Molecular mechanism of dynein-dynactin complex assembly by LIS1.

Cytoplasmic dynein is a microtubule motor vital for cellular organization and division. It functions as a ~4-megadalton complex containing its cofactor dynactin and a cargo-specific coiled-coil adaptor. However, how dynein and dynactin recognize diverse adaptors, how they interact with each other during complex formation, and the role of critical regulators such as lissencephaly-1 (LIS1) protein (LIS1) remain unclear. In this study, we determined the cryo-electron microscopy structure of dynein-dynactin on microtubules with LIS1 and the lysosomal adaptor JIP3. This structure reveals the molecular basis of interactions occurring during dynein activation. We show how JIP3 activates dynein despite its atypical architecture. Unexpectedly, LIS1 binds dynactin's p150 subunit, tethering it along the length of dynein. Our data suggest that LIS1 and p150 constrain dynein-dynactin to ensure efficient complex formation.

NUDC is critical for rod photoreceptor function, maintenance, and survival.

NUDC (nuclear distribution protein C) is a mitotic protein involved in nuclear migration and cytokinesis across species. Considered a cytoplasmic dynein (henceforth dynein) cofactor, NUDC was shown to associate with the dynein motor complex during neuronal migration. NUDC is also expressed in postmitotic vertebrate rod photoreceptors where its function is unknown. Here, we examined the role of NUDC in postmitotic rod photoreceptors by studying the consequences of a conditional NUDC knockout in mouse rods (rNudC-/- ). Loss of NUDC in rods led to complete photoreceptor cell death at 6 weeks of age. By 3 weeks of age, rNudC-/- function was diminished, and rhodopsin and mitochondria were mislocalized, consistent with dynein inhibition. Levels of outer segment proteins were reduced, but LIS1 (lissencephaly protein 1), a well-characterized dynein cofactor, was unaffected. Transmission electron microscopy revealed ultrastructural defects within the rods of rNudC-/- by 3 weeks of age. We investigated whether NUDC interacts with the actin modulator cofilin 1 (CFL1) and found that in rods, CFL1 is localized in close proximity to NUDC. In addition to its potential role in dynein trafficking within rods, loss of NUDC also resulted in increased levels of phosphorylated CFL1 (pCFL1), which would purportedly prevent depolymerization of actin. The absence of NUDC also induced an inflammatory response in Müller glia and microglia across the neural retina by 3 weeks of age. Taken together, our data illustrate the critical role of NUDC in actin cytoskeletal maintenance and dynein-mediated protein trafficking in a postmitotic rod photoreceptor.

Genome-scale requirements for dynein-based transport revealed by a high-content arrayed CRISPR screen.

The microtubule motor dynein plays a key role in cellular organization. However, little is known about how dynein's biosynthesis, assembly, and functional diversity are orchestrated. To address this issue, we have conducted an arrayed CRISPR loss-of-function screen in human cells using the distribution of dynein-tethered peroxisomes and early endosomes as readouts. From a genome-wide gRNA library, 195 validated hits were recovered and parsed into those impacting multiple dynein cargoes and those whose effects are restricted to a subset of cargoes. Clustering of high-dimensional phenotypic fingerprints revealed co-functional proteins involved in many cellular processes, including several candidate novel regulators of core dynein functions. Further analysis of one of these factors, the RNA-binding protein SUGP1, indicates that it promotes cargo trafficking by sustaining functional expression of the dynein activator LIS1. Our data represent a rich source of new hypotheses for investigating microtubule-based transport, as well as several other aspects of cellular organization captured by our high-content imaging.

Gene dosage of independent dynein arm motor preassembly factors influences cilia assembly in Chlamydomonas reinhardtii.

