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.DOI: http://dx.doi.org/10.7554/eLife.03205.001

The ability of cytoskeletal motors to move unidirectionally along filamentous tracks is central to their role in cargo transport, motility and cell division. While kinesin and myosin motor families have members that move in opposite directions 1 , 2 , all dyneins studied to date exclusively move towards the microtubule (MT) minus-end 3 . In order to understand the mechanism of dynein’s directionality, we sought to engineer a plus-end-directed dynein guided by cryo-electron microscopy and molecular dynamics simulations. As shown by single-molecule assays, elongation or shortening of the coiled-coil stalk that connects the motor to the MT controls helical directionality of S. cerevisiae dynein around MTs. By changing the length and angle of the stalk, we successfully reversed the motility towards the MT plus-end. These modifications act by altering the direction dynein’s linker swings relative to the MT, not by reversing the asymmetric unbinding of the motor from MT. Because the length and angle of dynein’s stalk are fully conserved among species, our findings provide an explanation for why all dyneins move towards the MT minus-end.

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.

SUMMARY Kinesin-1 is a two-headed motor that takes processive 8-nm hand-over-hand steps and transports intracellular cargos towards the plus end of microtubules. Processive motility requires a gating mechanism to coordinate the mechanochemical cycles of the two heads. Kinesin gating involves the 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 from 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.

Motor proteins take part in the organization and division of eukaryotic cells by using their ability to move unidirectionally along the cytoskeletal tracks. While kinesin and myosin motor families have members that move towards either end of actin and microtubules, respectively, all dynein motors exclusively move towards the minus-end of microtubules. Previous studies reported that dynein asymmetrically responds to external forces, moving faster when pulled forward, while resisting backward movement under hindering forces. I hypothesized that this asymmetry enables dynein to harness energy from external force fluctuations for faster movement towards the minus-end. In my doctoral work, I have shown that dynein can harness energy from cytoskeletal fluctuations. Using optical trapping techniques, I have characterized how external forces affect the velocity of dynein motility in the presence and absence of ATP. The results demonstrated that dynein forms an asymmetric slip bond with the microtubule. Using an oscillatory optical trapping assay, I showed that dynein can rectify force fluctuations to move towards the microtubule minus-end in the absence of ATP. Dynein was capable of moving towards the minus-end, even when the net force is in the plus-end direction. In the presence of ATP, dynein was able to move faster, generate power from force fluctuations, and stand against higher resistive forces. I developed a mathematical model that connects the force-induced release rate of dynein monomers with the force-velocity relationship of dynein dimers to describe dynein's reaction to force. i This dissertation is dedicated to my mom, Meryem Ezber, who has sacrificed all she has for her four children. She is the most patient person I know, and her unconditional love is the best gift I have received in my life. She will always be the person that I will seek the support of.