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Journal of Cell Science Apr 2024Primary cilia are essential eukaryotic organelles required for signalling and secretion. Dynein-2 is a microtubule-motor protein complex and is required for ciliogenesis...
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.
Topics: Cilia; Humans; Microtubule-Associated Proteins; Animals; Dyneins; Hedgehog Proteins; Signal Transduction; Mice; Flagella
PubMed: 38533689
DOI: 10.1242/jcs.261816 -
ELife Nov 2023Male infertility is common and complex, presenting a wide range of heterogeneous phenotypes. Although about 50% of cases are estimated to have a genetic component, the...
Male infertility is common and complex, presenting a wide range of heterogeneous phenotypes. Although about 50% of cases are estimated to have a genetic component, the underlying cause often remains undetermined. Here, from whole-exome sequencing on samples from 168 infertile men with asthenoteratozoospermia due to severe sperm flagellum, we identified homozygous variants in four unrelated patients. In sperm cells from these individuals, immunofluorescence revealed altered localization of DNAH1, DNALI1, WDR66, and TTC29. Axonemal localization of ZMYND12 ortholog TbTAX-1 was confirmed using the model. RNAi knock-down of TbTAX-1 dramatically affected flagellar motility, with a phenotype similar to the sperm from men bearing homozygous variants. Co-immunoprecipitation and ultrastructure expansion microscopy in revealed TbTAX-1 to form a complex with TTC29. Comparative proteomics with samples from and KO mice identified a third member of this complex: DNAH1. The data presented revealed that ZMYND12 is part of the same axonemal complex as TTC29 and DNAH1, which is critical for flagellum function and assembly in humans, and . ZMYND12 is thus a new asthenoteratozoospermia-associated gene, bi-allelic variants of which cause severe flagellum malformations and primary male infertility.
Topics: Humans; Male; Animals; Mice; Asthenozoospermia; Semen; Flagella; Infertility, Male; Fertility; Calcium-Binding Proteins; Dyneins
PubMed: 37934199
DOI: 10.7554/eLife.87698 -
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 -
The Journal of Biological Chemistry Jul 2023Asthenozoospermia characterized by decreased sperm motility is a major cause of male infertility, but the majority of the etiology remains unknown. Here, we showed that...
Asthenozoospermia characterized by decreased sperm motility is a major cause of male infertility, but the majority of the etiology remains unknown. Here, we showed that the cilia and flagella associated protein 52 (Cfap52) gene was predominantly expressed in testis and its deletion in a Cfap52 knockout mouse model resulted in decreased sperm motility and male infertility. Cfap52 knockout also led to the disorganization of the midpiece-principal piece junction of the sperm tail but had no effect on the axoneme ultrastructure in spermatozoa. Furthermore, we found that CFAP52 interacted with the cilia and flagella associated protein 45 (CFAP45) and knockout of Cfap52 decreased the expression level of CFAP45 in sperm flagellum, which further disrupted the microtubule sliding produced by dynein ATPase. Together, our studies demonstrate that CFAP52 plays an essential role in sperm motility by interacting with CFAP45 in sperm flagellum, providing insights into the potential pathogenesis of the infertility of the human CFAP52 mutations.
Topics: Animals; Humans; Male; Mice; Cilia; Flagella; Infertility, Male; Mice, Knockout; Proteins; Semen; Sperm Motility; Sperm Tail; Spermatozoa
PubMed: 37236356
DOI: 10.1016/j.jbc.2023.104858 -
Cell Death & Disease Jan 2024Cilia are highly conserved eukaryotic organelles that protrude from the cell surface and are involved in sensory perception, motility, and signaling. Their proper... (Review)
Review
Cilia are highly conserved eukaryotic organelles that protrude from the cell surface and are involved in sensory perception, motility, and signaling. Their proper assembly and function rely on the bidirectional intraflagellar transport (IFT) system, which involves motor proteins, including antegrade kinesins and retrograde dynein. Although the role of IFT-mediated transport in cilia has been extensively studied, recent research has highlighted the contribution of IFT-independent kinesins in ciliary processes. The coordinated activities and interplay between IFT kinesins and IFT-independent kinesins are crucial for maintaining ciliary homeostasis. In this comprehensive review, we aim to delve into the specific contributions and mechanisms of action of the IFT-independent kinesins in cilia. By shedding light on their involvement, we hope to gain a more holistic perspective on ciliogenesis and ciliopathies.
Topics: Flagella; Kinesins; Biological Transport; Cilia; Homeostasis; Dyneins
PubMed: 38218748
DOI: 10.1038/s41419-024-06428-9 -
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 -
The EMBO Journal Mar 2024The efficacy of current antimitotic cancer drugs is limited by toxicity in highly proliferative healthy tissues. A cancer-specific dependency on the microtubule motor...
The efficacy of current antimitotic cancer drugs is limited by toxicity in highly proliferative healthy tissues. A cancer-specific dependency on the microtubule motor protein KIF18A therefore makes it an attractive therapeutic target. Not all cancers require KIF18A, however, and the determinants underlying this distinction remain unclear. Here, we show that KIF18A inhibition drives a modest and widespread increase in spindle assembly checkpoint (SAC) signaling from kinetochores which can result in lethal mitotic delays. Whether cells arrest in mitosis depends on the robustness of the metaphase-to-anaphase transition, and cells predisposed with weak basal anaphase-promoting complex/cyclosome (APC/C) activity and/or persistent SAC signaling through metaphase are uniquely sensitive to KIF18A inhibition. KIF18A-dependent cancer cells exhibit hallmarks of this SAC:APC/C imbalance, including a long metaphase-to-anaphase transition, and slow mitosis overall. Together, our data reveal vulnerabilities in the cell division apparatus of cancer cells that can be exploited for therapeutic benefit.