Motile cilia assembly utilizes over 800 structural and cytoplasmic proteins. Variants in approximately 58 genes cause primary ciliary dyskinesia (PCD) in humans, including the dynein arm (pre)assembly factor (DNAAF) gene DNAAF4. In humans, outer dynein arms (ODAs) and inner dynein arms (IDAs) fail to assemble motile cilia when DNAAF4 function is disrupted. In Chlamydomonas reinhardtii, a ciliated unicellular alga, the DNAAF4 ortholog is called PF23. The pf23-1 mutant assembles short cilia and lacks IDAs, but partially retains ODAs. The cilia of a new null allele (pf23-4) completely lack ODAs and IDAs and are even shorter than cilia from pf23-1. In addition, PF23 plays a role in the cytoplasmic modification of IC138, a protein of the two-headed IDA (I1/f). As most PCD variants in humans are recessive, we sought to test if heterozygosity at two genes affects ciliary function using a second-site non-complementation (SSNC) screening approach. We asked if phenotypes were observed in diploids with pairwise heterozygous combinations of 21 well-characterized ciliary mutant Chlamydomonas strains. Vegetative cultures of single and double heterozygous diploid cells did not show SSNC for motility phenotypes. When protein synthesis is inhibited, wild-type Chlamydomonas cells utilize the pool of cytoplasmic proteins to assemble half-length cilia. In this sensitized assay, 8 double heterozygous diploids with pf23 and other DNAAF mutations show SSNC; they assemble shorter cilia than wild-type. In contrast, double heterozygosity of the other 203 strains showed no effect on ciliary assembly. Immunoblots of diploids heterozygous for pf23 and wdr92 or oda8 show that PF23 is reduced by half in these strains, and that PF23 dosage affects phenotype severity. Reductions in PF23 and another DNAAF in diploids affect the ability to assemble ODAs and IDAs and impedes ciliary assembly. Thus, dosage of multiple DNAAFs is an important factor in cilia assembly and regeneration.

Dynein Light Intermediate Chains Exhibit Different Arginine Methylation Patterns.

BACKGROUND: The motor protein dynein is integral to retrograde transport along microtubules and interacts with numerous cargoes through the recruitment of cargo-specific adaptor proteins. This interaction is mediated by dynein light intermediate chain subunits LIC1 (DYNC1LI1) and LIC2 (DYNC1LI2), which govern the adaptor binding and are present in distinct dynein complexes with overlapping and unique functions. METHODS: Using bioinformatics, we analyzed the C-terminal domains (CTDs) of LIC1 and LIC2, revealing similar structural features but diverse post-translational modifications (PTMs). The methylation status of LIC2 and the proteins involved in this modification were examined through immunoprecipitation and immunoblotting analyses. The specific methylation sites on LIC2 were identified through a site-directed mutagenesis analysis, contributing to a deeper understanding of the regulatory mechanisms of the dynein complex. RESULTS: We found that LIC2 is specifically methylated at the arginine 397 residue, a reaction that is catalyzed by protein arginine methyltransferase 1 (PRMT1). CONCLUSIONS: The distinct PTMs of the LIC subunits offer a versatile mechanism for dynein to transport diverse cargoes efficiently. Understanding how these PTMs influence the functions of LIC2, and how they differ from LIC1, is crucial for elucidating the role of dynein-related transport pathways in a range of diseases. The discovery of the arginine 397 methylation site on LIC2 enhances our insight into the regulatory PTMs of dynein functions.

Roles for CEP170 in cilia function and dynein-2 assembly.

Primary cilia are essential eukaryotic organelles required for signalling and secretion. Dynein-2 is a microtubule-motor protein complex and is required for ciliogenesis via its role in facilitating retrograde intraflagellar transport (IFT) from the cilia tip to the cell body. Dynein-2 must be assembled and loaded onto IFT trains for entry into cilia for this process to occur, but how dynein-2 is assembled and how it is recycled back into a cilium remain poorly understood. Here, we identify centrosomal protein of 170 kDa (CEP170) as a dynein-2-interacting protein in mammalian cells. We show that loss of CEP170 perturbs intraflagellar transport and hedgehog signalling, and alters the stability of dynein-2 holoenzyme complex. Together, our data indicate a role for CEP170 in supporting cilia function and dynein-2 assembly.

Skewed X-chromosome inactivation drives the proportion of DNAAF6-defective airway motile cilia and variable expressivity in primary ciliary dyskinesia.

