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Cell Reports Nov 2023Intraflagellar transport (IFT) trains, built around IFT-A and IFT-B complexes, are carried by opposing motors to import and export ciliary cargo. While transported by...
Intraflagellar transport (IFT) trains, built around IFT-A and IFT-B complexes, are carried by opposing motors to import and export ciliary cargo. While transported by kinesin-2 on anterograde IFT trains, the dynein-2 motor adopts an autoinhibitory conformation until it needs to be activated at the ciliary tip to power retrograde IFT. Growing evidence has linked the IFT-A complex to retrograde IFT; however, its roles in this process remain unknown. Here, we use CRISPR-Cas9-mediated genome editing to disable the dynein-2 autoinhibition mechanism in Caenorhabditis elegans and assess its impact on IFT with high-resolution live imaging and photobleaching analyses. Remarkably, this dynein-2 "hot-wiring" approach reignites retrograde motility inside IFT-A-deficient cilia without triggering tug-of-war events. In addition to providing functional evidence that multiple mechanisms maintain dynein-2 inhibited during anterograde IFT, our data establish key roles for IFT-A in mediating motor-train coupling during IFT turnaround, promoting retrograde IFT initiation, and modulating dynein-2 retrograde motility.
Topics: Animals; Dyneins; Biological Transport; Cilia; Caenorhabditis elegans; Caenorhabditis elegans Proteins; Flagella
PubMed: 37883232
DOI: 10.1016/j.celrep.2023.113337 -
The EMBO Journal Dec 2023The microtubule motor dynein mediates polarised trafficking of a wide variety of organelles, vesicles and macromolecules. These functions are dependent on the dynactin...
The microtubule motor dynein mediates polarised trafficking of a wide variety of organelles, vesicles and macromolecules. These functions are dependent on the dynactin complex, which helps recruit cargoes to dynein's tail and activates motor movement. How the dynein-dynactin complex orchestrates trafficking of diverse cargoes is unclear. Here, we identify HEATR5B, an interactor of the adaptor protein-1 (AP1) clathrin adaptor complex, as a novel player in dynein-dynactin function. HEATR5B was recovered in a biochemical screen for proteins whose association with the dynein tail is augmented by dynactin. We show that HEATR5B binds directly to the dynein tail and dynactin and stimulates motility of AP1-associated endosomal membranes in human cells. We also demonstrate that the Drosophila HEATR5B homologue is an essential gene that selectively promotes dynein-based transport of AP1-bound membranes to the Golgi apparatus. As HEATR5B lacks the coiled-coil architecture typical of dynein adaptors, our data point to a non-canonical process orchestrating motor function on a specific cargo. We additionally show that HEATR5B promotes association of AP1 with endosomal membranes independently of dynein. Thus, HEATR5B co-ordinates multiple events in AP1-based trafficking.
Topics: Humans; Dyneins; Dynactin Complex; Microtubule-Associated Proteins; Biological Transport; Microtubules; Endosomes
PubMed: 37872872
DOI: 10.15252/embj.2023114473 -
PLoS Biology Oct 2023The endoplasmic reticulum (ER) forms contacts with the lysosomal compartment, regulating lysosome positioning and motility. The movements of lysosomes are controlled by...
The endoplasmic reticulum (ER) forms contacts with the lysosomal compartment, regulating lysosome positioning and motility. The movements of lysosomes are controlled by the attachment of molecular motors to their surface. However, the molecular mechanisms by which ER controls lysosome dynamics are still elusive. Here, using mouse brain extracts and mouse embryonic fibroblasts, we demonstrate that spatacsin is an ER-resident protein regulating the formation of tubular lysosomes, which are highly dynamic. Screening for spatacsin partners required for tubular lysosome formation showed spatacsin to act by regulating protein degradation. We demonstrate that spatacsin promotes the degradation of its partner AP5Z1, which regulates the relative amount of spastizin and AP5Z1 at lysosomes. Spastizin and AP5Z1 contribute to regulate tubular lysosome formation, as well as their trafficking by interacting with anterograde and retrograde motor proteins, kinesin KIF13A and dynein/dynactin subunit p150Glued, respectively. Ultimately, investigations in polarized mouse cortical neurons in culture demonstrated that spatacsin-regulated degradation of AP5Z1 controls the directionality of lysosomes trafficking. Collectively, our results identify spatacsin as a protein regulating the directionality of lysosome trafficking.
