Motor proteins are fascinating molecular machines that convert chemical energy stored in ATP into mechanical work and move along cytoskeletal tracks. Kinesin and dynein are two types of motors that actively transport cargos along the microtubules (MTs) in cells towards the MT plus-end (cell periphery) and minus-end (cell nucleus), respectively, as illustrated in Fig. 1.
Figure 1. Schematic molecular structures of kinesin and dynein, and the microtubule that these motors move on. Image adpated from Cooper, "Cell, A Molecular Approach".
Much has been learned about these motors from in vitro experiments using purified kinesin and dynein. For example, kinesin normally takes an 8 nm step for every ATP molecule that it hydrolyzes. This distance matches the periodicity of the motor binding site on the MTs. Dynein, on the other hand, has been reported to take 8, 16, 24, and even 32 nm steps per ATP hydrolysis, with large step sizes at low external force loads, acting like a 'gear'. As a dimeric protein, kinesin has also been shown to walk with an asymmetric 'hand-over-hand' mechanism.
The above findings, however, are not sufficient to answer the question of how exactly the motors work in the cells. This is because in contrast to the clean in vitro situation, there are many complications in living cells, among which are (1) the presence multiple motors with the same and/or opposite directionality on a single cargo; (2) the crowdedness in the cellular environment; (3) the effects of regulatory and accessory proteins, e.g. the dynactin complex on dynein. The resolution of individual motor stepping events is crucial to our understanding of molecular motor dynamics in living cells, but the high cytoplasmic ATP concentration makes this difficult to achieve.
To this end, we need to image the kinesin- and dynein-mediated movements of cargos in living cells with sub-millisecond time resolution and nanometer spatial precision. Most recently, we developed a novel particle tracking scheme with 25 μs time resolution and 1.5 nm spatial precision made possible by the strong scattering from gold nanoparticles (GNPs) imaged with dark field microscopy. We employed an efficient detection scheme and a quadrant photodiode to record the positions of endocytosed GNPs. By integrating this tracking scheme with a positional feedback loop, we can record long (tens of microns), high time resolution and spatial precision trajectories of organelles enclosing GNPs as they are actively transported by kinesin and dynein in living cells.
Figure 2. Tracking GNPs in living A549 cells. A) Dark field image of endocytosed GNPs inside an A549 cell. Scale bar: 5μm. B) A 17 μm x-y trajectory obtained with the tracking system. C) Broad velocity distribution of the first 10 s of the trajectory in (B) with an average velocity of 1.6 μm/s.
The high time resolution and spatial precision of our tracking system enabled us to resolve individual steps (Figure 3) and to determine the step size for the entire range of cargo velocities (0-8 μm/s). The histograms of step sizes in the kinesin and dynein directions are shown in Figures 4A and B, respectively. While cargoes carried by kinesin exhibit exclusively 8 nm steps, regardless of velocity, cargoes carried by dynein exhibit both 8 nm and large step sizes at all organelle velocities. We also notice that in dynein trajectories, the large steps often occur consecutively, as shown by the conditional histogram (Figure 4C). The conditional histogram also reveals distinct peaks around 8, 12, 16, 20, and 24 nm which we attribute to the different possible step sizes of dynein.
Figure 3. Representative stepping trajectories of kinesin (A) and dynein (B) in living cells obtained by image the light scattered by endocytosed gold nanoparticles onto a quadrant photodiode and tracking with 25 μs time resolution and nanometer spatial precision. (C) Two-dimensional position trajectory of dynein stepping.
Figure 4. A) Kinesin step size histogram showing a peak a 8 nm. B) Dynein step size histogram showing a peak at 8 nm and a broad shoulder extending to 32 nm. C) Conditional step step size histogram for dynein trajectories in which all steps following a step that is at least 12 nm are counted. This histogram has distinct peaks with a periodicity of 4 nm starting at 8 nm.
Our data demonstrate that while cargoes carried by kinesin exhibit only 8 nm steps, those carried by dynein exhibit both 8 nm and larger (12, 16, 20, and 24 nm) steps. Although our understanding of how motors work individually and collectively in vivo is far from complete, the high resolution measurements required to address this complex problem are afforded by this technique.
References
X. Nan, P.A. Sims, X.S. Xie. ChemPhysChem. 9, 707-712 (2008).
