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Molecular Biology of the Cell Mar 2024Phagocytosis by macrophages is a highly polarized process to destroy large target cells. Binding to particles induces extensive cortical actin-generated forces that...
Phagocytosis by macrophages is a highly polarized process to destroy large target cells. Binding to particles induces extensive cortical actin-generated forces that drive the formation of elaborate pseudopods around the target particle. Postinternalization, the resultant phagosome is driven toward the cell interior on microtubules (MTs) by cytoplasmic dynein. However, it is unclear whether dynein and cargo-adaptors contribute to the earlier steps of particle internalization and phagosome formation. Here we reveal that ninein, a MT minus-end-associated protein that localizes to the centrosome, is also present at the phagocytic cup in macrophages. Ninein depletion impairs particle internalization by delaying the early F-actin recruitment to sites of particle engagement and cup formation, with no impact on F-actin dynamics beyond this initial step. Ninein forms membrane-bound clusters on phagocytic cups that do not nucleate acentrosomal MTs but instead mediate the assembly of dynein-dynactin complex at active phagocytic membranes. Both ninein depletion and pharmacological inhibition of dynein activity reduced inward displacement of bound particles into macrophages. We found that ninein and dynein motor activity were required for timely retrograde movement of phagosomes and for phagolysosome formation. Taken together, these data show that ninein, alone and with dynein, play significant roles during phagocytosis.
Topics: Actins; Carrier Proteins; Dyneins; Macrophages; Phagocytosis; Phagosomes; Humans; Nuclear Proteins; Cytoskeletal Proteins
PubMed: 38117588
DOI: 10.1091/mbc.E23-06-0216 -
Current Biology : CB Dec 2023Dyneins are a family of motor proteins that carry out motility and force generation functions towards the minus end of microtubule filaments. Cytoplasmic dynein...
Dyneins are a family of motor proteins that carry out motility and force generation functions towards the minus end of microtubule filaments. Cytoplasmic dynein (dynein-1) is responsible for transporting intracellular cargos in the retrograde direction in the cytoplasm, anchoring several organelles to the microtubule network, driving nuclear migration in developing neurons, and orienting the mitotic spindle in dividing cells. All other dyneins are localized to cilia. Similar to dynein-1, dynein-2 walks along microtubules and drives intraflagellar transport in the retrograde direction. Other ciliary dyneins are positioned between adjacent microtubule doublets of the axoneme and power ciliary beating by sliding microtubules relative to each other. In this primer, we first highlight the structure, mechanism, and regulation of dynein-1, which is the best-characterized member of the dynein motor family, and then describe the unique features and cellular roles of other dyneins. We also discuss accessory proteins that regulate the activation and motility of dynein motors in different cellular contexts.
Topics: Dyneins; Microtubules; Axoneme; Kinesins; Spindle Apparatus
PubMed: 38113834
DOI: 10.1016/j.cub.2023.10.064 -
Scientific Reports Dec 2023The preservation of liquid semen is pivotal for both industrial livestock production and genetic management/conservation of species with sperm that are not highly...
The preservation of liquid semen is pivotal for both industrial livestock production and genetic management/conservation of species with sperm that are not highly cryo-tolerant. Nevertheless, with regard to poultry semen, even brief in vitro storage periods can lead to a notable decline in fertility, despite the in vivo capacity to maintain fertility for several weeks when within the hen's sperm storage tubules. For fertility in sperm, intracellular calcium ions ([Ca]i) play a key role in signaling towards modifying energy metabolism. While reducing [Ca]i has been found to enhance the preservation of sperm fertility in some mammals, the connection between semen fertility and calcium availability in avian sperm has received limited attention. In this study, we demonstrate that the use of extracellular and intracellular calcium chelators in liquid semen extenders, specifically EGTA and EGTA-AM, has distinct effects on prolonging the fertility of chicken sperm. These results were validated through in vivo fertility tests. Mechanistically, the effects observed were linked to coordination of mitochondrial metabolism and ATP catabolism. Despite both calcium chelators inducing hypoxia, they differentially regulated mitochondrial respiration and ATP accumulation. This regulation was closely linked to a bimodal control of dynein ATPase activity; a direct initial activation with reduction in [Ca]i, and subsequent suppression by cytoplasmic acidification caused by lactic acid. These findings not only contribute to advancing poultry liquid semen preservation techniques, but also elucidates biologically relevant mechanisms that may underlie storage within the female reproductive tract in birds.
