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DYNC1H1 Mutations Associated with Neurological Diseases Compromise DYNC1H1 mutations associated with neurological PNAS PLUS diseases compromise processivity of dynein–dynactin– cargo adaptor complexes Ha Thi Hoanga, Max A. Schlagerb,1, Andrew P. Carterb, and Simon L. Bullocka,2 aDivision of Cell Biology, Medical Research Council (MRC) Laboratory of Molecular Biology, Cambridge CB2 0QH, United Kingdom; and bDivision of Structural Studies, MRC Laboratory of Molecular Biology, Cambridge CB2 0QH, United Kingdom Edited by Gary G. Borisy, The Forsyth Institute, Cambridge, MA, and approved January 24, 2017 (received for review December 7, 2016) Mutations in the human DYNC1H1 gene are associated with neuro- AAA1 is the major ATP hydrolysis site, a microtubule-binding logical diseases. DYNC1H1 encodes the heavy chain of cytoplasmic domain (MTBD), and an antiparallel coiled-coil stalk that mediates dynein-1, a 1.4-MDa motor complex that traffics organelles, vesicles, communication between the ring and the MTBD. In addition, there and macromolecules toward microtubule minus ends. The effects of is a buttress domain that emerges from AAA5 of the ring and the DYNC1H1 mutations on dynein motility, and consequently their provides support to the base of the stalk, a linker that is remodeled links to neuropathology, are not understood. Here, we address this to produce the powerstroke, and a C-terminal domain that regu- issue using a recombinant expression system for human dynein cou- lates force production and processivity (16). The heavy chain also pled to single-molecule resolution in vitro motility assays. We func- contains the tail domain, which mediates dimerization and provides tionally characterize 14 DYNC1H1 mutations identified in humans a platform for recruiting the other chains. The accessory chains are diagnosed with malformations in cortical development (MCD) or spi- important for linking the motor to cargos, either through direct nal muscular atrophy with lower extremity predominance (SMALED), interactions or by binding to an intermediary cargo adaptor (17). as well as three mutations that cause motor and sensory defects in At the time of writing, more than 30 heterozygous missense mice. Two of the human mutations, R1962C and H3822P, strongly mutations in the DYNC1H1 gene have been identified in patients interfere with dynein’s core mechanochemical properties. The diagnosed with spinal muscular atrophy with lower extremity remaining mutations selectively compromise the processive mode predominance (SMALED) [Online Mendelian Inheritance in of dynein movement that is activated by binding to the accessory Man (OMIM): 158600)] or malformations of cortical develop- complex dynactin and the cargo adaptor Bicaudal-D2 (BICD2). Muta- ment (MCD) (OMIM: 614563) (1–13). SMALED is characterized tions with the strongest effects on dynein motility in vitro are asso- by muscle weakness in the legs, which is caused by congenital or ciated with MCD. The vast majority of mutations do not affect early childhood-onset loss of spinal cord motor neurons. MCD binding of dynein to dynactin and BICD2 and are therefore expected arises from defective neuronal proliferation or migration during to result in linkage of cargos to dynein–dynactin complexes that development of the cerebral cortex and is often associated with have defective long-range motility. This observation offers an expla- severe intellectual disability and epilepsy. Whereas some muta- nation for the dominant effects of DYNC1H1 mutations in vivo. Col- tions segregate with disease in familial cases (2, 4, 6–8, 13, 18), lectively, our results suggest that compromised processivity of others have arisen de novo (2, 5, 7, 9–12) (SI Appendix, Table S1). cargo–motor assemblies contributes to human neurological disease For the vast majority of mutations, direct evidence of an effect on and provide insight into the influence of different regions of the dynein function has not been provided, and thus it has not been heavy chain on dynein motility. conclusively shown that they are pathogenic. dynein | DYNC1H1 | neurological disease | cargo adaptor | processivity Significance BIOCHEMISTRY icrotubule motors play a key role in the sorting of many Mutations in microtubule motors and their cofactors are associ- Mcellular constituents, including organelles, vesicles, aggregated