A cytoplasmic dynein tail mutation impairs motor processivity

Abstract

Mutations in the tail of the cytoplasmic dynein molecule have been reported to cause neurodegenerative disease in mice. The mutant mouse strain Legs at odd angles (Loa) has impaired retrograde axonal transport, but the molecular deficiencies in the mutant dynein molecule, and how they contribute to neurodegeneration, are unknown. To address these questions, we purified dynein from wild-type mice and the Legs at odd angles mutant mice. Using biochemical, single-molecule, and live-cell-imaging techniques, we find a marked inhibition of motor run-length in vitro and in vivo, and significantly altered motor domain coordination in the dynein from mutant mice. These results suggest a potential role for the dynein tail in motor function, and provide direct evidence for a link between single-motor processivity and disease.

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/+, and Loa/Loa vesicles, quantified at right (average of n = 3 experiments ± SD). P: membrane pellet; 0.6 and 1.5 M: sucrose steps; V: vesicles from sucrose interface; Syn: synaptotagmin. (b) Coomassie-stained gel of purified wild-type and Loa/+ brain cytoplasmic dynein. (c) ATPase activity of wild-type and Loa/+ dynein as a function of microtubule concentration at low and high ionic strength. Activities ± SD were determined from n = 3 experiments in Tris buffer containing 10 mM KCl (dotted lines), or from n = 6 experiments in Tris buffer containing 50 mM KCl (dashed lines) and fitted with Michaelis-Menten kinetics. (d) Microtubule (MT) cosedimentation of purified wild-type and Loa/+ dynein assayed by immunoblotting for IC and tubulin. Input (I) is 20% of total. In the absence of microtubules, dynein remains in the supernatant (S). Graph depicts the amount of dynein ± SD in the microtubule pellet (P) in the absence and presence of ATP from n = 3 different experiments per genotype, per ATP condition (P<0.001).

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 ATP. (a) Velocities for quantum dot runs > 200 nm for wild-type, Loa/+, and Loa/Loa dynein (n>111 quantum dots). (b) Average run-length at molar ratios of dynein:quantum dots of 1:35 and 1:50. A significant, graded reduction in run-length is observed with decreasing wild-type dynein dose (P<0.001). Error bars indicate SEM. (c) Kymographs of wild-type, Loa/+, and Loa/Loa quantum dots. The percentage of quantum dots that exhibited unidirectional and bidirectional behaviors is below the kymographs. Scale bars = 1 um (x-axis) and 5 sec (y-axis). Histograms to the right of each set of kymographs depict the range of net run-lengths and associated mean ± SEM.

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 to single motor levels and ≥ 50 % to multiple motor levels (See Supplementary Information). (a) Bead velocity of Loa/+ dynein is unchanged vs. wild-type (mean ± SEM, n > 40, P=0.97). (b) Sample single motor stall force traces reveal no significant difference between the genotypes (mean ± SD, n > 29, mean stall forces agree within systematic noise of optical trap, 0.3–0.4 pN). (c) Distribution of axial step sizes for wild-type and Loa/+ dynein under modest load (n > 1979, P=0.89). (d) Average run-lengths in modest multi-motor range of Loa/+ dyneins are significantly reduced from that of wild-type motors. Error bars represent the SEM (wild-type: n = 38, 64, and 127 runs for binding fraction of 50, 75, and 100 %, respectively; Loa/+: n = 39 and 296 runs for binding fraction of 75 and 100 %, respectively. P<0.03). Loa/+ run-length at 50 % binding fraction was below measurement limit under assay conditions used here (Fig. S4c).

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 wild-type data (blue arrow) to predict motor number (dotted line), and then predict mutant run-lengths for mutant dynein (orange and pink arrows) (mean ± SEM, n > 400, 300, 600 simulated runs for wild type, Loa/+ and Loa/Loa, respectively). We find striking agreement between predictions (using 7.7 as the number of available motors) and in vivo measurements (bottom, hatched vs. solid bars, respectively). Error bars represent the SEM (n = 68, 78, and 55 uninterrupted retrograde runs for wild type, Loa/+ and Loa/Loa, respectively). (c) Graph depicting instantaneous and average lysosome velocities ± SEM for each genotype (n = 102, 98, and 111 for wild type, Loa/+ and Loa/Loa, respectively, P<0.001 for run-length and average velocity).

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 for dynein (bottom) demonstrates a significant deviation of mutant dynein from wild-type (top) (n = 32 and 18 runs for wild-type and Loa/+, respectively; P<0.05). Error bars represent the SD. (c) ATP concentration dependence of wild-type and Loa/+ dynein ATPase activity at 10 µM microtubules. Low ATP concentration range is expanded in bottom graph. Activities ± SD were determined from three different experiments per genotype and fitted to Michaelis-Menten kinetics. (d) Proposed effect of Loa mutation on motor coordination. In wild-type dynein, the stepping head (green) inhibits the tightly bound head (red) from binding ATP. In Loa dynein, the stepping head (green) does not adequately inhibit the tightly bound head (red), which binds ATP prematurely and causes release from the microtubule.