Control of cytoplasmic dynein force production and processivity by its C-terminal domain

Cytoplasmic dynein is a microtubule motor involved in cargo transport, nuclear migration and cell division. Despite structural conservation of the dynein motor domain from yeast to higher eukaryotes, the extensively studied S. cerevisiae dynein behaves distinctly from mammalian dyneins, which produce far less force and travel over shorter distances. However, isolated reports of yeast-like force production by mammalian dynein have called interspecies differences into question. We report that functional differences between yeast and mammalian dynein are real and attributable to a C-terminal motor element absent in yeast, which resembles a ‘cap’ over the central pore of the mammalian dynein motor domain. Removal of this cap increases the force generation of rat dynein from 1 pN to a yeast-like 6 pN and greatly increases its travel distance. Our findings identify the CT-cap as a novel regulator of dynein function.

Pairwise distance distribution function (PDF) calculated from the filtered data in a. Groups of gridlines (red) demonstrate the predominance of ~8.3-nm separations between apparent peaks (shifting of peaks from exact 8.3 nm multiples and apparent missing peaks are likely due to instrument positional drift). (c) Step-size analysis of the raw data in a (0-1.5 s trace segment) using the step-finding algorithm developed by Kerssemakers et al. (4). The raw data are shown in black and the steps detected by the step-finding program in red. The analysis agrees with the predominance of ~8 nm steps as revealed by the PDF in b. Experiments were performed with AC-purified protein.

Notes on bidirectional motility observed at high motor concentration
Bidirectional motion was observed only at very high motor concentrations (100% of beads exhibiting motion), and never at the single-molecule level (50% beads exhibiting motion). The behavior occurred on multiple MTs and also on axonemes. However, it was not observed for every bead tested under these conditions, and backward movement was often seen only sporadically. This may indicate that reversal of movement only occurs for specific configurations of grouped motors. Kinesin contamination was highly unlikely given the expression and purification strategies, as well as the specific linkage of the motor to the bead via an anti-GST antibody. Careful visual inspection of the fluorescence-labeled MTs that supported bidirectional motion never revealed any evidence of MT bundling that might lead to antiparallel tracks for the motors to move upon.
Bidirectional movement was only observed once for MD-CT (again, at very high motor concentration). This may reflect an inherent difference between MD-WT and MD-CT.
However, we believe it is more likely explained by the relatively low fraction of active MD-CT motors in our preparations (see below), which would decrease the likelihood of active motors attaching near each other on the trapping bead surface. Consistent with this, even at relatively high motor concentrations, the behavior of MD-CT (e.g. velocity and stall force) was not noticeably changed from that at the single-molecule level, suggesting that even at high concentrations, single motors drove the vast majority of events ( Supplementary Fig. 10).

Notes on the proportions of active dynein motors
Our purified MD-WT and MD-ΔCT samples were well behaved as judged by the absence of degradation and aggregation ( Supplementary Fig. 1). However, multiple observations suggest that the MD-ΔCT construct is particularly vulnerable to loss of activity. The enzymatic rate k cat obtained from ATPase assays at saturating MT concentration was variable from one preparation to the next, ranging from ~13 ATP/s to ~17 ATP/s for MD-WT and ~1 ATP/s to ~9 ATP/s for MD-ΔCT (the lower ATPase activities of MD-ΔCT might in part reflect the very high basal and MT-activated K M (ATP) values for this construct determined enzymatically, see Supplementary   Table 1). Low measured ATPase activity correlated with a low fraction of motile beads in the optical trapping assay (even at high motor concentrations) and some preparations of both constructs failed to produce any motility. This was probably due to inactive motors occupying the majority of the binding sites on most beads. However, the contribution of preparative conditions, freeze/thaw, and freshness to protein stability remains incompletely resolved.  Fig. 7B). These findings might suggest that MD-CT is immotile, has a slow enzymatic rate, or has a very high affinity for MTs (even in the presence of 5 mM ATP and 200 mM KCl; this behavior is partly consistent with that of yeast MD dimers, which can be extracted with inclusion of high salt, though this still had no apparent effect on rat MD-CT extraction). However, the robust motility observed in the trap suggests that single MD-CT motors are highly active. Indeed, we measured the maximal velocity for the motor under low load to be ~700 nm/s ( Fig. 3E and Supplementary   Fig. 8). Given the 8-nm step size, this implies k cat  90 s -1 , which is at least 10-fold greater than the result calculated from MT-stimulated ATPase measurements. This could mean that less than 10% of the motors in such a sample are active depending on the preparation. It is also notable that, under identical experimental conditions, a GST-dimerized yeast dynein analogous to MD- Given these challenges, we opted to avoid detailed mechanochemical interpretation of ensemble assays for MD-WT and MD-CT, and we instead focused on unambiguous single-molecule measurements using the optical trap. It is notable that both MD-WT and MD-CT retained activity in this assay up to multiple hours, suggesting that the observed loss of activity may result from failure to properly fold during expression, damage experienced during subsequent purification steps, or denaturation during freezing/thawing of stored aliquots. This may also reflect improved enzyme stability when attached to the trapping beads. Alternatively, it is conceivable that individual motors transition between active and inactive states, spending most of their time in the latter.

Discussion of mechanisms for regulation of dynein force and processivity by the CT-cap
Considerable future work will be needed to define the precise mechanism by which removal of  Table 2). Moreover, the K M (ATP) reported for yeast dynein (1) (Supplementary Table 2) is nearly as small as the lowest reported values for higher eukaryotes, suggesting that removal of the CT-cap does not, by itself, drastically affect K M (ATP).
Nonetheless, the dramatic decrease in vanadate sensitivity caused by removal of the CT-cap (2) suggests that some step in the catalytic cycle is under the control of dynein's C-terminal domain.
In conclusion, future work is needed to determine whether the CT-cap alters AAA1 function directly or acts as a "mechanical" element that affects the allosteric communication between the AAA ring and the MTBD.