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. dynein | DYNC1H1 | neurological disease | cargo adaptor | processivity

C9orf72-derived arginine-containing dipeptide repeats associate with axonal transport machinery and impede microtubule-based motility

“…Full length human dynein with a SNAPf-tag on Dynein heavy chain was expressed in Spodoptera frugiperda Sf9 cells and purified and labelled with fluorophores as described previously (61,92). SNAPf::BICD2N (not labelled with a fluorophore) was also produced from Sf9 cells using published methods (61).…”

“…Biotinylated and fluorescently-labelled porcine microtubules stabilised with taxol and GMP-CPP, PEG-biotin passivated glass surfaces, and motility chambers were prepared as described previously (92,96).…”

“…Full length human dynein with a SNAPf-tag on Dynein heavy chain was expressed in Spodoptera frugiperda Sf9 cells and purified and labelled with fluorophores as described previously (61,91).…”

“…Biotinylated and fluorescently-labelled porcine microtubules stabilized with taxol and GMP-CPP, PEG-biotin passivated glass surfaces, and motility chambers were prepared as described previously (91,95). For assays with TMR-dynein, microtubules were polymerized in the presence of HiLyte488-tubulin, with the exception of the experiments documented in Fig.…”

DYNLRB1 is Essential for Dynein Mediated Transport and Neuronal Survival

“…Our findings show that Dynlrb1 is critical not only for embryonic development, but also for maintenance of sensory neurons in adulthood. Deficits in dynein functions have been implicated in the pathogenesis of neurodegenerative diseases such as Amyotrophic Lateral Sclerosis (ALS) [40][41][42][43][44][45], and an increasing number of human neurodegenerative pathologies have been linked to mutations of dynein complex genes [46]. In addition, mutations and/or disruption of the function of the dynactin complex has been shown to be connected to motor neuron degeneration and ALS [47][48][49][50][51].…”