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.

Holographic optical tweezers (HOT) are a versatile technology, with which complex arrays and movements of optical traps can be realized to manipulate multiple microparticles in parallel and to measure the forces affecting them in the piconewton range. We report on the combination of HOT with a fluorescence microscope and a stop-flow, multi-channel microfluidic device. The integration of a high-speed camera into the setup allows for the calibration of all the traps simultaneously both using Boltzmann statistics or the power spectrum density of the particle diffusion within the optical traps. This setup permits complete spatial, chemical and visual control of the microenvironment applicable to probing chemo-mechanical properties of cellular or subcellular structures. As an example we constructed a biomimetic, quasi-two-dimensional actin network on an array of trapped polystyrene microspheres inside the microfluidic chamber. During crosslinking of the actin filaments by Mg(2+) ions, we observe the build up of mechanical tension throughout the actin network. Thus, we demonstrate how our integrated HOT-microfluidics platform can be used as a reconfigurable force sensor array with piconewton resolution to investigate chemo-mechanical processes.

Bundles of filamentous actin are dominant cytoskeletal structures, which play a crucial role in various cellular processes. As yet quantifying the fundamental interaction between two individual actin filaments forming the smallest possible bundle has not been realized. Applying holographic optical tweezers integrated with a microfluidic platform, we were able to measure the forces between two actin filaments during bundle formation. Quantitative analysis yields forces up to 0.2 pN depending on the concentration of bundling agents.