Cytoplasmic dynein is a minus-end directed microtubule motor protein responsible for a variety of cellular functions, including retrograde axonal transport1
. Two dynein mutant mouse strains, Legs at Odd Angles (Loa) and Cramping1 (Cra1), were identified in a screen for genes involved in late onset motor neuron disease (MND)2
. This finding identified a new class of MND genes, which also includes the mouse and human p150Glued
gene encoding a subunit of the dynein regulatory complex dynactin3–5
, and significantly expanded the limited pool of familial Amyotrophic Lateral Sclerosis (ALS) candidate genes. Of the dynein mutations, Loa has received particular attention6–10
. Loa/+ mice were initially reported to exhibit lower motor neuron degeneration, but more recent studies have found severe loss of sensory neurons8,9
. Loa mutant mice also exhibited a decreased rate of retrograde axonal transport2,10
. The Loa mutation resides in the extreme N-terminal region of the dynein heavy chain (HC) polypeptide (F580Y), within the dynein "tail." This region is responsible for organizing the multiple dynein subunits into a complex and for binding to membranous cargo. Dynein generates force through its two motor domains, each located at the C-terminal end of the HC, 1,500 a.a. (15–20 nm) away from the site of the Loa mutation. Whether and how the Loa mutation affects cytoplasmic dynein function has remained untested.
To approach this problem, we first tested the ability of Loa mutant dynein to remain associated with membranous vesicles isolated by sucrose step gradient flotation from wild-type, Loa/+ and Loa/Loa brains, but found no clear difference (). The Loa mutation lies in the region of the dynein HC involved in HC-HC dimerization and in HC-intermediate chain (IC) binding (Supplementary Information
, Fig. S1
). We therefore tested for potential defects in the stability of the mutant complex. Fractionation of whole brain cytosol by sucrose density gradient centrifugation revealed a single major 20S dynein peak for both the wild-type and Loa/+ mutant animals. Loa/Loa dynein, in contrast, showed a small but reproducible decrease in dynein s-value, accompanied by the appearance of a free IC peak (12 ± 2 % of total ICs) at 6S (Supplementary Information
, Fig. S2a
). To test whether these observations reflect a more general reduction in mutant dynein stability, we exposed brain extracts to potassium iodide (KI), a chaotropic salt to which dynein is particularly sensitive11
. In the presence of KI, dynein dissociation increased both as a function of KI concentration and of the proportion of mutant dynein HC (Supplementary Information
, Fig. S2b–d
). These results indicate that the Loa mutation may impair the interactions between subunits.
Figure 1 Purification and biochemical analysis of wild-type and mutant cytoplasmic dynein. (a) Association of dynein with membrane vesicles isolated from wild-type and mutant mouse brain. Immunoblot shows comparable levels of dynein HC and IC with wild-type, Loa/+, (more ...)
To gain further insight into the molecular effects of the Loa mutation, we purified cytoplasmic dynein from wild-type and Loa/+ adult mouse brains1
. As we observed in brain cytosol, the purified mutant dynein remained intact by sucrose density gradient centrifugation (data not shown). The purified Loa/+ dynein showed no differences in subunit composition from the wild-type mouse protein, and neither preparation showed detectable levels of the processivity factor dynactin by Coomassie Blue staining or immunoblotting (; Supplementary Information
, Fig. S3a–b
). Because the homozygous mutant mice die shortly after birth, the amounts of brain tissue we could obtain were inadequate to purify biochemical amounts of dynein by this procedure. Nonetheless, we were still able to purify small amounts of Loa/Loa dynein for single molecule analysis using a modification of this method (–, Supplementary Information
, Fig. S3c–d
Figure 2 Single molecule behavior of wild-type and mutant cytoplasmic dynein. Dynein was linked to quantum dots using an antibody to the IC, applied to microtubules in the absence of ATP, then monitored by fluorescence microscopy in the presence of 500 µM (more ...)
Figure 3 Optical trap analysis of wild-type and mutant cytoplasmic dynein behavior in single- and multi-motor regions. Bead-microtubule binding fractions were used as an indicator for the average number of available motors per bead: ≤ 30 % corresponding (more ...)
