Myosin-V is a linear molecular motor that hydrolyzes ATP to move processively toward the plus end of actin filaments. Motion of this motor under low forces has been studied recently in various single-molecule assays. In this paper we show that myosin-V reacts to high forces as a mechanical ratchet. High backward loads can induce rapid and processive backward steps along the actin filament. This motion is completely independent of ATP binding and hydrolysis. In contrast, forward forces cannot induce ATP-independent forward steps. We can explain this pronounced mechanical asymmetry by a model in which the strength of actin binding of a motor head is modulated by the lever arm conformation. Knowledge of the complete force-velocity dependence of molecular motors is important to understand their function in the cellular environment.backward movement ͉ molecular motor ͉ optical tweezers ͉ asymmetry ͉ kinesin C lass-V myosins are two-headed linear molecular motors involved in various intracellular transport processes that move processively and directionally toward the plus end of actin filaments (1-4). The energy required for forward motion is supplied by hydrolysis of ATP (1, 5). During each forward step both heads of a myosin-V motor undergo a coordinated chemomechanical cycle, which results in a hand-over-hand stepping mechanism (6, 7). After release of ADP in the trailing head, ATP can bind, and this head detaches from the filament. Now, the leading head can perform the power stroke of its lever arm (8). Subsequently, the now-forward head can rebind to the filament. The mechanism that prevents premature ADP release from the leading head in a two-head-bound myosin is believed to be based on intramolecular strain (9-11).Apart from myosin-V, forward motion has been studied extensively for many linear and rotary motors (12)(13)(14)(15)(16)(17)(18)(19). For most of these motors tight coupling of forward motion to ATP hydrolysis has been reported (6,20,21). In contrast, the modes of force-induced backward motion seem to be quite diverse in the different motor systems. Whereas for the F 1 -ATPase the hydrolysis cycle is completely reversible, and forced backward rotation can lead to ATP synthesis (22, 23), backward steps of the linear motor kinesin have been shown to be tightly coupled to ATP binding (24,25). In kinesin, backward forces lead to a decrease of the intrinsic forward bias of a step. In the present study, we investigate force-driven motion of myosin-V by using an optical trap with force feedback control in which we observe a forceinduced mode of backward motion distinct from kinesin and completely independent from the ATP cycle. ResultsIn a first set of experiments we tested motor velocities under various high loads in forward and backward direction at 1 M ATP. Forces in the direction of unloaded movement (forward) of myosin-V and forces opposing the unloaded movement (backward) of a motor attached to a trapped polystyrene bead were applied by moving a piezo-driven microscope stage parallel to a surface-anchor...

Myosin-V is a processive two-headed actin-based motor protein involved in many intracellular transport processes. A key question for understanding myosin-V function and the communication between its two heads is its behavior under load. Since in vivo myosin-V colocalizes with other much stronger motors like kinesins, its behavior under superstall forces is especially relevant. We used optical tweezers with a long-range force feedback to study myosin-V motion under controlled external forward and backward loads over its full run length. We find the mean step size remains constant at approximately 36 nm over a wide range of forces from 5 pN forward to 1.5 pN backward load. We also find two force-dependent transitions in the chemomechanical cycle. The slower ADP-release is rate limiting at low loads and depends only weakly on force. The faster rate depends more strongly on force. The stronger force dependence suggests this rate represents the diffusive search of the leading head for its binding site. In contrast to kinesin motors, myosin-V's run length is essentially independent of force between 5 pN of forward to 1.5 pN of backward load. At superstall forces of 5 pN, we observe continuous backward stepping of myosin-V, indicating that a force-driven reversal of the power stroke is possible.

Hereditary spastic paraplegia (HSP) is a neurodegenerative disease caused by motoneuron degeneration. It is linked to at least 30 loci, among them SPG10, which causes dominant forms and originates in point mutations in the neuronal Kinesin-1 gene (KIF5A). Here, we investigate the motility of KIF5A and four HSP mutants. All mutations are single amino-acid exchanges and located in kinesin's motor or neck domain. The mutation in the neck (A361V) did not change the gliding properties in vitro, the others either reduced microtubule affinity or gliding velocity or both. In laser-trapping assays, none of the mutants moved more than a few steps along microtubules. Motility assays with mixtures of homodimeric wild-type, homodimeric mutant and heterodimeric wild-type/mutant motors revealed that only one mutant (N256S) reduces the gliding velocity at ratios present in heterozygous patients, whereas the others (K253N, R280C) do not. Attached to quantum dots as artificial cargo, mixtures involving N256S mutants produced slower cargo populations lagging behind in transport, whereas mixtures with the other mutants led to populations of quantum dots that rarely bound to microtubules. These differences indicate that the dominant inheritance of SPG10 is caused by two different mechanisms that both reduce the gross cargo flux, leading to deficient supply of the synapse.

Kinesin-1 is a dimeric motor protein that moves stepwise along microtubules. A two-stranded alpha-helical coiled-coil formed by the neck domain links the two heads of the molecule, and forces the motor heads to alternate. By exchanging the particularly soft neck region of the conventional kinesin from the fungus Neurospora crassa with an artificial, highly stable coiled-coil we investigated how this domain affects motor kinetics and motility. Under unloaded standard conditions, both motor constructs developed the same gliding velocity. However, in a force-feedback laser trap the mutant showed increasing motility defects with increasing loads, and did not reach wild-type velocities and run lengths. The stall force dropped significantly from 4.1 to 3.0 pN. These results indicate the compliance of kinesin's neck is important to sustain motility under load, and reveal a so far unknown constrain on the imperfect coiled-coil heptad pattern of Kinesin-1. We conclude that coiled-coil structures, a motif encountered in various types of molecular motors, are not merely a clamp for linking two heavy chains to a functional unit but may have specifically evolved to allow motor progression in a viscous, inhomogeneous environment or when several motors attached to a transported vesicle are required to cooperate efficiently.

The protein family of kinesins contains processive motor proteins that move stepwise along microtubules. This mechanism requires the precise coupling of the catalytic steps in the two heads, and their precise mechanical coordination. Here we show that these functionalities can be uncoupled in chimera of processive and non-processive kinesins. A chimera with the motor domain of Kinesin-1 and the dimerization domain of a non-processive Kinesin-3 motor behaves qualitatively as conventional kinesin and moves processively in TIRF and bead motility assays, suggesting that spatial proximity of two Kinein-1 motor domains is sufficient for processive behavior. In the reverse chimera, the non-processive motor domains are unable to step along microtubules, despite the presence of the Kinesin-1 neck coiled coil. Still, ATP-binding to one head of these chimera induces ADP-release from the partner head, a characteristic feature of alternating site catalysis. These results show that processive movement of kinesin dimers requires elements in the motor head that respond to ADP-release and induce stepping, in addition to a proper spacing of the motor heads via the neck coiled coil.