DYNC1H1 mutations associated with neurological diseases compromise processivity of dynein-dynactin-cargo adaptor complexes

Abstract

Mutations in the human DYNC1H1 gene are associated with neurological diseases. DYNC1H1 encodes the heavy chain of cytoplasmic dynein-1, a 1.4-MDa motor complex that traffics organelles, vesicles, and macromolecules toward microtubule minus ends. The effects of the DYNC1H1 mutations on dynein motility, and consequently their links to neuropathology, are not understood. Here, we address this issue using a recombinant expression system for human dynein coupled to single-molecule resolution in vitro motility assays. We functionally characterize 14 DYNC1H1 mutations identified in humans diagnosed with malformations in cortical development (MCD) or spinal muscular atrophy with lower extremity predominance (SMALED), as well as three mutations that cause motor and sensory defects in mice. Two of the human mutations, R1962C and H3822P, strongly interfere with dynein's core mechanochemical properties. The remaining mutations selectively compromise the processive mode of dynein movement that is activated by binding to the accessory complex dynactin and the cargo adaptor Bicaudal-D2 (BICD2). Mutations with the strongest effects on dynein motility in vitro are associated with MCD. The vast majority of mutations do not affect binding of dynein to dynactin and BICD2 and are therefore expected to result in linkage of cargos to dynein-dynactin complexes that have defective long-range motility. This observation offers an explanation for the dominant effects of DYNC1H1 mutations in vivo. Collectively, our results suggest that compromised processivity of cargo-motor assemblies contributes to human neurological disease and provide insight into the influence of different regions of the heavy chain on dynein motility.

Keywords: DYNC1H1; cargo adaptor; dynein; neurological disease; processivity.

Fig. 1.

Positions of DYNC1H1 mutations and accessory chain composition of purified mutant dynein complexes. (A) Architecture of the dynein complex. The linker is behind the AAA+ ring in this view. C-term, C-terminal domain. (B) Positions in the DYNC1H1 polypeptide of the human and mouse mutations characterized in this study. Mutations discovered in mouse are numbered according to the equivalent residues in human DYNC1H1. H306R has also been associated with Charcot–Marie–Tooth disease type 2O in one pedigree (4). (C) Coomassie-stained denaturing gels of human recombinant dynein complexes. The same amount of total protein was loaded per well. None of the DYNC1H1 mutations resulted in an overt change in accessory chain composition of purified dynein complexes compared with the WT. This conclusion was confirmed with dynein samples expressed and purified from an independent construct. K671E DYNC1H1 could not be purified from insect cells in three independent experiments using two different expression constructs.

Fig. 2.

Effects of DYNC1H1 mutations on microtubule gliding by ensembles of human dynein complexes. (A) Diagram of the microtubule gliding assay. Dyneins are absorbed nonspecifically onto the glass surface. MT, microtubule. (B) Stills from image series in Movie S1 showing microtubules being translocated by WT dynein but not by dynein containing R1962C or H3822P DYNC1H1. In each condition, the starting position of two microtubule ends are labeled with arrows. (C) Quantification of gliding velocities in the presence of WT and mutant dynein complexes. Each magenta circle represents the mean value for an individual chamber (>30 microtubules analyzed per chamber). Gray bars show means of the individual chamber values for each condition; error bars represent SEM. Statistical significance, compared with WT (value illustrated by dashed gray line), was evaluated with a one-way ANOVA with Sidak’s multiple comparison test [***P < 0.001; **P < 0.01; *P < 0.05; n.a., not applicable because of the absence (R1962C) or extreme rarity (H3822P) of microtubule gliding].

Fig. 3.