To test the effects of the Loa mutation on dynein mechanochemical activity, we measured the ATPase activity of wild-type and Loa/+ dynein in the presence and absence of microtubules. The basal ATPase activity was similar for both wild-type and Loa/+ dynein: 51.3 ± 5.3 and 51.7 ± 3.3 nmole Pi/min/mg, respectively (). However, in the presence of microtubules, the ATPase activities of wild-type and mutant dynein differed markedly. The Michaelis constant for microtubules, KmMT
, was 1.5 ± 0.2 µM for the purified wild-type dynein, similar to the value for bovine cytoplasmic dynein12
, whereas the KmMT
for Loa/+ dynein was considerably higher, 11.8 ± 3.4 µM (P
<0.001). In contrast, the maximum rate (Vmax
) values were very similar (wild-type = 255 ± 9 nmol/min/mg; Loa/+ = 263 ± 43 nmol/min/mg). The latter results indicate that the maximal ATP turnover rate is unaffected by the Loa mutation. The increased KmMT
, however, suggests a pronounced decrease in the effective affinity of the mutant dynein for microtubules during ATP hydrolysis. In support of this possibility, the KmMT
for Loa/+ was partially rescued at reduced ionic strength (4.0 ± 0.6 µM vs.
1.2 ± 0.2 µM for wild-type), which increases dynein-microtubule affinity12
. Furthermore, microtubule binding by the purified mutant dynein was markedly reduced relative to wild-type dynein in the presence of ATP, though not in its absence (apo state) (). This surprising result suggests that much of the dynein fraction normally seen to cosediment with microtubules in the presence of ATP is engaged in active, processive movement along the microtubule. These results together suggest an effect of the Loa mutation on the interaction of dynein with microtubules during the ATPase and force-generating portion of the crossbridge cycle, though not in the strong microtubule binding (apo) state.
To gain insight into the underlying molecular defects, we used quantum dot and optical trap assays under single molecule conditions. We attached dynein to quantum dots using antibodies to the tail of the wild-type and mutant molecules and analyzed their velocity and run-length. Movements in the two dynein preparations were ATP-dependent and at a velocity similar to that reported for mouse dynein-dynactin complexes (; Supplementary Information
, Fig. S4a
. The purified wild-type and mutant dyneins showed predominantly unidirectional movements, but some bidirectional events were also observed, as previously reported ()13–15
. A clear difference, however, was observed in the wild-type vs
. mutant dynein run-lengths (). Wild-type dynein exhibited an average run-length of 339 ± 33 nm (), about half that of mouse dynein-dynactin complexes13
, a reflection of the absence of dynactin from our dynein preparations. Loa/+ dynein displayed fewer long runs and a 23 % shorter average run-length (259 ± 9 nm). Loa/Loa dynein was even more impaired, with an average run-length approximately half that of wild-type dynein (175 ± 4 nm) (). Optical trap experiments using the same dynein preparations adsorbed to polystyrene beads gave comparable results, with no difference in average velocity between Loa/+ and wild-type dynein, but with a similar reduction in Loa/+ single motor processivity (; Supplementary Information
, Fig. S4b–c
). Based on the motility data, we calculate a 31–37 % increase in the average off-rate for Loa/+ vs.
wild-type dynein at 0.5 – 1 mM ATP, respectively (see Supplementary Information
), consistent with the enhanced dissociation of Loa/+ dynein from microtubules in the presence of ATP (). As for bovine cytoplasmic dynein, the stall force for single wild-type mouse dynein molecules was 1.4 ± 0.3 pN, and within experimental error of those measured for Loa/+ (1.7 ± 0.4 pN) and Loa/Loa dynein (1.6 ± 0.4 pN) ()14,16
. We also observed no difference in the step size of the wild-type or mutant dynein motors while they moved along microtubules under load (; Supplementary Information
, Fig. S5a–c
Physiological cargoes typically use multiple motors. To assess how the observed defects in dynein processivity translate to the in vivo
condition, we tested the run-lengths for multiple wild-type and Loa mutant dyneins in vitro
and found an increase in run-length for multi-motor conditions (as reported in 17
); however, this increase was attenuated for the Loa/+ dynein (). We also carried out computational modeling of dynein run-lengths at more extensive motor/cargo ratios, and found the mean travel defect to persist to three or more dynein molecules/cargo, with and without assuming potential effects of in vivo
factors such as dynactin13,18,19
(; Supplementary Information
, Fig. S6a–b
). We find here that an additional consequence of decreased single molecule processivity is a reduction in the number of instantaneously engaged motors per cargo in the multiple motor range, which in turn further limits cargo travel (Fig. S6b
). These simulations reveal a previously unappreciated sensitivity of multiple-motor run-lengths to changes in single-motor processivity, especially apparent here where the single-motor processivity is low in comparison to bovine dynein.
Figure 4 Analysis of retrograde transport of lysosomes in wild-type and mutant axons, with theoretical comparison. (a) Kymographs of retrograde transport of lysosomes in axons, representative of more processive lysosome runs. (b) Theory (top) was constrained with (more ...)
