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...

Summary The microtubule motors kinesin and dynein function collectively to drive vesicular transport. High resolution tracking of vesicle motility in the cell indicates that transport is often bidirectional, characterized by frequent directional changes. However, the mechanisms coordinating the collective activities of oppositely-oriented motors bound to the same cargo are not well understood. To examine motor coordination, we purified neuronal transport vesicles and analyzed their motility using automated particle tracking with nanometer resolution. The motility of purified vesicles reconstituted in vitro closely models the movement of Lysotracker-positive vesicles in primary neurons, where processive bidirectional motility is interrupted with frequent directional switches, diffusional movement and pauses. Quantitative analysis indicates that vesicles co-purify with a low number of stably-bound motors: 1–5 dynein and 1–4 kinesin motors. These observations compare well to predictions from a stochastic tug-of-war model, where transport is driven by the force-dependent kinetics of teams of opposing motors in the absence of external regulation. Together, these observations indicate that vesicles move robustly with a small complement of tightly-bound motors, and suggest an efficient regulatory scheme for bidirectional motility where small changes in the number of engaged motors manifest in large changes in the motility of cargo.

Cytoplasmic dynein is the primary molecular motor responsible for transport of vesicles, organelles, proteins and RNA cargoes from the periphery of the cell towards the nucleus along the microtubule cytoskeleton of eukaryotic cells. Dynactin, a large multi-subunit activator of dynein, docks cargo to the motor and may enhance dynein processivity. Here, we show that individual fluorescently labelled dynein-dynactin complexes exhibit bidirectional and processive motility towards both the plus and minus ends of microtubules. The dependence of this activity on substrate ATP concentration, nucleotide analogues and inhibitors suggests that bidirectional motility is an active energy-transduction property of dynein-dynactin motor mechano-chemistry. The unique motility characteristics observed may reflect the flexibility of the dynein structure that leads to an enhanced ability to navigate around obstacles in the cell.

SummaryIntracellular cargo transport requires microtubule-based motors, kinesin and cytoplasmic dynein, and the actin-based myosin motors to maneuver through the challenges presented by the filamentous meshwork that comprises the cytoskeleton. Recent in vitro single molecule biophysical studies have begun to explore this process by characterizing what occurs as these tiny molecular motors happen upon an intersection between two cytoskeletal filaments. These studies, in combination with in vivo work, define the mechanism by which molecular motors exchange cargo while traveling between filamentous tracks and deliver it to its destination when going from the cell center to the periphery and back again.

microtubule motility ͉ membrane trafficking ͉ molecular motors ͉ bidirectional motility ͉ Huntington's disease