Directionality of dynein is controlled by the angle and length of its stalk.

The ability of cytoskeletal motors to move unidirectionally along filamentous tracks is central to their role in cargo transport, motility and cell division. Kinesin and myosin motor families have a subclass that moves towards the opposite end of the microtubule or actin filament with respect to the rest of the motor family1,2, whereas all dynein motors that have been studied so far exclusively move towards the minus end of the microtubule3. Guided by cryo-electron microscopy and molecular dynamics simulations, we sought to understand the mechanism that underpins the directionality of dynein by engineering a Saccharomyces cerevisiae dynein that is directed towards the plus end of the microtubule. Here, using single-molecule assays, we show that elongation or shortening of the coiled-coil stalk that connects the motor to the microtubule controls the helical directionality of dynein around microtubules. By changing the length and angle of the stalk, we successfully reversed the motility towards the plus end of the microtubule. These modifications act by altering the direction in which the dynein linker swings relative to the microtubule, rather than by reversing the asymmetric unbinding of the motor from the microtubule. Because the length and angle of the dynein stalk are fully conserved among species, our findings provide an explanation for why all dyneins move towards the minus end of the microtubule.

Dynein Harnesses Active Fluctuations of Microtubules for Faster Movement.

The cytoskeleton forms a dynamic network that generates fluctuations larger than thermal agitation of the cytoplasm1. Here, we tested whether dynein, a minus-end-directed microtubule (MT) motor2, can harness energy from these fluctuations using optical trapping in vitro. We show that dynein forms an asymmetric slip bond with MTs, where its detachment rate increases more slowly under hindering forces than assisting forces. This asymmetry enables dynein to generate unidirectional motility towards the minus-end from force fluctuations. Consistent with our model, oscillatory forces exerted by the trap drive dynein stepping without net force and ATP. Dynein is capable of ratcheting towards the minus-end even when the net force is in the plus-end direction. With ATP, force oscillations increase the velocity and stall force of dynein as it transports cargos and glides MTs. Therefore, dynein is a mechanical ratchet that rectifies cytoskeletal fluctuations to move faster and resists higher forces along MTs.

Kinesin and dynein use distinct mechanisms to bypass obstacles.

Kinesin-1 and cytoplasmic dynein are microtubule (MT) motors that transport intracellular cargoes. It remains unclear how these motors move along MTs densely coated with obstacles of various sizes in the cytoplasm. Here, we tested the ability of single and multiple motors to bypass synthetic obstacles on MTs in vitro. Contrary to previous reports, we found that single mammalian dynein is highly capable of bypassing obstacles. Single human kinesin-1 motors fail to avoid obstacles, consistent with their inability to take sideways steps on to neighboring MT protofilaments. Kinesins overcome this limitation when working in teams, bypassing obstacles as effectively as multiple dyneins. Cargos driven by multiple kinesins or dyneins are also capable of rotating around the MT to bypass large obstacles. These results suggest that multiplicity of motors is required not only for transporting cargos over long distances and generating higher forces, but also for maneuvering cargos on obstacle-coated MT surfaces.

Measurement of Force-Dependent Release Rates of Cytoskeletal Motors.

Optical tweezers permit measuring motor-filament rupture forces with piconewton sensitivity. For deeper structural and mechanistic understanding of motors, different structural constraints can be induced by pulling motor proteins at various positions and manipulating the direction of the exerted force. Here, we present an optical-trapping approach to investigate the effect of the magnitude and direction of tension applied to the linker element of cytoskeletal motors on motor-filament interactions. Using this approach, force-dependent microtubule release rates of monomeric kinesins can be directly measured by pulling on kinesin's "neck linker" with a constant force.

Kinesin's front head is gated by the backward orientation of its neck linker.

Kinesin-1 is a two-headed motor that takes processive 8-nm hand-over-hand steps and transports intracellular cargos toward the plus-end of microtubules. Processive motility requires a gating mechanism to coordinate the mechanochemical cycles of the two heads. Kinesin gating involves neck linker (NL), a short peptide that interconnects the heads, but it remains unclear whether gating is facilitated by the NL orientation or tension. Using optical trapping, we measured the force-dependent microtubule release rate of kinesin monomers under different nucleotide conditions and pulling geometries. We find that pulling NL in the backward direction inhibits nucleotide binding and subsequent release from the microtubule. This inhibition is independent of the magnitude of tension (2-8 pN) exerted on NL. Our results provide evidence that the front head of a kinesin dimer is gated by the backward orientation of its NL until the rear head releases from the microtubule.

Bidirectional helical motility of cytoplasmic dynein around microtubules.

Cytoplasmic dynein is a molecular motor responsible for minus-end-directed cargo transport along microtubules (MTs). Dynein motility has previously been studied on surface-immobilized MTs in vitro, which constrains the motors to move in two dimensions. In this study, we explored dynein motility in three dimensions using an MT bridge assay. We found that dynein moves in a helical trajectory around the MT, demonstrating that it generates torque during cargo transport. Unlike other cytoskeletal motors that produce torque in a specific direction, dynein generates torque in either direction, resulting in bidirectional helical motility. Dynein has a net preference to move along a right-handed helical path, suggesting that the heads tend to bind to the closest tubulin binding site in the forward direction when taking sideways steps. This bidirectional helical motility may allow dynein to avoid roadblocks in dense cytoplasmic environments during cargo transport.