Effects of DYNC1H1 mutations on processive movement of individual dynein complexes in the presence of dynactin and BICD2N. (A) Diagram of the assay for processive movement of dynein. Microtubules (MTs) are immobilized on biotin (orange)/streptavidin (pink)-coated glass using biotin present on a fraction of tubulins. (B) Kymographs (time–distance plots) showing examples of the effect of DYNC1H1 mutations on processive movement of dynein (green) in the presence of dynactin and BICD2N. White, yellow, and blue arrowheads indicate examples of processive, diffusive, and static behavior, respectively. (C) Quantification of effects of DYNC1H1 mutations on percentage of microtubule-associated dyneins that move processively in the presence of dynactin and BICD2N. Each magenta circle represents the mean value for an individual chamber (>70 dynein complexes analyzed per chamber). Throughout the study, each chamber used an independent assembly mix of dynein, dynactin, and BICD2N. Gray bars show means of the individual chamber values for each condition; error bars represent SEM. Statistical significance, compared with WT dynein in the presence of dynactin and BICD2N (value illustrated by dashed gray line), was evaluated with a one-way ANOVA with Sidak’s multiple comparison test (***P < 0.001; *P < 0.05; ^[*^]P < 0.05 but a significant difference was not reproduced in a second experimental series (SI Appendix, Fig. S3).

Fig. 4.

Effects of DYNC1H1 mutations on the assembly and processivity of dynein–dynactin–BICD2N complexes. (A) Kymographs showing examples of frequent colocalization of mutant TMR–dynein complexes (green in merged images) with Alexa 647–BICD2N (magenta in merged images) on microtubules in the presence of dynactin (see SI Appendix, Figs. S4 and S5 for examples of other mutant complexes). Colocalization indicates formation of a dynein–dynactin–BICD2N complex. White, blue, and yellow arrowheads indicate examples of colocalization of Alexa 647–BICD2N with TMR–dynein exhibiting processive, static, and diffusive behavior, respectively. Red arrowheads indicate examples of TMR–dynein complexes without an Alexa 647–BICD2N signal. (B) Quantification of the percentage of all microtubule-associated dyneins that, in the presence of dynactin, colocalizes with BICD2N. (C–E) Quantification of the percentage of microtubule-associated dynein–dynactin–BICD2N (DDB) complexes that exhibits processive (C), static (D), and diffusive (E) behavior. In B–E, each magenta circle represents the mean value for an individual chamber [>70 dynein complexes (B) or 50 dynein–dynactin–BICD2N (C–E) complexes analyzed per chamber]. Gray bars show means of the individual chamber values for each condition; error bars represent SEM. Statistical significance, compared with WT (value illustrated by dashed gray line), was evaluated with a one-way ANOVA with Sidak’s multiple comparison test (***P < 0.001; *P < 0.05).

Fig. 5.

Effects of DYNC1H1 mutations on the run length (A) and velocity (B) of processive dynein–dynactin–BICD2N complexes. Each magenta circle represents the mean value for an individual chamber (>50 runs or velocity segments were analyzed per chamber). Gray bars show means of the individual chamber values for each condition; error bars represent SEM. Statistical significance, compared with WT (values illustrated by dashed gray line), was evaluated with a one-way ANOVA with Sidak’s multiple comparison test (***P < 0.001; **P < 0.01). See SI Appendix, Fig. S8 for data on run length and velocity distributions.

Fig. 6.

Summary of the consequences of DYNC1H1 mutations on dynein motility. Mutations are clustered based on similarities of their effects on purified dynein complexes, with the severity of each cluster decreasing from Top to Bottom. Within each cluster, mutations are ordered in an N–C-terminal direction. MT, microtubule; DDB, dynein–dynactin–BICD2N. Note that K671E DYNC1H1 could not be purified.

Fig. 7.

Structural analysis of mutated residues in the DYNC1H1 motor domain. (A) Cartoon of a microtubule-bound dynein motor domain, showing positions of DYNC1H1 mutations (red circles). Individual AAA+ domains, which are divided into large and small subdomains, are labeled with a colored number. MTBD, microtubule binding domain. Eye shows the viewpoint for F. (B–F) Structural analysis of the positions of mutated residues in the DYNC1H1 motor domain. Domains are colored as in A; side chains of mutated residues are shown in magenta in B–E and G, and yellow in F. See text for details. Homology models are based on the structures of the motor domain of Dictyostelium dynein-1 bound to ADP [Protein Data Bank (PDB): 3VKG] (B, D, F, and G), human cytoplasmic dynein-2 bound to ADP.vanadate (PDB:4RH7) (C), and a pseudoatomic model from a 9-Å cryo-EM structure of the MTBD of mouse DYNC1H1 bound to microtubules (PDB: 3J1T) (E). ADP.vanadate is a nucleotide analog that mimics the transition state of ATP hydrolysis.