BACKGROUND: Primary ciliary dyskinesia (PCD) is a rare airway disorder caused by defective motile cilia. Only male patients have been reported with pathogenic mutations in X-linked DNAAF6, which result in the absence of ciliary dynein arms, whereas their heterozygous mothers are supposedly healthy. Our objective was to assess the possible clinical and ciliary consequences of X-chromosome inactivation (XCI) in these mothers. METHODS: XCI patterns of six mothers of male patients with DNAAF6-related PCD were determined by DNA-methylation studies and compared with their clinical phenotype (6/6 mothers), as well as their ciliary phenotype (4/6 mothers), as assessed by immunofluorescence and high-speed videomicroscopy analyses. The mutated X chromosome was tracked to assess the percentage of cells with a normal inactivated DNAAF6 allele. RESULTS: The mothers' phenotypes ranged from absence of symptoms to mild/moderate or severe airway phenotypes, closely reflecting their XCI pattern. Analyses of the symptomatic mothers' airway ciliated cells revealed the coexistence of normal cells and cells with immotile cilia lacking dynein arms, whose ratio closely mirrored their XCI pattern. CONCLUSION: This study highlights the importance of searching for heterozygous pathogenic DNAAF6 mutations in all female relatives of male PCD patients with a DNAAF6 defect, as well as in females consulting for mild chronic respiratory symptoms. Our results also demonstrate that about one-third-ranging from 20% to 50%-normal ciliated airway cells sufficed to avoid severe PCD, a result paving the way for gene therapy.

Dynein and dynactin move long-range but are delivered separately to the axon tip.

Axonal transport is essential for neuronal survival. This is driven by microtubule motors including dynein, which transports cargo from the axon tip back to the cell body. This function requires its cofactor dynactin and regulators LIS1 and NDEL1. Due to difficulties imaging dynein at a single-molecule level, it is unclear how this motor and its regulators coordinate transport along the length of the axon. Here, we use a neuron-inducible human stem cell line (NGN2-OPTi-OX) to endogenously tag dynein components and visualize them at a near-single molecule regime. In the retrograde direction, we find that dynein and dynactin can move the entire length of the axon (>500 µm). Furthermore, LIS1 and NDEL1 also undergo long-distance movement, despite being mainly implicated with the initiation of dynein transport. Intriguingly, in the anterograde direction, dynein/LIS1 moves faster than dynactin/NDEL1, consistent with transport on different cargos. Therefore, neurons ensure efficient transport by holding dynein/dynactin on cargos over long distances but keeping them separate until required.

Novel lissencephaly-associated NDEL1 variant reveals distinct roles of NDE1 and NDEL1 in nucleokinesis and human cortical malformations.

The development of the cerebral cortex involves a series of dynamic events, including cell proliferation and migration, which rely on the motor protein dynein and its regulators NDE1 and NDEL1. While the loss of function in NDE1 leads to microcephaly-related malformations of cortical development (MCDs), NDEL1 variants have not been detected in MCD patients. Here, we identified two patients with pachygyria, with or without subcortical band heterotopia (SBH), carrying the same de novo somatic mosaic NDEL1 variant, p.Arg105Pro (p.R105P). Through single-cell RNA sequencing and spatial transcriptomic analysis, we observed complementary expression of Nde1/NDE1 and Ndel1/NDEL1 in neural progenitors and post-mitotic neurons, respectively. Ndel1 knockdown by in utero electroporation resulted in impaired neuronal migration, a phenotype that could not be rescued by p.R105P. Remarkably, p.R105P expression alone strongly disrupted neuronal migration, increased the length of the leading process, and impaired nucleus-centrosome coupling, suggesting a failure in nucleokinesis. Mechanistically, p.R105P disrupted NDEL1 binding to the dynein regulator LIS1. This study identifies the first lissencephaly-associated NDEL1 variant and sheds light on the distinct roles of NDE1 and NDEL1 in nucleokinesis and MCD pathogenesis.

Dynein directs prophase centrosome migration to control the stem cell division axis in the developing Caenorhabditis elegans epidermis.

The microtubule motor dynein is critical for the assembly and positioning of mitotic spindles. In Caenorhabditis elegans, these dynein functions have been extensively studied in the early embryo but remain poorly explored in other developmental contexts. Here, we use a hypomorphic dynein mutant to investigate the motor's contribution to asymmetric stem cell-like divisions in the larval epidermis. Live imaging of seam cell divisions that precede formation of the seam syncytium shows that mutant cells properly assemble but frequently misorient their spindle. Misoriented divisions misplace daughter cells from the seam cell row, generate anucleate compartments due to aberrant cytokinesis, and disrupt asymmetric cell fate inheritance. Consequently, the seam becomes disorganized and populated with extra cells that have lost seam identity, leading to fatal epidermal rupture. We show that dynein orients the spindle through the cortical GOA-1Gα-LIN-5NuMA pathway by directing the migration of prophase centrosomes along the anterior-posterior axis. Spindle misorientation in the dynein mutant can be partially rescued by elongating cells, implying that dynein-dependent force generation and cell shape jointly promote correct asymmetric division of epithelial stem cells.

