 1971, Huxley and Simmons (3) deduced an ingenious mechanical model to fit the kinetics of these transients. In this model, the force produced by the crossbridges is transmitted to thick and thin filaments by a series elastic element. The model assumed for simplification that the mechanical compliance (reciprocal of the stiffness) in the sarcomere is mostly localized in the crossbridges and that the thin and thick filaments are rigid to transmit the force produced by the crossbridges to the next sarcomere along the muscle fiber axis. This assumption has allowed us to lump the mechanical behavior of the crossbridges distributed along the thin filaments. Indeed, many subsequent experiments have been interpreted on the basis of this simplifying assumption.The assumption that compliance resides primarily within the crossbridges has been tested by many mechanical and structural studies. Ford et al. (4) measured the instantaneous stiffness of isometrically contracting muscle fibers at various sarcomere lengths and concluded that .80% of the sarcomere compliance is due to the crossbridges. A similar result was obtained from the dependence of the rigor stiffness on the sarcomere length (6). Huxley and coworkers (7,8) showed that the strong 5.9-nm x-ray diffraction peak from actin filaments did not change spacing significantly during contraction. These results suggested that the thin filaments are rigid, but the experiments are not conclusive (see Discussion). On the other hand, measurements of the elastic modulus for bending of actin filaments have suggested that the actin filament is quite flexible and, hence, might contribute significantly to muscle compliance (see ref. 9 for review).Recently, we have developed a nanomanipulation technique for single actin filaments that involves attach...

We have developed a technique that allows mechanical and ligand-binding events in a single myosin molecule to be monitored simultaneously. We describe how steps in the ATPase reaction are temporally related to mechanical events at the single molecule level. The results show that the force generation does not always coincide with the release of bound nucleotide, presumably ADP. Instead the myosin head produces force several hundreds of milliseconds after bound nucleotide is released. This finding does not support the widely accepted view that force generation is directly coupled to the release of bound ligands. It suggests that myosin has a hysteresis or memory state, which stores chemical energy from ATP hydrolysis.

We have analyzed the mechanics of individual kinesin molecules by optical trapping nanometry. A kinesin molecule was adsorbed onto a latex bead, which was captured by an optical trap and brought into contact with an axoneme that was bound to a glass surface. The displacement of kinesin during force generation was determined by measuring the position of the beads with nanometer accuracy. As the displacement of kinesin was attenuated because of the compliance of the kinesin-to-bead and kinesin-to-microtubule linkages, the compliance was monitored during force generation and was used to correct the displacement of kinesin. Thus the velocity and the unitary steps could be obtained accurately over a wide force range. The force-velocity curves were linear from 0 to a maximum force at 10 microM and 1 mM ATP, and the maximum force was approximately 7 pN, which is larger by approximately 30% than values previously reported. Kinesin exhibited forward and occasionally backward stepwise displacements with a size of approximately 8 nm. The histograms of step dwell time show a monotonic decrease with time. Model calculations indicate that each kinesin head steps by 16-nm, whereas kinesin molecule steps by 8-nm.

Intracellular transport is thought to be achieved by teams of motor proteins bound to a cargo. However, the coordination within a team remains poorly understood as a result of the experimental difficulty in controlling the number and composition of motors. Here, we developed an experimental system that links together defined numbers of motors with defined spacing on a DNA scaffold. By using this system, we linked multiple molecules of two different types of kinesin motors, processive kinesin-1 or nonprocessive Ncd (kinesin-14), in vitro. Both types of kinesins markedly increased their processivities with motor number. Remarkably, despite the poor processivity of individual Ncd motors, the coupling of two Ncd motors enables processive movement for more than 1 μm along microtubules (MTs). This improvement was further enhanced with decreasing spacing between motors. Force measurements revealed that the force generated by groups of Ncd is additive when two to four Ncd motors work together, which is much larger than that generated by single motors. By contrast, the force of multiple kinesin-1s depends only weakly on motor number. Numerical simulations and single-molecule unbinding measurements suggest that this additive nature of the force exerted by Ncd relies on fast MT binding kinetics and the large drag force of individual Ncd motors. These features would enable small groups of Ncd motors to crosslink MTs while rapidly modulating their force by forming clusters. Thus, our experimental system may provide a platform to study the collective behavior of motor proteins from the bottom up.

Motor proteins and microtubule-associated proteins (MAPs) play important roles in cellular transport, regulation of shape and polarity of cells. While motor proteins generate motility, MAPs are thought to stabilize the microtubule tracks. However, the proteins also interfere with each other, such that MAPs are able to inhibit transport of vesicles and organelles in cells. In order to investigate the mechanism of MAP±motor interference in molecular detail, we have studied single kinesin molecules by total internal re¯ection¯uorescence microscopy in the presence of different neuronal MAPs (tau, MAP2c). The parameters observed included run-length (a measure of processivity), velocity and frequency of attachment. The main effect of MAPs was to reduce the attachment frequency of motors. This effect was dependent on the concentration, the af®nity to microtubules and the domain composition of MAPs. In contrast, once attached, the motors did not show a change in speed, nor in their run-length. The results suggest that MAPs can regulate motor activity on the level of initial attachment, but not during motion.