Single-Molecule Analysis of Dynein Processivity and Stepping Behavior

Abstract

Cytoplasmic dynein, the 1.2 MDa motor driving minus-end-directed motility, has been reported to move processively along microtubules, but its mechanism of motility remains poorly understood. Here, using S. cerevisiae to produce recombinant dynein with a chemically controlled dimerization switch, we show by structural and single-molecule analysis that processivity requires two dynein motor domains but not dynein's tail domain or any associated subunits. Dynein advances most frequently in 8 nm steps, although longer as well as side and backward steps are observed. Individual motor domains show a different stepping pattern, which is best explained by the two motor domains shuffling in an alternating manner between rear and forward positions. Our results suggest that cytoplasmic dynein moves processively through the coordination of its two motor domains, but its variable step size and direction suggest a considerable diffusional component to its step, which differs from Kinesin-1 and is more akin to myosin VI.

In vitro reconstitution of a highly processive recombinant human dynein complex

“…The DYNC1H1 gene was fused to a His‐ZZ‐LTLT tag (Reck‐Peterson et al , 2006) and inserted into pACEBac1 (Vijayachandran et al , 2013). Ligation‐independent infusion (Clontech) cloning was used to seamlessly insert a SNAPf tag (New England Biolabs) to generate pDyn1.…”

The Kinesin-8 Kip3 Switches Protofilaments in a Sideward Random Walk Asymmetrically Biased by Force

“…Sideward motion of other motors has been detected via rotations of filaments driven by multiple motors in gliding assays and by off-axis movement of motor-attached microspheres or quantum dots used as tracking probes. Probe and microtubule rotations imply torque generation for all cytoskeletal motors: myosin (11), dynein (9,(12)(13)(14)(15)(16)(17) and kinesin (kinesin-1 monomers (18) and dimers (10), kinesin-2 (19,20), kinesin-5 (21), kinesin-8 (16,22), and kinesin-14 (23)). Occasional directed sideward steps-as suggested for kinesin-8-may explain microtubule rotations of motor-ensemble gliding assays (22) or the spiralling motion of multimotor-coated microspheres around microtubules (20).…”

Molecular motors: not quite like clockwork

“…The conformational change in each motor domain is reversed after detachment (between steps 4 and 5) in response to further steps in the ATPase cycle, so that rebinding is likely to occur closer to the minus end. Note the 16nm shift of the tubulin-binding domains between steps 4 and 6, though the cargo is shifted only 8nm [54,55]. The centre of the head would appear to move an even shorter distance; 3.7nm has been measured for axonemal dynein [48].…”

“…Both sliding and the use of secondary "tethers" to aid processivity are understandable in a thermal model context. In contrast to dynein c, cytoplasmic dynein needs to be dimeric to move processively [54,55] and is far less processive than kinesin-1. It even fails to follow individual protofilaments faithfully [54,56].…”

“…In contrast to dynein c, cytoplasmic dynein needs to be dimeric to move processively [54,55] and is far less processive than kinesin-1. It even fails to follow individual protofilaments faithfully [54,56]. To achieve an adequate level of processivity in cells, it makes a secondary, indirect contact with the MT via the dynactin complex [57,58].…”