Topics: Animals; Mice; Dyneins; Fibroblasts; Lysosomes; Neurons; Proteins; Adaptor Proteins, Vesicular Transport
PubMed: 37871017
DOI: 10.1371/journal.pbio.3002337 -
Communications Biology Oct 2023How recently originated gene copies become stable genomic components remains uncertain as high sequence similarity of young duplicates precludes their functional...
How recently originated gene copies become stable genomic components remains uncertain as high sequence similarity of young duplicates precludes their functional characterization. The tandem multigene family Sdic is specific to Drosophila melanogaster and has been annotated across multiple reference-quality genome assemblies. Here we show the existence of a positive correlation between Sdic copy number and total expression, plus vast intrastrain differences in mRNA abundance among paralogs, using RNA-sequencing from testis of four strains with variable paralog composition. Single cell and nucleus RNA-sequencing data expose paralog expression differentiation in meiotic cell types within testis from third instar larva and adults. Additional RNA-sequencing across synthetic strains only differing in their Y chromosomes reveal a tissue-dependent trans-regulatory effect on Sdic: upregulation in testis and downregulation in male accessory gland. By leveraging paralog-specific expression information from tissue- and cell-specific data, our results elucidate the intraspecific functional diversification of a recently expanded tandem gene family.
Topics: Animals; Male; Drosophila melanogaster; Drosophila Proteins; Spermatogenesis; Testis; RNA; Axonemal Dyneins
PubMed: 37864070
DOI: 10.1038/s42003-023-05427-4 -
World Journal of Surgical Oncology Oct 2023Genetic variants of outer dynein arm docking complex subunit 2 (ODAD2) have been reported to be closely associated with primary ciliary dyskinesia and colorectal cancer...
Outer dynein arm docking complex subunit 2 polymorphism rs7893462 modulates hepatocellular carcinoma susceptibility and can serve as an overall survival biomarker for hepatitis B virus-related hepatocellular carcinoma after hepatectomy: a cohort study with a long-term follow-up.
BACKGROUND
Genetic variants of outer dynein arm docking complex subunit 2 (ODAD2) have been reported to be closely associated with primary ciliary dyskinesia and colorectal cancer in previous studies, but the association of genetic variants of ODAD2 with hepatocellular carcinoma (HCC) has not been reported.
METHODS
We enrolled 80 healthy subjects and 468 Guangxi hepatitis B virus (HBV)-related HCC patients in this study. A case-control study method was used to explore the association of different ODAD2-rs7893462 genotypes with hepatocarcinogenesis. A comprehensive survival analysis was used to explore the association of rs7893462 with the prognosis of HBV-related HCC in Guangxi.
RESULTS
Through a case-control study, we observed that patients carrying the G allele of rs7893462 had a markedly increased susceptibility to hepatocarcinogenesis (odds ratio = 1.712, 95% confidence interval = 1.032-2.839, P = 0.037). We found that there were significant prognosis differences among three different genotypes of rs7893462. Nomogram analysis suggested that the contribution of rs7893462 polymorphisms to the prognosis of HBV-related HCC was second only to the BCLC stage. Stratified survival analysis suggested that the AG genotype of rs7893462 was an independent prognostic risk factor for HBV-related HCC. Joint effect survival analysis also observed that the AG genotype of rs7893462 combined with clinical parameters could significantly identify HBV-related HCC patients with different prognostic outcomes more accurately, and the AG genotype was also observed to be independent of clinical factors in HBV-related HCC survival.
