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Cytoplasmic dynein-1 (dynein) is an essential molecular motor in eukaryotic cells. Dynein primarily exists in an autoinhibited Phi state and requires conformational changes to assemble with its cofactors and form active transport complexes. LIS1, a key dynein regulator, enhances dynein activation and assembly. Using cryo-EM and a human dynein-LIS1 sample incubated with ATP, we map the conformational landscape of dynein activation by LIS1 and identify an early intermediate state that we propose precedes the previously identified dynein-LIS1 Chi state. Mutations that disrupt this species, which we termed “Pre-Chi”, lead to motility defects in vitro, emphasizing its functional importance. Together, our findings provide insights into how LIS1 relieves dynein autoinhibition during the activation pathway. The molecular motor dynein is modulated by several protein regulators, including LIS1. Here, authors use cryo-EM to show how LIS1 activates dynein by disrupting its autoinhibition and stabilizing intermediate states required to assemble the complexes that mediate transport.

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.

Polarised mRNA transport is a prevalent mechanism for spatial control of protein synthesis. However, the composition of transported ribonucleoprotein particles (RNPs) and the regulation of their movement are poorly understood. We have reconstituted microtubule minus end-directed transport of mRNAs using purified components. A Bicaudal-D (BicD) adaptor protein and the RNA-binding protein Egalitarian (Egl) are sufficient for long-distance mRNA transport by the dynein motor and its accessory complex dynactin, thus defining a minimal transport-competent RNP. Unexpectedly, the RNA is required for robust activation of dynein motility. We show that a cis-acting RNA localisation signal promotes the interaction of Egl with BicD, which licenses the latter protein to recruit dynein and dynactin. Our data support a model for BicD activation based on RNA-induced occupancy of two Egl-binding sites on the BicD dimer. Scaffolding of adaptor protein assemblies by cargoes is an attractive mechanism for regulating intracellular transport. In our cells, tiny molecular motors transport the components necessary for life’s biological processes from one location to another. They do so by loading their cargo, and burning up chemical fuel to carry it along pathways made of filaments. For example, one such motor, called dynein, can move molecules of messenger RNA (mRNA) to specific locations within the cell. There, the mRNA will be used as a template to create proteins, which will operate at exactly the right place. Transporting mRNA in this way is critical in processes such as embryonic development and the formation of memories; yet, this mechanism is still poorly understood. Previous work suggested that the mRNA is simply a passenger of the dynein motor, but McClintock et al. asked if this is really the case. Instead, could mRNA regulate its own sorting by controlling the activity of dynein? Studying mRNA trafficking within the complex molecular environment of a cell is challenging, so mRNA transporting machinery was recreated in the laboratory. Only the proteins necessary to build a working system were included in the experiments. In addition to the filaments, the components included dynein and a complex of proteins known as dynactin, which allows the motor to move together with a protein called BICD2. A protein named Egalitarian was used to link the mRNA to BICD2. By filming fluorescently labelled proteins and mRNAs, McClintock et al. discovered that mRNA strongly promotes the movement of the dynein motor. A structured section in the mRNA acts as a docking area for two copies of Egalitarian. This activates BICD2, which then binds to dynein and dynactin, thereby completing the transport machinery. According to these results, the mRNA directs the assembly of the system that will carry it within the cell. Viruses such as HIV and herpesvirus hijack dynein motors to have their genetic information moved around a cell in order to propagate infection. Understanding precisely how mRNA is transported may help to develop new strategies to fight these viruses.

Spindle orientation depends on the tethering of microtubules to the cell cortex through LGN, NuMA and dynein/dynactin. Cheeseman and colleagues find that spindle-pole-associated Plk1 activity restricts polar dynein whereas chromosomal RanGTP negatively regulates LGN localization at the lateral cell cortex, thus identifying two differentially localized signals that modulate spindle positioning by acting on dynein-mediated forces. Mitotic spindle positioning by cortical pulling forces 1 defines the cell division axis and location 2 , which is critical for proper cell division and development 3 . Although recent work has identified developmental and extrinsic cues that regulate spindle orientation 4 , 5 , 6 , the contribution of intrinsic signals to spindle positioning and orientation remains unclear. Here, we demonstrate that cortical force generation in human cells is controlled by distinct spindle-pole- and chromosome-derived signals that regulate cytoplasmic dynein localization. First, dynein exhibits a dynamic asymmetric cortical localization that is negatively regulated by spindle-pole proximity, resulting in spindle oscillations to centre the spindle within the cell. We find that this signal comprises the spindle-pole-localized polo-like kinase (Plk1), which regulates dynein localization by controlling the interaction between dynein–dynactin and its upstream cortical targeting factors NuMA and LGN. Second, a chromosome-derived RanGTP gradient restricts the localization of NuMA–LGN to the lateral cell cortex to define and maintain the spindle orientation axis. RanGTP acts in part through the nuclear localization sequence of NuMA to locally alter the ability of NuMA–LGN to associate with the cell cortex in the vicinity of chromosomes. We propose that these chromosome- and spindle-pole-derived gradients generate an intrinsic code to control spindle position and orientation.

