THE MECHANICS OF DYNEIN STEPPING AND DIRECTIONALITY
- Author(s): Can, Sinan
- Advisor(s): Yildiz, Ahmet
- et al.
The ability of cytoskeletal motors to move unidirectionally along linear tracks is central to their cellular roles. While kinesin and myosin motor families have members that move in opposite directions, all dyneins studied to date exclusively move towards the microtubule minus-end. The source of dynein’s directionality remains as the central unresolved question about the mechanism of its motility. In my doctoral work, I focused on understanding the underlying mechanism of dynein’s directional motility along the microtubules in three dimensions. I used a protein engineering approach, guided by all-atom molecular dynamics simulations and high-resolution cryoEM imaging, along with the tools of single-molecule biophysics to dissect the elements of dynein motility. We successfully engineered a plus-end-directed dynein for the first time and revealed the mechanism of its directionality. This work has three major outcomes. First, by altering the length of the coiled-coil stalk that connects the dynein motor domain to the microtubule, we controlled the handedness of the helical motility of dynein around the circumference of the microtubule. This experiment showed that the stalk length of native dynein is critical for restricting sideways movement and directing dynein motility along the microtubule axis. Second, we altered the angle and length of dynein’s stalk. Remarkably, these modifications reversed the direction by which the linker swings relative to the microtubule and directed the motility towards the plus-end. Finally, similar to native dynein, the plus-end directed mutant maintains its preference to release from the microtubule when pulled towards the minus-end by an optical trap. Our results provide direct evidence for the linker swing model in which the direction the linker swings determines the direction of dynein motility. Our results also rule out the asymmetric release model that the directionality is proposed to be determined by the direction dynein favors for release from the microtubule under tension. Two critical features of dynein’s stalk, the length of its antiparallel coiled-coils and two proline residues located at its base, control the directionality by directing the linker swing towards the MT minus-end. Because both features of the stalk are fully conserved in cytoplasmic and ciliary dyneins across species, this mechanism explains the minus-end directionality of all dyneins.