Walking the walk: how kinesin and dynein coordinate their steps

Gennerich, Arne; Vale, Ronald D.

doi:10.1016/j.ceb.2008.12.002

Cited by 274 publications

(253 citation statements)

References 60 publications

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“…The notion that ATP binding by the MT-bound head induces the attachment of its tethered partner to the MT (10, 31, 32), which is a key feature of the prevailing model (2,3,10,14,15,17) (Fig. 3B), is consistent with evidence from biochemical measurements for the release of the fluorescent ADP analog, mantADP, from the tethered head upon nucleotide binding its MT-bound partner (31,(33)(34)(35).…”

Section: Discussionsupporting

confidence: 68%

“…Starting from a 1-HB ATPwaiting state [1] (26-29), we propose that ATP binding by the bound head triggers partial NL docking, biasing the tethered (ADP-bound) partner head forward [2]. ATP hydrolysis then completes the docking of the NL against the bound head, enabling productive MT binding by the tethered head at the forward binding site [3]. This is the point in the cycle where processivity is gated.…”

Section: Discussionmentioning

confidence: 99%

“…The completion of an 8.2-nm step (18) entails the binding of this tethered head to the MT, ATP hydrolysis, and detachment of the trailing head, thereby returning the motor to the ATP-waiting state (2,3,(10)(11)(12)(13)(14)(15)(16)(17). Prevailing models of the kinesin mechanochemical cycle (2,3,10,14,15,17), which invoke NL docking upon ATP binding, explain the highly directional nature of kinesin motility and offer a compelling outline of the sequence of events following ATP binding. Nevertheless, these abstractions do not speak directly to the branching transitions that determine whether kinesin dissociates from the MT (off-pathway) or continues its processive reaction cycle (onpathway).…”

mentioning

confidence: 99%

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Kinesin processivity is gated by phosphate release

Milic

Andreasson

Hancock

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

122

296

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Kinesin-1 is a dimeric motor protein, central to intracellular transport, that steps hand-over-hand toward the microtubule (MT) plus-end, hydrolyzing one ATP molecule per step. Its remarkable processivity is critical for ferrying cargo within the cell: over 100 successive steps are taken, on average, before dissociation from the MT. Despite considerable work, it is not understood which features coordinate, or "gate," the mechanochemical cycles of the two motor heads. Here, we show that kinesin dissociation occurs subsequent to, or concomitant with, phosphate (P i ) release following ATP hydrolysis. In optical trapping experiments, we found that increasing the steady-state population of the posthydrolysis ADP·P i state (by adding free P i ) nearly doubled the kinesin run length, whereas reducing either the ATP binding rate or hydrolysis rate had no effect. The data suggest that, during processive movement, tethered-head binding occurs subsequent to hydrolysis, rather than immediately after ATP binding, as commonly suggested. The structural change driving motility, thought to be neck linker docking, is therefore completed only upon hydrolysis, and not ATP binding. Our results offer additional insights into gating mechanisms and suggest revisions to prevailing models of the kinesin reaction cycle.single molecule | mechanochemistry | optical tweezers | molecular motors S ince its discovery nearly 30 years ago (1), kinesin-1-the founding member of the kinesin protein superfamily-has emerged as an important model system for studying biological motors (2, 3). During "hand-over-hand" stepping, kinesin dimers alternate between a two-heads-bound (2-HB) state, with both heads attached to the microtubule (MT), and a one-head-bound (1-HB) state, where a single head, termed the tethered head, remains free of the MT (4, 5). The catalytic cycles of the two heads are maintained out of phase by a series of gating mechanisms, thereby enabling the dimer to complete, on average, over 100 steps before dissociating from the . A key structural element for this coordination is the neck linker (NL), a ∼14-aa segment that connects each catalytic head to a common stalk (9). In the 1-HB state, nucleotide binding is thought to induce a structural reconfiguration of the NL, immobilizing it against the MT-bound catalytic domain (2,3,(10)(11)(12)(13)(14)(15)(16)(17). This transition, called "NL docking," is believed to promote unidirectional motility by biasing the position of the tethered head toward the next MT binding site (2,3,(10)(11)(12)(13)(14)(15)(16)(17). The completion of an 8.2-nm step (18) entails the binding of this tethered head to the MT, ATP hydrolysis, and detachment of the trailing head, thereby returning the motor to the ATP-waiting state (2,3,(10)(11)(12)(13)(14)(15)(16)(17). Prevailing models of the kinesin mechanochemical cycle (2,3,10,14,15,17), which invoke NL docking upon ATP binding, explain the highly directional nature of kinesin motility and offer a compelling outline of the sequence of events following ATP binding....

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Section: Discussionsupporting

confidence: 68%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

See 1 more Smart Citation

Kinesin processivity is gated by phosphate release

Milic

Andreasson

Hancock

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

122

296

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“…Both dynein and kinesin motors take small steps (8-32 nm) relative to the distances between the intersecting microtubules that we observed (23). Thus, it seems unlikely that switching is mediated by an individual motor but, rather, by other excess motors present on the same cargo.…”

Section: Conclusion and Discussionmentioning

confidence: 80%

Correlative live-cell and superresolution microscopy reveals cargo transport dynamics at microtubule intersections