The lissencephaly 1 gene, LIS1 , is mutated in patients with the neurodevelopmental disease lissencephaly. The Lis1 protein is conserved from fungi to mammals and is a key regulator of cytoplasmic dynein-1, the major minus-end-directed microtubule motor in many eukaryotes. Lis1 is the only dynein regulator known to bind directly to dynein’s motor domain, and by doing so alters dynein’s mechanochemistry. Lis1 is required for the formation of fully active dynein complexes, which also contain essential cofactors: dynactin and an activating adaptor. Here, we report the first high-resolution structure of the yeast dynein–Lis1 complex. Our 3.1 Å structure reveals, in molecular detail, the major contacts between dynein and Lis1 and between Lis1’s ß-propellers. Structure-guided mutations in Lis1 and dynein show that these contacts are required for Lis1’s ability to form fully active human dynein complexes and to regulate yeast dynein’s mechanochemistry and in vivo function.

Impaired cytoplasmic dynein function has been implicated in amyotrophic lateral sclerosis (ALS) pathogenesis, yet the contributions of spinal interneurons to disease phenotypes remain unclear. We tested the hypothesis that hypomorphic dynein function in cholinergic neurons disrupts the development, survival, or positioning of inhibitory interneuron populations in the lumbar spinal cord. Using ChAT-Cre recombination, we generated four mouse genotypes with graded reductions in dynein activity in ChAT + cells: Dync1h1 +/+ (wildtype), Dync1h1 −/+ (hemizygous wildtype), Dync1h1 +/Loa (heterozygous Loa mutation), and Dync1h1 −/Loa (hemizygous Loa). At 52 weeks of age, lumbar spinal cords (L3–L6) were harvested, cryosectioned, and immunostained for ChAT, GAD-67, Parvalbumin, and Calbindin. Cell counts were performed on confocal images from eight sections per mouse (N = 3 male mice/genotype), and radial distances from the central canal were normalised to gray matter width. Angular distributions were analysed via circular statistics. There were no significant genotype-dependent differences in the numbers of ChAT + , GAD-67 + , Parvalbumin + , or Calbindin + cells, nor in ChAT + subpopulations (motor neurons versus interneurons) or double‐positive interneuron subsets (e.g., ChAT + –GAD-67 + , Parvalbumin + –GAD-67 + , Parvalbumin + –Calbindin + ). Radial positioning relative to the central canal was similarly preserved across all markers and genotypes. Circular‐median tests revealed statistically significant shifts in mean angle for ChAT + , GAD-67 + , and certain double‐positive cells, but these amounted to only 5–10° displacements, translating to lateral shifts of ~10–20 µm, well within single laminar bands, and are unlikely to impact circuit connectivity. Despite substantial motor deficits and hallmark TDP-43 pathology previously seen in these models, impaired dynein function does not precipitate interneuron loss or gross migratory defects in the lumbar spinal cord. Instead, our findings suggest that the primary contributions of dynein to ALS-like phenotypes likely arise from functional disruptions in axonal transport, synaptic maintenance, and neuronal physiology rather than from structural alterations or loss of interneuron populations.

Jamel Chelly, Nicholas Cowan and colleagues report mutations in TUBG1 , DYNC1H1 , KIF2A and KIF5C in individuals with malformations of cortical development and microcephaly. Their findings emphasize the importance of centrosomal and microtubule-related proteins for normal brain development. The genetic causes of malformations of cortical development (MCD) remain largely unknown. Here we report the discovery of multiple pathogenic missense mutations in TUBG1, DYNC1H1 and KIF2A , as well as a single germline mosaic mutation in KIF5C , in subjects with MCD. We found a frequent recurrence of mutations in DYNC1H1 , implying that this gene is a major locus for unexplained MCD. We further show that the mutations in KIF5C, KIF2A and DYNC1H1 affect ATP hydrolysis, productive protein folding and microtubule binding, respectively. In addition, we show that suppression of mouse Tubg1 expression in vivo interferes with proper neuronal migration, whereas expression of altered γ-tubulin proteins in Saccharomyces cerevisiae disrupts normal microtubule behavior. Our data reinforce the importance of centrosomal and microtubule-related proteins in cortical development and strongly suggest that microtubule-dependent mitotic and postmitotic processes are major contributors to the pathogenesis of MCD.