Topics: Female; Animals; Male; Semen; Calcium; Poultry; Chickens; Calcium Chelating Agents; Sperm Motility; Spermatozoa; Calcium, Dietary; Fertility; Adenosine Triphosphate; Mammals
PubMed: 38066036
DOI: 10.1038/s41598-023-48550-2 -
Genome Biology Dec 2023Identifying host factors is key to understanding RNA virus pathogenicity. Besides proteins, RNAs can interact with virus genomes to impact replication.
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.
Topics: Humans; Zika Virus; Zika Virus Infection; RNA, Viral; 3' Untranslated Regions; Dengue Virus; Virus Replication; Dengue; Antiviral Agents; Dyneins
PubMed: 38053173
DOI: 10.1186/s13059-023-03110-9 -
Frontiers in Immunology 2023Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related deaths worldwide and has a poor prognosis. Thus, there is a need for an effective biomarker...
BACKGROUND
Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related deaths worldwide and has a poor prognosis. Thus, there is a need for an effective biomarker to improve and predict the prognosis of HCC.
METHODS
RNA sequencing data, simple nucleotide variation data, and clinical data of HCC patients from The Cancer Genome Atlas (TCGA) to identify mutant genes, simple nucleotide variation data, and clinical data of HCC patients from the International Cancer Genome Consortium (ICGC) to validate the prognostic value of mutant genes were the data sources of the present study. To identify the overall survival (OS)-related mutant genes, a Kaplan-Meier (KM) analysis was conducted. We carried out univariate Cox and multivariate Cox regression analyses to identify the independent prognostic factors. We also conducted a correlation analysis of immune cells and mutant genes. To explore the molecular mechanisms of mutant genes, we conducted a gene set enrichment analysis (GSEA). A nomogram was constructed to help predict the prognosis of HCC. In addition, we explored the expression profile of mutant genes in HCC based on a TCGA dataset, an ICGC dataset, and our own HCC tissue samples.
RESULTS
We identified and validated a mutant gene, dynein axonemal heavy chain 5 (), which was negatively related to the OS of HCC patients. Univariate Cox and multivariate Cox regression analyses revealed that the mutant gene could act as an independent prognostic factor for HCC. Most pathways of the mutant gene were involved in cancer development and progression based on GSEA analysis. The mutant gene was negatively correlated with monocytes, naive CD4 T cells, activated dendritic cells, and activated mast cells. In addition, the mRNA and protein levels of had a significantly higher level of expression in the tissue samples of patients with HCC. A nomogram consisting of the pathological stage, , and tumor mutation burden (TMB) performed well.
CONCLUSION
The mutant gene had a significantly higher level of expression in the tissue samples of patients with HCC, could act as an independent prognostic factor for HCC, and is a potential new immunotherapy target for HCC.
Topics: Humans; Carcinoma, Hepatocellular; Prognosis; Liver Neoplasms; Nomograms; Nucleotides; Axonemal Dyneins
PubMed: 38022557
DOI: 10.3389/fimmu.2023.1236995 -
Molecular Biology of the Cell Jan 2024Chromosome segregation relies on the correct assembly of a bipolar spindle. Spindle pole self-organization requires dynein-dependent microtubule (MT) transport along...
Chromosome segregation relies on the correct assembly of a bipolar spindle. Spindle pole self-organization requires dynein-dependent microtubule (MT) transport along other MTs. However, during M-phase RanGTP triggers MT nucleation and branching generating polarized arrays with nonastral organization in which MT minus ends are linked to the sides of other MTs. This raises the question of how branched-MT nucleation and dynein-mediated transport cooperate to organize the spindle poles. Here, we used RanGTP-dependent MT aster formation in () egg extract to study the interplay between these two seemingly conflicting organizing principles. Using temporally controlled perturbations of MT nucleation and dynein activity, we found that branched MTs are not static but instead dynamically redistribute over time as poles self-organize. Our experimental data together with computer simulations suggest a model where dynein together with dynactin and NuMA directly pulls and move branched MT minus ends toward other MT minus ends.
Topics: Animals; Dyneins; Xenopus laevis; Spindle Apparatus; Microtubules; Dynactin Complex; Microtubule-Associated Proteins; Xenopus Proteins
PubMed: 37991893
DOI: 10.1091/mbc.E23-10-0407 -
Nature Communications Nov 2023Intracellular vesicular transport along cytoskeletal filaments ensures targeted cargo delivery. Such transport is rarely unidirectional but rather bidirectional, with...
