Myosin V is a dimeric molecular motor that moves processively on actin, with the center of mass moving approximately 37 nanometers for each adenosine triphosphate hydrolyzed. We have labeled myosin V with a single fluorophore at different positions in the light-chain domain and measured the step size with a standard deviation of <1.5 nanometers, with 0.5-second temporal resolution, and observation times of minutes. The step size alternates between 37 + 2x nm and 37 - 2x, where x is the distance along the direction of motion between the dye and the midpoint between the two heads. These results strongly support a hand-over-hand model of motility, not an inchworm model.

Dynein and kinesin motor proteins transport cellular cargos toward opposite ends of microtubule tracks. In neurons, microtubules are abundantly decorated with microtubule-associated proteins (MAPs) such as tau. Motor proteins thus encounter MAPs frequently along their path. To determine the effects of tau on dynein and kinesin motility, we conducted single molecule studies of motor proteins moving along tau-decorated microtubules. Dynein tended to reverse direction whereas kinesin tended to detach at patches of bound tau. Kinesin was inhibited at ~ 10-fold lower tau concentration than dynein and the microtubule-binding domain of tau was sufficient to inhibit motor activity. The differential modulation of dynein and kinesin motility suggests that MAPs can spatially regulate the balance of microtubule-dependent axonal transport.Active transport of cytoplasmic material along microtubules is critical for cell organization and function, and defects in this process are associated with dysfunction and disease (1). Much of the active transport in cells depends on the molecular motor proteins cytoplasmic dynein and kinesin-1, which transport cargo toward the minus-end (toward the cell center) and plusend of microtubules (toward the cell periphery), respectively. Dynein and kinesin have very different structures and translocation mechanisms (2). Kinesin has a compact motor domain and walks unidirectionally along single protofilaments with 8-nm steps (2). In contrast, dynein has a larger, more complex motor domain and is capable of variable step sizes, lateral steps across the microtubule surface, and processive runs toward both the minus-and plus-end of the microtubule (3-5). Cytoplasmic dynein function in vivo also requires an accessory complex, dynactin. This large, multiprotein complex is thought to facilitate dynein processivity (6) and may also regulate dynein activity (5). Within the cell, the balance between oppositely directed transport determines the steady-state distribution of organelles and biomolecules.In the crowded cell environment, dynein and kinesin compete with non-motile microtubuleassociated proteins (MAPs) for binding to the microtubule surface. MAPs bound to microtubules might also block the path of motor proteins. Thus, MAPs can provide spatiotemporal regulation of motor proteins in vivo. Tau, a neuronal MAP, inhibits kinesin activity in vivo and in vitro (7-10); however, its effect on dynein activity is not well understood. Our aim was to directly observe individual encounters between single dynein or kinesin motors and tau on microtubule tracks to determine how structurally distinct motors respond to obstacles in their path. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptTau is expressed in neurons as multiple splice forms in a developmentally regulated manner (11). These isoforms differ in the number of microtubule binding repeats and the length of the projection domain (Fig. 1A). Here we focused on the shortest and longest tau isoforms, tau23 and tau40, to comp...

SUMMARY1. Orthophosphate (Pi, 041-2-0 mM) was photogenerated within the filament lattice of isometrically contracting glycerinated fibres of rabbit psoas muscle at 10 and 200C. The Pi was produced by laser flash photolysis of the photolabile compound 1-(2-nitrophenyl)ethylphosphate (caged Pi). Caged Pi caused a depression of tension that was much smaller than that caused by Pi.2. Photolysis of caged P1 produced a decline in isometric force composed of four phases: phase I, a lag phase (e.g. 1-4 ms at 1000) during which force did not change; phase II, an exponential decline by as much as 20 % of the pre-pulse force; phase III, a partial force recovery (0-3 % of the pre-pulse force); and phase IV, a further slow (0 5-3 s) decline to the steady value. Phases I, III and IV were largely independent of [Pi] and are likely to be indirect effects caused by the caged Pi photolysis.3. Both the rate and amplitude of phase II depended markedly on [Pi]. The amplitude of phase II was similar to the reduction of steady-state force by Pi.

The structural change that generates force and motion in actomyosin motility has been proposed to be tilting of the myosin light chain domain, which serves as a lever arm. Several experimental approaches have provided support for the lever arm hypothesis; however, the extent and timing of tilting motions are not well defined in the motor protein complex of functioning actomyosin. Here we report three-dimensional measurements of the structural dynamics of the light chain domain of brain myosin V using a single-molecule fluorescence polarization technique that determines the orientation of individual protein domains with 20-40-ms time resolution. Single fluorescent calmodulin light chains tilted back and forth between two well-defined angles as the myosin molecule processively translocated along actin. The results provide evidence for lever arm rotation of the calmodulin-binding domain in myosin V, and support a 'hand-over-hand' mechanism for the translocation of double-headed myosin V molecules along actin filaments. The technique is applicable to the study of real-time structural changes in other biological systems.