Topics: Humans; Anaphase-Promoting Complex-Cyclosome; Dyneins; Kinesins; Kinetochores; Mitosis; Neoplasms
PubMed: 38279026
DOI: 10.1038/s44318-024-00031-6 -
Experimental & Molecular Medicine Apr 2024Intracellular retrograde transport in eukaryotic cells relies exclusively on the molecular motor cytoplasmic dynein 1. Unlike its counterpart, kinesin, dynein has a... (Review)
Review
Intracellular retrograde transport in eukaryotic cells relies exclusively on the molecular motor cytoplasmic dynein 1. Unlike its counterpart, kinesin, dynein has a single isoform, which raises questions about its cargo specificity and regulatory mechanisms. The precision of dynein-mediated cargo transport is governed by a multitude of factors, including temperature, phosphorylation, the microtubule track, and interactions with a family of activating adaptor proteins. Activating adaptors are of particular importance because they not only activate the unidirectional motility of the motor but also connect a diverse array of cargoes with the dynein motor. Therefore, it is unsurprising that dysregulation of the dynein-activating adaptor transport machinery can lead to diseases such as spinal muscular atrophy, lower extremity, and dominant. Here, we discuss dynein motor motility within cells and in in vitro, and we present several methodologies employed to track the motion of the motor. We highlight several newly identified activating adaptors and their roles in regulating dynein. Finally, we explore the potential therapeutic applications of manipulating dynein transport to address diseases linked to dynein malfunction.
Topics: Humans; Cytoplasmic Dyneins; Animals; Biological Transport; Microtubules; Dyneins
PubMed: 38556551
DOI: 10.1038/s12276-024-01200-7 -
Nature Structural & Molecular Biology Mar 2024Dynein and kinesin motors mediate long-range intracellular transport, translocating towards microtubule minus and plus ends, respectively. Cargoes often undergo...
Dynein and kinesin motors mediate long-range intracellular transport, translocating towards microtubule minus and plus ends, respectively. Cargoes often undergo bidirectional transport by binding to both motors simultaneously. However, it is not known how motor activities are coordinated in such circumstances. In the Drosophila female germline, sequential activities of the dynein-dynactin-BicD-Egalitarian (DDBE) complex and of kinesin-1 deliver oskar messenger RNA from nurse cells to the oocyte, and within the oocyte to the posterior pole. We show through in vitro reconstitution that Tm1-I/C, a tropomyosin-1 isoform, links kinesin-1 in a strongly inhibited state to DDBE-associated oskar mRNA. Nuclear magnetic resonance spectroscopy, small-angle X-ray scattering and structural modeling indicate that Tm1-I/C suppresses kinesin-1 activity by stabilizing its autoinhibited conformation, thus preventing competition with dynein until kinesin-1 is activated in the oocyte. Our work reveals a new strategy for ensuring sequential activity of microtubule motors.
Topics: Animals; Kinesins; Drosophila Proteins; Dyneins; Tropomyosin; Drosophila; Microtubules; RNA, Messenger
PubMed: 38297086
DOI: 10.1038/s41594-024-01212-x -
Aging Jul 2023Heart failure (HF) remains a huge medical burden worldwide. Pathological cardiac hypertrophy is one of the most significant phenotypes of HF. Several studies have...
BACKGROUND
Heart failure (HF) remains a huge medical burden worldwide. Pathological cardiac hypertrophy is one of the most significant phenotypes of HF. Several studies have reported that the TGF-β pathway plays a double-sided role in HF. Therefore, TGF-β-related genes (TRGs) may be potential therapeutic targets for cardiac hypertrophy and HF. However, the roles of TRGs in HF at the single-cell level remain unclear.
METHOD
In this study, to analyze the expression pattern of TRGs during the progress of cardiac hypertrophy and HF, we used three public single-cell RNA sequencing datasets for HF (GSE161470, GSE145154, and GSE161153), one HF transcriptome data (GSE57338), and one hypertrophic cardiomyopathy transcriptome data (GSE141910). Weighted gene co-expression network analysis (WGCNA), functional enrichment analysis and machine learning algorithms were used to filter hub genes. Transverse aortic constriction mice model, CCK-8, wound healing assay, quantitative real-time PCR and western blotting were used to validate bioinformatics results.
RESULTS
We observed that cardiac fibroblasts (CFs) and endothelial cells showed high TGF-β activity during the progress of HF. Three modules (royalblue, brown4, and darkturquoize) were identified to be significantly associated with TRGs in HF. Six hub genes (TANC2, ADAMTS2, DYNLL1, MRC2, EGR1, and OTUD1) showed anomaly trend in cardiac hypertrophy. We further validated the regulation of the TGF-β-MYC-ADAMTS2 axis on CFs activation .
CONCLUSIONS
This study identified six hub genes (TANC2, ADAMTS2, DYNLL1, MRC2, EGR1, and OTUD1) by integrating scRNA and transcriptome data. These six hub genes might be therapeutic targets for cardiac hypertrophy and HF.
Topics: Mice; Animals; Transforming Growth Factor beta; Endothelial Cells; Heart Failure; Cardiomegaly; Sequence Analysis, RNA; Cytoplasmic Dyneins
PubMed: 37498303
DOI: 10.18632/aging.204901