Myosin VI is a reverse direction myosin motor that, as a dimer, moves processively on actin with an average center-of-mass movement of ϳ30 nm for each step. We labeled myosin VI with a single fluorophore on either its motor domain or on the distal of two calmodulins (CaMs) located on its putative lever arm. Using a technique called FIONA (fluorescence imaging with one nanometer accuracy), step size was observed with a standard deviation of <1.5 nm, with 0.5-s temporal resolution, and observation times of minutes. Irrespective of probe position, the average step size of a labeled head was ϳ60 nm, strongly supporting a hand-over-hand model of motility and ruling out models in which the unique myosin VI insert comes apart. However, the CaM probe displayed large spatial fluctuations (presence of ATP but not ADP or no nucleotide) around the mean position,whereas the motor domain probe did not. This supports a model of myosin VI motility in which the lever arm is either mechanically uncoupled from the motor domain or is undergoing reversible isomerization for part of its motile cycle on actin.The myosin superfamily is composed of 18 classes of molecular motor proteins, the vast majority of which traffic toward the barbed (ϩ) end of actin filaments (1). Class VI myosins were the first of the superfamily identified to traffic toward the pointed (Ϫ) end of the actin filament (2). They were first identified in Drosophila melanogaster (3) and are expressed from Caenorhabditis elegans to human (4 -6). In addition to its unusual directionality, myosin VI has a number of additional unusual features. Single molecules of two-headed myosin VI, like myosin V, are capable of taking multiple steps (processive movement) on an actin filament without detachment (7). However, while myosin V has been demonstrated to move along actin filaments in 36-nm steps using its long lever arm (containing six calmodulins (CaMs) 1 ) via a hand-over-hand mechanism (8 -12), myosin VI does not appear to use a simple lever arm mechanism for its motility (7, 13). Myosin VI has been shown recently to have only two CaMs bound to each head (14), which should result in a short effective lever arm and small step size. Surprisingly, myosin VI has a step size that is highly variable (7) but on average nearly as large as that of myosin V, which has a lever arm that is three times longer. The variability of the step size has led to the postulate that myosin VI contains a long elastic element that allows it to undergo biased diffusion on an actin filament. The elastic element could be attached to the short lever arm, and/or the lever arm itself could become loosely attached to the motor at some point in the motile ATPase cycle. It was initially postulated that the unique insert of 39 amino acids in myosin VI that precedes the IQ motif (CaM-binding site) might come apart to form this flexible linker during the motile cycle on actin (7). This would result in the CaM being significantly displaced from the motor domain.EM images of two-headed myosin V bound to actin s...

Neurotransmission depends on movements of transmitter-laden synaptic vesicles, but accurate, nanometer-scale monitoring of vesicle dynamics in presynaptic terminals has remained elusive. Here, we report three-dimensional, real-time tracking of quantum dot-loaded single synaptic vesicles with an accuracy of 20 to 30 nanometers, less than a vesicle diameter. Determination of the time, position, and mode of fusion, aided by trypan blue quenching of Qdot fluorescence, revealed that vesicles starting close to their ultimate fusion sites tended to fuse earlier than those positioned farther away. The mode of fusion depended on the prior motion of vesicles, with long-dwelling vesicles preferring kiss-and-run rather than full-collapse fusion. Kiss-and-run fusion events were concentrated near the center of the synapse, whereas full-collapse fusion events were broadly spread.

Myosin VI is a reverse direction actin-based motor capable of taking large steps (30-36 nm) when dimerized. However, all dimeric myosin VI molecules so far examined have included non-native coiled-coil sequences, and reports on full-length myosin VI have failed to demonstrate the existence of dimers. Herein, we demonstrate that full-length myosin VI is capable of forming stable, processive dimers when monomers are clustered, which move up to 1-2 mum in approximately 30 nm, hand-over-hand steps. Furthermore, we present data consistent with the monomers being prevented from dimerizing unless they are held in close proximity and that dimerization is somewhat inhibited by the cargo binding tail. A model thus emerges that cargo binding likely clusters and initiates dimerization of full-length myosin VI molecules. Although this mechanism has not been previously described for members of the myosin superfamily, it is somewhat analogous to the proposed mechanism of dimerization for the kinesin Unc104.

Myosin X has features not found in other myosins. Its structure must underlie its unique ability to generate filopodia, which are essential for neuritogenesis, wound healing, cancer metastasis and some pathogenic infections. By determining high-resolution structures of key components of this motor, and characterizing the in vitro behaviour of the native dimer, we identify the features that explain the myosin X dimer behaviour. Single-molecule studies demonstrate that a native myosin X dimer moves on actin bundles with higher velocities and takes larger steps than on single actin filaments. The largest steps on actin bundles are larger than previously reported for artificially dimerized myosin X constructs or any other myosin. Our model and kinetic data explain why these large steps and high velocities can only occur on bundled filaments. Thus, myosin X functions as an antiparallel dimer in cells with a unique geometry optimized for movement on actin bundles.