The Loa mutation has been linked to defects in retrograde axonal transport2
. To compare the effects of the Loa mutation in vivo
with our in vitro
data, we performed live cell imaging analysis of lysosome/late endosome behavior at high temporal resolution (5 frames/sec; ). We then used custom analysis software to precisely identify periods of uninterrupted motion (“runs”, see Supplemental Information
). We imaged the far distal region of the axon (>100 um from cell body), where we observed predominantly unidirectional, retrograde runs as previously described20
. Retrograde run-lengths were reduced by 53 % and 83 % in Loa/+ and Loa/Loa neurons, respectively (; Supplementary Information
, Fig. S6c
). The wild-type in vivo
run-length (5.27 ± 0.34 µm) allowed us to use our theoretical model to estimate an average of 7.7 dynein molecules per cargo (), similar to recent values based on stall force and biochemical isolation21,22
. We then predicted the expected mutant run-lengths at the same 7.7 dyneins/cargo, adjusting only the single-motor processivity to reflect the measured in vitro
processivity defects. The predicted run-lengths were in excellent agreement with those measured in vivo
(2.49 ± 0.13 vs. 2.43 ± 0.21 µm for Loa/+; 0.89 ± 0.05 vs. 0.86 ± 0.07 µm for Loa/Loa, ). As in our in vitro
experiments (–), we see no change in instantaneous lysosome velocity in the Loa mutant neurons (, left). However, the overall average velocity was reduced by 22 % in Loa/+ and 43 % in Loa/Loa neurons vs.
wild-type (, right), consistent with an increase in run terminations, and comparable to theoretical prediction (11 % decrease in average velocity for Loa/+ and 37 % for Loa/Loa, see Supplemental Information
). Together, these results argue strongly that the Loa dynein processivity defect we identify in vitro
can account for the observed impairment in retrograde axonal transport.
To gain further insight into the mechanism responsible for altered Loa dynein processivity, we tested for the reported tendency of cytoplasmic dynein to step laterally on the microtubule surface, in contrast to the strict linear travel of kinesin15,23
. We confirmed lateral stepping in mouse dynein, and observed a significant increase in its frequency for the Loa/+ mutant protein (). One potential explanation for this result is a disrupted coordination between the two motor domains within the native dynein complex. A gating mechanism between dimeric motor domains is well established for kinesins and myosins, but is not well understood for dynein24–26
. Gating contributes to processive motion by ensuring that, as one motor domain advances, the other remains strongly associated with its track in the apo state, thereby preventing premature detachment26
. As a test for altered gating in the Loa mutant dynein, we measured its KmATP
. Despite the somewhat decreased Vmax
for Loa/+ dynein due to its lower KmMT
(), the KmATP
was clearly decreased (18.8 ± 4.1 µM vs.
27.0 ± 6.5 µM for wild-type, P
<0.05) (). This result is consistent with a defect in communication between motor domains, allowing premature ATP binding by the apo motor domain, and subsequent release from the microtubule (), though further kinetic analysis will be needed to confirm this model.
Figure 5 Biophysical and biochemical evidence of altered motor coordination in mutant dynein. (a) Sample lateral position traces of beads carried by a single kinesin, wild-type or Loa/+ dynein. (b) Distributions of the instantaneous change in lateral position (more ...)
These results have novel implications for intramolecular regulation of cytoplasmic dynein motor activity. Despite the location of the Loa mutation within the dynein tail, we find several lines of evidence for an altered interaction between dynein and microtubules, the first direct indication for communication between the dynein motor and tail domains. The clear defect we observed in mutant dynein processivity raised the possibility of motor domain miscoordination. Supporting this, we identified novel defects in the Loa mutant dynein: both an increase in lateral stepping on the microtubule lattice, and an increased affinity for ATP apparently leading to premature run termination. Consistent with the subtly decreased stability of the Loa dynein complex, we propose that an abnormal linkage within the base of the Loa dynein molecule disrupts coordination between the two motor domains by altering their relative positioning and ability to interact laterally.
Our results also suggest a novel role for processivity defects in disease causation. We argue that neurodegeneration in the Loa/+ mutant mouse is unlikely to result from dynein subunit dissociation, which we do not detect for the Loa/+ complex in cytosolic extracts or following purification. (Only dynein from the Loa/Loa mouse, which dies shortly after birth, shows evidence of dissociation: Supplementary Information
, Fig. S2
). Our data also argue against a loss in the association of dynein with membranous cargo, which persisted in our biochemical analysis. The most dramatic change we observed to result from the Loa mutation was a decrease in dynein run-length. This effect quantitatively accounted for the observed defect in axonal transport as indicated by a combination of empirical analysis with computational modeling. The neurons that are most likely to be affected by this defect in processivity and impairment in transport would be those with the longest axons, such as motor and sensory neurons. Our study therefore provides the first evidence that altered motor protein processivity can have pronounced consequences for in vivo
transport, the impairment of which clearly correlates with neuronal death and disease.