Tension on the linker gates the ATP-dependent release of dynein from microtubules

Abstract

Cytoplasmic dynein is a dimeric motor that transports intracellular cargoes towards the minus-end of microtubules (MTs). In contrast to other processive motors, stepping of the dynein motor domains (heads) is not precisely coordinated. Therefore, the mechanism of dynein processivity remains unclear. Here, by engineering the mechanical and catalytic properties of the motor, we show that dynein processivity minimally requires a single active head and a second inert MT binding domain. Processivity arises from a high ratio of MT-bound to unbound time, and not from interhead communication. Additionally, nucleotide-dependent microtubule release is gated by tension on the linker domain. Intramolecular tension sensing is observed in dynein’s stepping motion at high interhead separations. We developed a quantitative model for the stepping characteristics of dynein and its response to chemical and mechanical perturbation.

Cytoplasmic dynein regulates its attachment to microtubules via nucleotide state-switched mechanosensing at multiple AAA domains

“…Our results are consistent with our own (11) and others' reports (12,13,20) that less force is required to break the dynein-MT bond when pulling the motor forward than backward. Interestingly, in the apo state, whether tension is applied via the linker vs. the C terminus has little effect on unbinding forces, implying that linker conformation and/or tension transmitted through the dynein ring are not responsible for the anisotropy.…”

“…Under backward load, catch bonding (diminished unbinding rate with applied force) has been reported (31-33), but we instead found that slip bonding occurs up to ∼2 pN, above which the unbinding rate is insensitive to force (Fig. 1 F and G), characteristic of ideal bonding (these findings agree with those from constant-force assays (20) that directly measure unbinding rates). Because the behavior seen here under rearward force exhibits features of both slip bonding (at low force) and ideal bonding (at higher force), we term it slip-ideal bonding.…”

“…1B) of a tail-truncated, single-headed "wildtype" (WT) dynein (Dyn1 331kDa or VY137 dynein; see SI Appendix, SI Materials and Methods) in the nucleotide-free (apo) state ( Fig. 1 C-E), similar to Cleary et al (20). As expected, forces required to unbind dynein monomers from MTs were significantly larger when pulling backward ( Fig.…”

“…Interestingly, in the apo state, whether tension is applied via the linker vs. the C terminus has little effect on unbinding forces, implying that linker conformation and/or tension transmitted through the dynein ring are not responsible for the anisotropy. Cleary et al observed similar results for a stalk/MTBD construct lacking the entire dynein ring (20). The molecular mechanism for the anisotropy remains to be elucidated and could include tension-induced reconfiguration of the coiled-coil stalk [which allosterically regulates MT affinity (5,41,42)], direct force-induced changes in the MTBD, geometrical reorientations of the binding interface, or even strain-induced effects on the MT lattice (43).…”

“…Less force was required to induce forward than backward movement (11). More recently, Cleary et al showed that the lifetimes of single monomeric dynein-MT bonds depend on the direction of applied force (20). These results imply that mechanical tension contributes to control of dynein motion along the MT.…”