Overlapping hand-over-hand mechanism of single molecular motility of cytoplasmic dynein

Abstract

Structural differences between dynein and kinesin suggest a unique molecular mechanism of dynein motility. Measuring the mechanical properties of a single molecule of dynein is crucial for revealing the mechanisms underlying its movement. We measured the step size and force produced by single molecules of active cytoplasmic dynein by using an optical trap and fluorescence imaging with a high temporal resolution. The velocity of dynein movement, 800 nm͞s, is consistent with that reported in cells. The maximum force of 7-8 pN was independent of the ATP concentration and similar to that of kinesin. Dynein exhibited forward and occasional backwards steps of Ϸ8 nm, independent of load. It is suggested that the large dynein heads take 16-nm steps by using an overlapping hand-over-hand mechanism.microtubules ͉ motor protein ͉ optical tweezers ͉ step size ͉ nanotechnology D ynein is a molecular motor that moves along microtubules to the direction of the minus end. Cytoplasmic dynein transports cellular organelles toward the minus end of microtubules, whereas most kinesin molecules transport organelles toward the plus end (1-3). Hirakawa et al. (4) have reported that purified axonemal dynein produced maximum forces of 5 pN, and they also showed stepwise displacements of 8 nm. In contrast, another study (5) has demonstrated that single molecules of purified cytoplasmic dynein move with short steps (8 nm) at high loads (Ͼ0.8 pN) and long steps (16-32 nm) at low forces (Ͻ0.4 pN), and from these findings a molecular gear mechanism was proposed. The values for maximum force and the size of the steps differ between those two reports. Moreover, the very low velocity (Ͻ50 nm͞s) of movement of the purified cytoplasmic dynein (5) is in direct contrast to the high velocity (Ϸ1 m͞s) of dynein movement in a cell and in an in vitro motility assay (6-8). These discrepancies possibly result from inactive dynein molecules being included with the purified native dynein in the analysis.In this study a method of coating the beads with dynein was developed to keep dynein active and allow the force and step size produced by single molecules of dynein to be measured. The step size of active cytoplasmic dynein was 8 nm and was independent of both force and ATP concentration. The stall force was 7-8 pN. These values are very similar to those recorded for kinesin (9); however, the stepping manner was different from that of kinesin. ResultsActive Dynein Bound to Protein A-Coated Beads. The method used to purify cytoplasmic dynein without its accessory protein, dynactin, has been described (Fig. 1a, lane D) (10, 11). Dynein was further purified by allowing it to bind to the microtubules (Fig. 1a, lane AMP-PNP) (12). Dynein was then released from microtubules in the presence of 0.1-10 mM ATP (Fig. 1a, lane ATP).The method used to bind dynein to the coverslips for in vitro motility assay and beads for optical trap assay was modified from previously reported methods (5, 8). Beads coated with protein A were suitable for motility and force generation....

In vivo analysis of MT‐based vesicle transport by confocal reflection microscopy

“…The remarkably great speed implies an involvement of the MT-based motor molecules, most likely the classical Kinesin-I and the cytoplasmic dynein for the plus-and the minus-end directed runs, respectively. Interestingly, the speed measured for both the plus (848 6 17 nm/s) and the minus end directed motions (975 6 25 nm/s) is as high as determined for the conventional kinesin (600-1800 nm/sec) [Vale et al, 1985;von Massow et al, 1989;Bohm et al, 1999] and cytoplasmic dynein (800-1250 nm/s) [Paschal et al, 1987;Toba et al, 2006] in vitro, and about twice as high as measured in early syncytial embryos during phase I (407 6 49 nm/s and 475 6 42 nm/s for plus and minus end, respectively) [Welte et al, 1998]. This suggests that the MT-based transport machinery during the stages of the fast ooplasmic streaming is, not surprisingly, in a fully activated state.…”

On the use of in vivo cargo velocity as a biophysical marker

“…A more specialized version of the model further proposes that run lengths are determined by random detachment of engaged motors, promoted by this load. As mentioned above, in vitro molecular motor studies have shown that motor velocity depends on load [19][20][21] such that motors move faster when under less load. Further, there is evidence that when multiple motors work together under negligible opposing load, the catalytic rate of each individual motor is not altered [26].…”

“…For neuronal vesicle transport velocities were normalized on a per-run basis by the authors. For the general VENoM model to be viable, the FVCs obtained in this fashion should at least satisfy one wellestablished property of molecular motors: a decrease in load should correspond to an increase in velocity [19][20][21]. Figure 1 shows the various velocities as a function of the relative load per motor -the predicted in vivo FVC.…”

“…biochemical regulation, are of secondary importance. This proposal stems from in vitro observations [19][20][21] showing that load on a motor decreases its processivity and velocity. Thus, if motors share the load opposing cargo motion, then removal of a motor from the engaged motor pool will increase load on a per-motor basis, and thus decrease cargo velocity.…”

Molecular motors: not quite like clockwork

“…Under a high load, the dynein dimers moved forwards in 8nm steps if plenty of ATP was available but larger steps were reported for small or zero loads. It is not yet clear whether the observed 16nm and 24nm steps were actually 2 or 3 × 8nm steps in rapid succession, since other groups [60,61] did not observe any steps longer than 8nm, or whether under some circumstances the stalk is flexible enough to swing up to 24nm between specific binding sites on tubulin dimers (Fig. 2).…”