Bálint

Vilanova

Álvarez

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

198

230

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Intracellular transport plays an essential role in maintaining the organization of polarized cells. Motor proteins tether and move cargos along microtubules during long-range transport to deliver them to their proper location of function. To reach their destination, cargo-bound motors must overcome barriers to their forward motion such as intersection points between microtubules. The ability to visualize how motors navigate these barriers can give important information about the mechanisms that lead to efficient transport. Here, we first develop an all-optical correlative imaging method based on single-particle tracking and superresolution microscopy to map the transport trajectories of cargos to individual microtubules with high spatiotemporal resolution. We then use this method to study the behavior of lysosomes at microtubulemicrotubule intersections. Our results show that the intersection poses a significant hindrance that leads to long pauses in transport only when the separation distance of the intersecting microtubules is smaller than ∼100 nm. However, the obstructions are typically overcome by the motors with high fidelity by either switching to the intersecting microtubule or eventually passing through the intersection. Interestingly, there is a large tendency to maintain the polarity of motion (anterograde or retrograde) after the intersection, suggesting a high degree of regulation of motor activity to maintain transport in a given direction. These results give insights into the effect of the cytoskeletal geometry on cargo transport and have important implications for the mechanisms that cargo-bound motors use to maneuver through the obstructions set up by the complex cytoskeletal network.C ells rely on a two-way transport system to deliver important proteins and organelles to their location of function. Kinesin and dynein motors are responsible for long-range transport along microtubules (1). Although dynein walks toward the (−) end of the microtubule (retrograde), carrying cargo toward the cell nucleus, most kinesins walk toward the (+) end (anterograde), carrying cargo toward the cell periphery (1). Microtubules organize into a complex, 3D network inside cells, and the intersections between microtubule filaments or between microtubules and other cytoskeletal filaments (actin, intermediate filaments) likely have important consequences on the efficiency and accuracy of cargo transport (2). For example, microtubule-microtubule intersections can serve as switching points or barriers that disrupt continuous transport in a given direction.The effect of microtubule-microtubule intersections on the movement of individual motors and motor-decorated beads has been studied using in vitro reconstituted microtubules deposited on top of each other (3, 4). These studies showed that although single motors can have varied behavior (passing, dissociation, switching), at high motor densities dynein-decorated beads stop and tether at the intersection (3). On the basis of these results, it was suggested that microtubul...

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“…These motors work by coupling high and low affinity binding to microtubule or actin filaments, with force producing conformational changes driven by an ATP catalytic cycle (3)(4)(5). The coordination between these steps is critical for efficient linear movement.…”

mentioning

confidence: 99%

A Low Affinity Ground State Conformation for the Dynein Microtubule Binding Domain

McNaughton

Tikhonenko

Banavali

et al. 2010

Journal of Biological Chemistry

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Dynein interacts with microtubules through a dedicated binding domain that is dynamically controlled to achieve high or low affinity, depending on the state of nucleotide bound in a distant catalytic pocket. The active sites for microtubule binding and ATP hydrolysis communicate via conformational changes transduced through a ϳ10-nm length antiparallel coiled-coil stalk, which connects the binding domain to the roughly 300-kDa motor core. Recently, an x-ray structure of the murine cytoplasmic dynein microtubule binding domain (MTBD) in a weak affinity conformation was published, containing a covalently constrained ␤ ؉ registry for the coiled-coil stalk segment (Carter, A. P., Garbarino, J. E., Wilson-Kubalek, E. M., Shipley, W. E., Cho, C., Milligan, R. A., Vale, R. D., and Gibbons, I. R. (2008) Science 322, 1691-1695). We here present an NMR analysis of the isolated MTBD from Dictyostelium discoideum that demonstrates the coiled-coil ␤ ؉ registry corresponds to the low energy conformation for this functional region of dynein. Addition of sequence encoding roughly half of the coiled-coil stalk proximal to the binding tip results in a decreased affinity of the MTBD for microtubules. In contrast, addition of the complete coiled-coil sequence drives the MTBD to the conformationally unstable, high affinity binding state. These results suggest a thermodynamic coupling between conformational free energy differences in the ␣ and ␤ ؉ registries of the coiled-coil stalk that acts as a switch between high and low affinity conformations of the MTBD. A balancing of opposing conformations in the stalk and MTBD enables potentially modest long-range interactions arising from ATP binding in the motor core to induce a relaxation of the MTBD into the stable low affinity state.Dyneins comprise one of the three families of cytoskeletonbased molecular motors that generate force and translocate cargo in eukaryotic cells (2). These motors work by coupling high and low affinity binding to microtubule or actin filaments, with force producing conformational changes driven by an ATP catalytic cycle (3-5). The coordination between these steps is critical for efficient linear movement. Force production occurs after tight binding is achieved, in a manner that both moves cargo forward and facilitates motor repositioning to advance another step. Although ATP hydrolysis and product release provide the thermodynamic driving force for motility, binding to the microtubule or actin filaments also provides feedback to the catalytic pocket and influences the catalytic rate.An understanding of how substrate affinity is achieved and how it is coupled to nucleotide hydrolysis remains an important problem for all three families of motors (dynein, kinesin, and myosin). For dynein, these issues are particularly complex. In contrast to the close proximity of the substrate-binding domains and catalytic sites in kinesin or myosin-type motors (5), the ATP-sensitive interaction of dynein with microtubules occurs through contacts within a relatively small (ϳ125 ...

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Walking the walk: how kinesin and dynein coordinate their steps

Cited by 274 publications

References 60 publications

Kinesin processivity is gated by phosphate release

Kinesin processivity is gated by phosphate release

Correlative live-cell and superresolution microscopy reveals cargo transport dynamics at microtubule intersections

A Low Affinity Ground State Conformation for the Dynein Microtubule Binding Domain

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