Skip to main content
eScholarship
Open Access Publications from the University of California

Scholarly Works (4 results)

  • Thesis
  • Peer Reviewed
UC Berkeley Electronic Theses and Dissertations (2018)

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.

    Cover page: THE MECHANICS OF DYNEIN STEPPING AND DIRECTIONALITY
    • Article
    • Peer Reviewed
    UC Berkeley Previously Published Works (2014)
    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.
      Cover page: Bidirectional helical motility of cytoplasmic dynein around microtubules
      • Article
      • Peer Reviewed
      UC Berkeley Previously Published Works (2015)
      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.
        Cover page: Kinesin’s Front Head Is Gated by the Backward Orientation of Its Neck Linker
        • Article
        • Peer Reviewed
        UC Berkeley Previously Published Works (2019)
        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.
          Cover page: Directionality of dynein is controlled by the angle and length of its stalk
          Top