Role of BicDR in bristle shaft construction and support of BicD functions.

Cell polarization requires asymmetric localization of numerous mRNAs, proteins and organelles. The movement of cargo towards the minus end of microtubules mostly depends on cytoplasmic dynein motors. In the dynein-dynactin-Bicaudal-D transport machinery, Bicaudal-D (BicD) links the cargo to the motor. Here, we focus on the role of Drosophila BicD-related (BicDR, CG32137) in the development of the long bristles. Together with BicD, it contributes to the organization and stability of the actin cytoskeleton in the not-yet-chitinized bristle shaft. BicD and BicDR also support the stable expression and distribution of Rab6 and Spn-F in the bristle shaft, including the distal tip localization of Spn-F, pointing to the role of microtubule-dependent vesicle trafficking for bristle construction. BicDR supports the function of BicD, and we discuss the hypothesis whereby BicDR might transport cargo more locally, with BicD transporting cargo over long distances, such as to the distal tip. We also identified embryonic proteins that interact with BicDR and appear to be BicDR cargo. For one of them, EF1γ (also known as eEF1γ), we show that the encoding gene EF1γ interacts with BicD and BicDR in the construction of the bristles.

Multimerization of a disordered kinetochore protein promotes accurate chromosome segregation by localizing a core dynein module.

Kinetochores connect chromosomes and spindle microtubules to maintain genomic integrity through cell division. Crosstalk between the minus-end directed motor dynein and kinetochore-microtubule attachment factors promotes accurate chromosome segregation by a poorly understood pathway. Here, we identify a linkage between the intrinsically disordered protein Spc105 (KNL1 orthologue) and dynein using an optogenetic oligomerization assay. Core pools of the checkpoint protein BubR1 and the adaptor complex RZZ contribute to the linkage. Furthermore, a minimal segment of Spc105 with a propensity to multimerize and which contains protein binding motifs is sufficient to link Spc105 to RZZ/dynein. Deletion of the minimal region from Spc105 compromises the recruitment of its binding partners to kinetochores and elevates chromosome missegregation due to merotelic attachments. Restoration of normal chromosome segregation and localization of BubR1 and RZZ requires both protein binding motifs and oligomerization of Spc105. Together, our results reveal that higher-order multimerization of Spc105 contributes to localizing a core pool of RZZ that promotes accurate chromosome segregation.

A splice donor variant of GAS8 induces structural disorganization of the axoneme in sperm flagella and leads to nonsyndromic male infertility.

Motile cilia and flagella are closely related organelles structured around a highly conserved axoneme whose formation and maintenance involve proteins from hundreds of genes. Defects in many of these genes have been described to induce primary ciliary dyskinesia (PCD) mainly characterized by chronic respiratory infections, situs inversus and/or infertility. In men, cilia/flagella-related infertility is usually caused by asthenozoospermia due to multiple morphological abnormalities of the sperm flagella (MMAF). Here, we investigated a cohort of 196 infertile men displaying a typical MMAF phenotype without any other PCD symptoms. Analysis of WES data identified a single case carrying a deleterious homozygous GAS8 variant altering a splice donor consensus site. This gene, also known as DRC4, encodes a subunit of the Nexin-Dynein Regulatory Complex (N-DRC), and has been already associated to male infertility and mild PCD. Confirming the deleterious effect of the candidate variant, GAS8 staining by immunofluorescence did not evidence any signal from the patient's spermatozoa whereas a strong signal was present along the whole flagella length in control cells. Concordant with its role in the N-DRC, transmission electron microscopy evidenced peripheral microtubule doublets misalignments. We confirm here the importance of GAS8 in the N-DRC and observed that its absence induces a typical MMAF phenotype not necessarily accompanied by other PCD symptoms.

Overexpression of JNK-associated leucine zipper protein induces chromosomal instability through interaction with dynein light intermediate chain 1.