ated with several neurological diseases in humans. How disease- proteins, and macromolecules. Because of their elongated processes, associated mutations affect motor function, and consequently neurons are particularly reliant on a fully operational microtubule- have neuropathological effects, is largely unknown. We take based transport system. This point is underscored by the association advantage of recent advances in the ability to activate transport of several mutations in microtubule motors and their cofactors with of the human dynein complex in vitro to functionally characterize human neurological diseases (1–3). How these mutations affect 17 disease-associated mutations in the gene encoding its heavy transport mechanisms is poorly understood. Addressing this issue is chain. The vast majority of mutations do not affect the ability of expected to provide insight into the cellular basis of neurological dynein to bind a cargo adaptor but do compromise long-range disease and also inform diagnostic and therapeutic efforts. motion of the motor complex. Our findings suggest that de- Mutations in the gene encoding the heavy chain of the cyto- fective motility of cargo–motor complexes contributes to dynein- plasmic dynein-1 (dynein) motor have repeatedly been implicated in associated neurological phenotypes and highlight several regions neurological diseases (1–13). Dynein is a 1.4-MDa complex that is of the enigmatic motor that orchestrate its ability to walk over responsible for the vast majority of cargo transport toward the mi- long distances. nus ends of microtubules (14, 15). The complex consists of two copies each of the 530 kDa heavy chain (DYNC1H1), an in- Author contributions: H.T.H., M.A.S., A.P.C., and S.L.B. designed research; H.T.H. and M.A.S. termediate chain (either the DYNC1I1 or DYNC1I2 isoform), a performed research; H.T.H. analyzed data; and H.T.H., A.P.C., and S.L.B. wrote the paper. light intermediate chain (either the DYNC1LI1 or DYNC1LI2 The authors declare no conflict of interest. isoform), and three different light chains [DYNLT1 (Tctex1), This article is a PNAS Direct Submission. DYNLL1 (LC8), and DYNLRB1 (Robl)] (Fig. 1 A and B). The Freely available online through the PNAS open access option. heavy chain contains a motor domain, which translocates the pro- 1Present address: Roche Diagnostics, 68305 Mannheim, Germany. tein complex toward the minus ends of microtubules using the en- 2To whom correspondence should be addressed. Email: [email protected]. ergy derived from ATP hydrolysis. Key elements within the motor This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. domain include a ring formed by six AAA+ domains, of which 1073/pnas.1620141114/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1620141114 PNAS | Published online February 14, 2017 | E1597–E1606 Downloaded by guest on September 25, 2021 A B Tail Motor K3336N Dimerization domain E1518K R3344Q DYNC1I (DIC) R1567Q R1962C K3241T R3384QH3822P 1 4646 DYNLRB1 (RobI) Linker AAA1 AAA2 AAA3 AAA4 AAA5 AAA6 C-term. DYNLL1 (LC8) DYNC1LI (DLIC) Stalk Buttress DYNLT1 MTBD (Tctex1) Linker 1 6 2 C-term. 1 200 560 770 900 990 1165 1450 3 5 4 Buttress DLIC- Dimerization DIC-binding binding Stalk K129I H306R F582Y (Loa) K671E Y970C Y1057C (Cra1) I584L Δ659-662 Swl MTBD Δ1042-1045+A ( ) MCD SMALED Mouse (Swl) ) ) Cra1 C Loa 7Q 659-662 WT K129I H306R F582Y ( I584L Δ Y970C Δ1042-1045+AY1057C ( WT E1518K R156 R1962C K3241TK3336N R3344QR3384Q H3822P kDa DYNC1H1 250 150 100 75 DYNC1I2 DYNC1LI2 50 37 25 20 15 DYNLT1 12 DYNLL1 DYNLRB1 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 char- acterized 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. The links between DYNC1H1 mutations and neurological dis- study robust minus-end–directed movement of individual dynein ease are further strengthened by the discovery of three mutations complexes in vitro. Whereas individual mammalian dyneins in vitro that, when heterozygous, cause overlapping neuromuscular and very rarely exhibit processive behavior (the ability to take repeated sensory deficits in adult mice (1) (SI Appendix,TableS2). The steps along microtubules) (25–28), dynein complexes bound to the DYNC1H1 mutant strains were identified in genetic screens due to accessory complex dynactin and a cargo adaptor frequently move hind limb clenching
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