Cytoplasmic dynein participates in transport functions and is essential in spermatogenesis. KM23 belongs to the dynein light chain family. The TGFβ signaling pathway is indispensable in spermatogenesis, and Smad2 is an important member of this pathway. We cloned PTKM23 and PTSMAD2 from Portunus trituberculatus and measured their expression during spermatogenesis. PTKM23 may be related to cell division, acrosome formation, and nuclear remodeling, and PTSMAD2 may participate in regulating the expression of genes related to spermatogenesis. We assessed the localization of PTKM23 with PTDHC and α-tubulin, and the results suggested that PTKM23 functions in intracellular transport during spermatogenesis. We knocked down PTKM23 in vivo, and the expression of p53, B-CATAENIN and CYCLIN B decreased significantly, further suggesting a role of PTKM23 in transport and cell division. The localization of PTDIC with α-tubulin and that of PTSMAD2 with PTDHC changed after PTKM23 knockdown. We transfected PTKM23 and PTSMAD2 into HEK-293 T cells and verified their colocalization. These results indicate that PTKM23 is involved in the assembly of cytoplasmic dynein and microtubules during spermatogenesis and that PTKM23 mediates the participation of cytoplasmic dynein in the transport of PTSMAD2 during spermatogenesis. Summary Sentence This study demonstrated that PTKM23 transports PTSMAD2 during spermatogenesis and provides a theoretical molecular biological basis for the breeding of P. trituberculatus. Graphical Abstract

All animal cells use the motor cytoplasmic dynein 1 (dynein) to transport diverse cargo toward microtubule minus ends and to organize and position microtubule arrays such as the mitotic spindle. Cargo-specific adaptors engage with dynein to recruit and activate the motor, but the molecular mechanisms remain incompletely understood. Here, we use structural and dynamic nuclear magnetic resonance (NMR) analysis to demonstrate that the C-terminal region of human dynein light intermediate chain 1 (LIC1) is intrinsically disordered and contains two short conserved segments with helical propensity. NMR titration experiments reveal that the first helical segment (helix 1) constitutes the main interaction site for the adaptors Spindly (SPDL1), bicaudal D homolog 2 (BICD2), and Hook homolog 3 (HOOK3). In vitro binding assays show that helix 1, but not helix 2, is essential in both LIC1 and LIC2 for binding to SPDL1, BICD2, HOOK3, RAB-interacting lysosomal protein (RILP), RAB11 family-interacting protein 3 (RAB11FIP3), ninein (NIN), and trafficking kinesin-binding protein 1 (TRAK1). Helix 1 is sufficient to bind RILP, whereas other adaptors require additional segments preceding helix 1 for efficient binding. Point mutations in the C-terminal helix 1 of Caenorhabditis elegans LIC, introduced by genome editing, severely affect development, locomotion, and life span of the animal and disrupt the distribution and transport kinetics of membrane cargo in axons of mechanosensory neurons, identical to what is observed when the entire LIC C-terminal region is deleted. Deletion of the C-terminal helix 2 delays dynein-dependent spindle positioning in the one-cell embryo but overall does not significantly perturb dynein function. We conclude that helix 1 in the intrinsically disordered region of LIC provides a conserved link between dynein and structurally diverse cargo adaptor families that is critical for dynein function in vivo.

Primary ciliary dyskinesia (PCD) is a rare multi-system cilia-related disorder, and approximately 50% of individuals with PCD exhibit left-right asymmetry disorder. The dynein axonemal heavy chain 11 gene ( DNAH11 ) pathogenic variants are responsible for primary ciliary dyskinesia 7, with or without left-right asymmetry disorder. This study aimed to detect the pathogenic variants in three unrelated patients diagnosed with PCD or left-right asymmetry disorder based on the clinical and imaging examinations. Whole exome sequencing, Sanger sequencing, and comprehensive bioinformatics analyses were performed. Seven DNAH11 heterozygous variants, which involved evolutionarily conserved residues and were predicted to exert deleterious effects, reduce protein stability, change protein conformation, and affect non-covalent residue’s interactions, were identified as potential pathogenic factors responsible for these patients, respectively. In patient 1, three variants in compound heterozygotes, c.[3541A > G];[4334G > A;12428T > C] (p.[(Ser1181Gly)];[(Arg1445Gln;Met4143Thr)]), were confirmed. In patient 2, two variants in potential compound heterozygotes, c.2912A > G(;)7980A > T (p.(Asp971Gly)(;)(Gln2660His)), were detected. In patient 3, two variants in compound heterozygotes, c.[845T > C];[11402C > G] (p.[(Met282Thr)];[(Pro3801Arg)]), were confirmed. The phenotypes observed in these patients are consistent with typical/probably atypical PCD or DNAH11 -associated ciliopathy, although functional validation is needed to confirm variant pathogenicity. These findings expand the phenotypic spectrum of DNAH11 variants and may facilitate more accurate genetic diagnosis and counseling.