CONCLUSION
The ODAD2-rs7893462 polymorphisms can be used as an independent prognostic indicator of HBV-related HCC overall survival and are significantly associated with susceptibility to hepatocarcinogenesis.
Topics: Humans; Carcinoma, Hepatocellular; Hepatitis B virus; Liver Neoplasms; Dyneins; Case-Control Studies; Cohort Studies; Hepatectomy; Follow-Up Studies; Genetic Predisposition to Disease; Polymorphism, Single Nucleotide; China; Genotype; Biomarkers; Hepatitis B
PubMed: 37833735
DOI: 10.1186/s12957-023-03205-4 -
Methods in Molecular Biology (Clifton,... 2024Cytoskeletal motor proteins are essential molecular machines that hydrolyze ATP to generate force and motion along cytoskeletal filaments. Members of the dynein and...
Cytoskeletal motor proteins are essential molecular machines that hydrolyze ATP to generate force and motion along cytoskeletal filaments. Members of the dynein and kinesin superfamilies play critical roles in transporting biological payloads (such as proteins, organelles, and vesicles) along microtubule pathways, cause the beating of flagella and cilia, and act within the mitotic and meiotic spindles to segregate replicated chromosomes to progeny cells. Understanding the underlying mechanisms and behaviors of motor proteins is critical to provide better strategies for the treatment of motor protein-related diseases. Here, we provide detailed protocols for the recombinant expression of the Kinesin-1 motor KIF5C using a baculovirus/insect cell system and provide updated protocols for performing single-molecule studies using total internal reflection fluorescence microscopy and optical tweezers to study the motility and force generation of the purified motor.
Topics: Kinesins; Cytoskeletal Proteins; Microtubules; Spindle Apparatus; Dyneins
PubMed: 37824000
DOI: 10.1007/978-1-0716-3377-9_4 -
Nature Communications Oct 2023YTHDF2 has been extensively studied and typified as an RNA-binding protein that specifically recognizes and destabilizes RNAs harboring N-methyladenosine (mA), the most...
YTHDF2 has been extensively studied and typified as an RNA-binding protein that specifically recognizes and destabilizes RNAs harboring N-methyladenosine (mA), the most prevalent internal modification found in eukaryotic RNAs. In this study, we unravel the mA-independent role of YTHDF2 in the formation of an aggresome, where cytoplasmic protein aggregates are selectively sequestered upon failure of protein homeostasis mediated by the ubiquitin-proteasome system. Downregulation of YTHDF2 in HeLa cells reduces the circularity of aggresomes and the rate of movement of misfolded polypeptides, inhibits aggresome formation, and thereby promotes cellular apoptosis. Mechanistically, YTHDF2 is recruited to a misfolded polypeptide-associated complex composed of UPF1, CTIF, eEF1A1, and DCTN1 through its interaction with UPF1. Subsequently, YTHDF2 increases the interaction between the dynein motor protein and the misfolded polypeptide-associated complex, facilitating the diffusion dynamics of the movement of misfolded polypeptides toward aggresomes. Therefore, our data reveal that YTHDF2 is a cellular factor involved in protein quality control.
Topics: Humans; Cytoplasm; Dyneins; HeLa Cells; Peptides; Protein Folding; RNA Helicases; RNA-Binding Proteins; Trans-Activators; Transcription Factors; Proteolysis; Organelles
PubMed: 37803021
DOI: 10.1038/s41467-023-42015-w -
Development (Cambridge, England) Nov 2023Actin-related proteins (Arps) are classified according to their similarity to actin and are involved in diverse cellular processes. ACTL7B is a testis-specific Arp, and...