The c-Jun N-terminal kinase-associated leucine zipper protein (JLP), a scaffold protein of mitogen-activated protein kinase signaling pathways, is a multifunctional protein involved in a variety of cellular processes. It has been reported that JLP is overexpressed in various types of cancer and is expected to be a potential therapeutic target. However, whether and how JLP overexpression affects non-transformed cells remain unknown. Here, we aimed to investigate the effect of JLP overexpression on chromosomal stability in human non-transformed cells and the mechanisms involved. We found that aneuploidy was induced in JLP-overexpressed cells. Moreover, we established JLP-inducible cell lines and observed an increased frequency of chromosome missegregation, reduced time from nuclear envelope breakdown to anaphase onset, and decreased levels of the spindle assembly checkpoint (SAC) components at the prometaphase kinetochore in cells overexpressing the wild-type JLP. In contrast, we observed that a point mutant JLP lacking the ability to interact with dynein light intermediate chain 1 (DLIC1) failed to induce chromosomal instability. Our results suggest that overexpression of the wild-type JLP facilitates premature SAC silencing through interaction with DLIC1, leading to aneuploidy. This study provides a novel insight into the mechanism through which JLP overexpression is associated with cancer development and progression.

CCDC65, encoding a component of the axonemal Nexin-Dynein regulatory complex, is required for sperm flagellum structure in humans.

Sperm flagella share an evolutionary conserved microtubule-based structure with motile cilia expressed at the surface of several cell types, such as the airways epithelial cells. As a result, male infertility can be observed as an isolated condition or a syndromic trait, illustrated by Primary Cilia Dyskinesia (PCD). We report two unrelated patients showing multiple morphological abnormalities of the sperm flagella (MMAF) and carrying distinct homozygous truncating variants in the PCD-associated gene CCDC65. We characterized one of the identified variants (c.1208del; p.Asn403Ilefs*9), which induces the near absence of CCDC65 protein in patient sperm. In Chlamydomonas, CCDC65 ortholog (DRC2, FAP250) is a component of the Nexin-Dynein Regulatory complex (N-DRC), which interconnects microtubule doublets and coordinates dynein arms activity. In sperm cells from the patient, we also show the loss of GAS8, another component of the N-DRC, supporting a structural/functional link between the two proteins. Our work indicates that, similarly to ciliary axoneme, CCDC65 is required for sperm flagellum structure. Importantly, our work provides first evidence that mutations in the PCD-associated gene CCDC65 also cause asthenozoospermia.

Novel biallelic variants in DNAH1 cause multiple morphological abnormalities of sperm flagella with favorable outcomes of fertility after ICSI in Han Chinese males.

BACKGROUND: Multiple morphological abnormalities of sperm flagella is an idiopathic asthenoteratozoospermia characterized by absent, short, coiled, angulation, and irregular-caliber flagella. Genetic variants of DNAH1 gene have been identified as a causative factor of multiple morphological abnormalities of sperm flagella and intracytoplasmic sperm injection is an available strategy for infertile males with dynein axonemal heavy chain 1 defects to conceive. OBJECTIVES: To identify novel variants and candidate mutant hotspots of DNAH1 gene related to multiple morphological abnormalities of sperm flagella and male infertility in humans. MATERIALS AND METHODS: The DNAH1 variants were identified by whole exome sequencing and confirmed with Sanger sequencing. Papanicolaou staining, scanning and transmission electron microscopy, and immunostaining were performed to investigate the morphological and ultrastructural characteristics of spermatozoa. Intracytoplasmic sperm injection was applied for the assisted reproductive therapy of males harboring biallelic DNAH1 variants. RESULTS: We identified 18 different DNAH1 variants in 11 unrelated families, including nine missense variants (p.A2564T, p.T3657R, p.G1862R, p.L2296P, p.T4041I, p.L611P, p.A913D, p.R1932Q, p.R2356W) and nine loss-of-function variants (c.2301-1G>T, p.Q1518*, p.R1702*, p.D2845Mfs*2, p.P3909Rfs*33, p.Q4040Dfs*33, p.Q4058*, p.E4060Pfs*61, p.V4071Cfs*54). A total of 66.7% (12/18) of the identified variants were novel. Morphological analysis based on Papanicolaou staining and scanning electron microscopy demonstrated the typical multiple morphological abnormalities of sperm flagella characteristics of dynein axonemal heavy chain 1-deficient spermatozoa. Immunostaining further revealed the absence of inner dynein arms but not outer dynein arms, which induced a general ultrastructural disorganization, such as the loss of central pair and mis-localization of the microtubule doublets and outer dense fibers. To date, seven affected couples have accepted the intracytoplasmic sperm injection treatment, and three of them have given birth to five healthy babies. DISCUSSION AND CONCLUSION: These findings further expand the variant spectrum of DNAH1 gene related to multiple morphological abnormalities of sperm flagella and male infertility in humans, thus providing new information for the molecular diagnosis of asthenoteratozoospermia. The favorable fertility outcomes of intracytoplasmic sperm injection will facilitate the genetic counseling and clinical treatment of infertile males with multiple morphological abnormalities of sperm flagella in the future.