Intracellular vesicular transport along cytoskeletal filaments ensures targeted cargo delivery. Such transport is rarely unidirectional but rather bidirectional, with frequent directional reversals owing to the simultaneous presence of opposite-polarity motors. So far, it has been unclear whether such complex motility pattern results from the sole mechanical interplay between opposite-polarity motors or requires regulators. Here, we demonstrate that a minimal system, comprising purified Dynein-Dynactin-BICD2 (DDB) and kinesin-3 (KIF16B) attached to large unilamellar vesicles, faithfully reproduces in vivo cargo motility, including runs, pauses, and reversals. Remarkably, opposing motors do not affect vesicle velocity during runs. Our computational model reveals that the engagement of a small number of motors is pivotal for transitioning between runs and pauses. Taken together, our results suggest that motors bound to vesicular cargo transiently engage in a tug-of-war during pauses. Subsequently, stochastic motor attachment and detachment events can lead to directional reversals without the need for regulators.
Topics: Dyneins; Kinesins; Biological Transport; Cytoskeleton; Dynactin Complex; Microtubules
PubMed: 37985763
DOI: 10.1038/s41467-023-42605-8 -
The EMBO Journal Dec 2023Chromosome biorientation on the mitotic spindle is prerequisite to errorless genome inheritance. CENP-E (kinesin-7) and dynein-dynactin (DD), microtubule motors with...
Chromosome biorientation on the mitotic spindle is prerequisite to errorless genome inheritance. CENP-E (kinesin-7) and dynein-dynactin (DD), microtubule motors with opposite polarity, promote biorientation from the kinetochore corona, a polymeric structure whose assembly requires MPS1 kinase. The corona's building block consists of ROD, Zwilch, ZW10, and the DD adaptor Spindly (RZZS). How CENP-E and DD are scaffolded and mutually coordinated in the corona remains unclear. Here, we show that when corona assembly is prevented through MPS1 inhibition, CENP-E is absolutely required to retain RZZS at kinetochores. An RZZS phosphomimetic mutant bypasses this requirement, demonstrating the existence of a second receptor for polymeric RZZS. With active MPS1, CENP-E is dispensable for corona expansion, but strictly required for physiological kinetochore accumulation of DD. Thus, we identify the corona as an integrated scaffold where CENP-E kinesin controls DD kinetochore loading for coordinated bidirectional transport of chromosome cargo.
Topics: Dyneins; Kinetochores; Kinesins; Cell Cycle Proteins; Spindle Apparatus; Microtubules; Dynactin Complex; Mitosis; Chromosome Segregation
PubMed: 37984321
DOI: 10.15252/embj.2023114838 -
Biochemical and Biophysical Research... Dec 2023Phagosomes are dynamic organelles formed by macrophages to capture and destroy microbial pathogens. Phagosome transport from the cell periphery to the perinuclear...
Phagosomes are dynamic organelles formed by macrophages to capture and destroy microbial pathogens. Phagosome transport from the cell periphery to the perinuclear region, is essential for fusion with lysosomes and the elimination of pathogens. Molecular motors, kinesin and dynein, generate opposing forces, transporting the phagosome away from and towards the lysosome, respectively. Luminal acidification plays a crucial role in determining the net directional movement of the phagosome. The mechanics of this regulation are not known. In this study, we used the sodium proton exchanger NHE9 to selectively modulate phagosomal acidification in macrophages. We then investigated its impact on the mechanical properties of kinesin and dynein motors through optical trapping experiments. We observed a negative correlation between the tenacity of dynein motors and pH under high resistive forces. Reduced luminal acidification impaired generation of dynein cooperative forces, which are crucial for transporting the phagosome to the lysosome. Conversely, the kinesin-powered motility of phagosomes is enabled by a decrease in phagosomal acidification. Given the various methods pathogens employ to limit phagosomal acidification, our findings are highly significant in the context of host-pathogen interactions.
Topics: Dyneins; Kinesins; Phagosomes; Lysosomes; Hydrogen-Ion Concentration
PubMed: 37979328
DOI: 10.1016/j.bbrc.2023.149236 -
Clinical Genetics Mar 2024Sperm flagella share an evolutionary conserved microtubule-based structure with motile cilia expressed at the surface of several cell types, such as the airways...
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.
Topics: Humans; Male; Sperm Tail; Axoneme; Seeds; Microtubule-Associated Proteins; Mutation; Dyneins; Infertility, Male; Glycoproteins
PubMed: 37975235
DOI: 10.1111/cge.14459