Actin-related proteins (Arps) are classified according to their similarity to actin and are involved in diverse cellular processes. ACTL7B is a testis-specific Arp, and is highly conserved in rodents and primates. ACTL7B is specifically expressed in round and elongating spermatids during spermiogenesis. Here, we have generated an Actl7b-null allele in mice to unravel the role of ACTL7B in sperm formation. Male mice homozygous for the Actl7b-null allele (Actl7b-/-) were infertile, whereas heterozygous males (Actl7b+/-) were fertile. Severe spermatid defects, such as detached acrosomes, disrupted membranes and flagella malformations start to appear after spermiogenesis step 9 in Actl7b-/- mice, finally resulting in spermatogenic arrest. Abnormal spermatids were degraded and levels of autophagy markers were increased. Co-immunoprecipitation with mass spectrometry experiments identified an interaction between ACTL7B and the LC8 dynein light chains DYNLL1 and DYNLL2, which are first detected in step 9 spermatids and mislocalized when ACTL7B is absent. Our data unequivocally establish that mutations in ACTL7B are directly related to male infertility, pressing for additional research in humans.
Topics: Animals; Humans; Male; Mice; Actins; Cytoplasmic Dyneins; Dyneins; Semen; Spermatids; Spermatogenesis; Spermatozoa; Testis
PubMed: 37800308
DOI: 10.1242/dev.201593 -
Molecular & Cellular Proteomics : MCP Nov 2023The fragile X messenger ribonucleoprotein 1 (FMRP) is a multifunctional RNA-binding protein implicated in human neurodevelopmental and neurodegenerative disorders. FMRP...
The fragile X messenger ribonucleoprotein 1 (FMRP) is a multifunctional RNA-binding protein implicated in human neurodevelopmental and neurodegenerative disorders. FMRP mediates the localization and activity-dependent translation of its associated mRNAs through the formation of phase-separated condensates that are trafficked by microtubule-based motors in axons. Axonal transport and localized mRNA translation are critical processes for long-term neuronal survival and are closely linked to the pathogenesis of neurological diseases. FMRP dynein-mediated axonal trafficking is still largely unexplored but likely to constitute a key process underlying FMRP spatiotemporal translational regulation. Here, we show that dynein light chain roadblock 1 (Dynlrb1), a subunit of the dynein complex, is a critical regulator of FMRP function. In sensory axons, FMRP associates with endolysosomal organelles, likely through annexin A11, and is retrogradely trafficked by the dynein complex in a Dynlrb1-dependent manner. Moreover, Dynlrb1 silencing induced FMRP granule accumulation and repressed the translation of microtubule-associated protein 1b, one of its primary mRNA targets. Our findings suggest that Dynlrb1 regulates FMRP function through the control of its transport and targeted degradation.
Topics: Humans; Dyneins; Fragile X Mental Retardation Protein; Axons; Sensory Receptor Cells; Microtubules; RNA, Messenger
PubMed: 37739344
DOI: 10.1016/j.mcpro.2023.100653 -
Neuroscience Research Dec 2023Kinesin motor proteins play crucial roles in anterograde transport of cargo vesicles in neurons, moving them along axons from the cell body towards the synaptic region.... (Review)
Review
Kinesin motor proteins play crucial roles in anterograde transport of cargo vesicles in neurons, moving them along axons from the cell body towards the synaptic region. Not only the transport force and velocity of single motor protein, but also the number of kinesin molecules involved in transporting a specific cargo, is pivotal for synapse formation. This collective transport by multiple kinesins ensures stable and efficient cargo transport in neurons. Abnormal increases or decreases in the number of engaged kinesin molecules per cargo could potentially act as biomarkers for neurodegenerative diseases such as Alzheimer's, Parkinson's, amyotrophic lateral sclerosis (ALS), spastic paraplegia, polydactyly syndrome, and virus transport disorders. We review here a model constructed using physical measurements to quantify the number of kinesin molecules associated with their cargo, which could shed light on the molecular mechanisms of neurodegenerative diseases related to axonal transport.
Topics: Humans; Kinesins; Axonal Transport; Axons; Dyneins; Amyotrophic Lateral Sclerosis
PubMed: 37734449
DOI: 10.1016/j.neures.2023.09.004