Enucleation of the C. elegans embryo revealed dynein-dependent spacing between microtubule asters.

The intracellular positioning of the centrosome, a major microtubule-organizing center, is important for cellular functions. One of the features of centrosome positioning is the spacing between centrosomes; however, the underlying mechanisms are not fully understood. To characterize the spacing activity in Caenorhabditis elegans embryos, a genetic setup was developed to produce enucleated embryos. The centrosome was duplicated multiple times in the enucleated embryo, which enabled us to characterize the chromosome-independent spacing activity between sister and non-sister centrosome pairs. We found that the timely spacing depended on cytoplasmic dynein, and we propose a stoichiometric model of cortical and cytoplasmic pulling forces for the spacing between centrosomes. We also observed dynein-independent but non-muscle myosin II-dependent movement of centrosomes in the later cell cycle phase. The spacing mechanisms revealed in this study are expected to function between centrosomes in general, regardless of the presence of a chromosome/nucleus between them, including centrosome separation and spindle elongation.

Dengue and Zika RNA-RNA interactomes reveal pro- and anti-viral RNA in human cells.

BACKGROUND: Identifying host factors is key to understanding RNA virus pathogenicity. Besides proteins, RNAs can interact with virus genomes to impact replication. RESULTS: Here, we use proximity ligation sequencing to identify virus-host RNA interactions for four strains of Zika virus (ZIKV) and one strain of dengue virus (DENV-1) in human cells. We find hundreds of coding and non-coding RNAs that bind to DENV and ZIKV viruses. Host RNAs tend to bind to single-stranded regions along the virus genomes according to hybridization energetics. Compared to SARS-CoV-2 interactors, ZIKV-interacting host RNAs tend to be downregulated upon virus infection. Knockdown of several short non-coding RNAs, including miR19a-3p, and 7SK RNA results in a decrease in viral replication, suggesting that they act as virus-permissive factors. In addition, the 3'UTR of DYNLT1 mRNA acts as a virus-restrictive factor by binding to the conserved dumbbell region on DENV and ZIKV 3'UTR to decrease virus replication. We also identify a conserved set of host RNAs that interacts with DENV, ZIKV, and SARS-CoV-2, suggesting that these RNAs are broadly important for RNA virus infection. CONCLUSIONS: This study demonstrates that host RNAs can impact virus replication in permissive and restrictive ways, expanding our understanding of host factors and RNA-based gene regulation during viral pathogenesis.

Axonal transport of autophagosomes is regulated by dynein activators JIP3/JIP4 and ARF/RAB GTPases.

Neuronal autophagosomes form and engulf cargos at presynaptic sites in the axon and are then transported to the soma to recycle their cargo. Autophagic vacuoles (AVs) mature en route via fusion with lysosomes to become degradatively competent organelles; transport is driven by the microtubule motor protein cytoplasmic dynein, with motor activity regulated by a sequential series of adaptors. Using lysate-based single-molecule motility assays and live-cell imaging in primary neurons, we show that JNK-interacting proteins 3 (JIP3) and 4 (JIP4) are activating adaptors for dynein that are regulated on autophagosomes and lysosomes by the small GTPases ARF6 and RAB10. GTP-bound ARF6 promotes formation of the JIP3/4-dynein-dynactin complex. Either knockdown or overexpression of RAB10 stalls transport, suggesting that this GTPase is also required to coordinate the opposing activities of bound dynein and kinesin motors. These findings highlight the complex coordination of motor regulation during organelle transport in neurons.