Differential requirement for centriolar satellites in cilium formation and ciliary signaling among different vertebrate cells
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PROVIDER: PRJNA507257 | ENA |
REPOSITORIES: ENA
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Project description:Centriolar satellites are an array of membrane-less granules that localize and move around the vertebrate centrosome/cilium complex. They have recently emerged as key regulators of the biogenesis and function of the centrosome/cilium-complex and their mutations are linked to ciliopathies. Although centriolar satellites are ubiquitous structures of the vertebrate cells, their precise function and molecular mechanism of action in different cell types remain poorly understood. Here, we generated kidney and retinal epithelial cells that lack centriolar satellites by genetically ablating their scaffolding protein PCM1 and investigated the cellular and molecular consequences of satellite loss in cells. We showed that centriolar satellites are required for cilium assembly, regulation of ciliary content, timely response to Hedgehog signals and three- dimensional epithelial cell organization, but not for cell proliferation, cell cycle progression and centriole duplication. Importantly, the requirement for centriolar satellites in cilium assembly varied between retinal and kidney epithelial cells and we identified the differences in the efficiency of targeting key ciliogenesis factors to the centrosome including Mib1 and Talpid3 as the likely molecular basis for this phenotypic variability. Quantitative global transcriptomic and proteomic profiling of satellite-less cells showed that loss of centriolar satellites does not lead to a major transcriptional response, but leads to a significant rearrangement of the global proteome. Together, our findings identify important roles for centriolar satellites in key cilium-related cellular processes through regulating the proteostasis and centrosomal/ciliary targeting of proteins and provide insight into the disease mechanisms of ciliopathies.
2018-11-28 | GSE123017 | GEO
Project description:Glioblastoma multiforme (GBM) possesses glioma stem cells (GSCs) that promote self-renewal, tumor propagation, and relapse. Understanding the mechanisms of GSCs self-renewal can offer targeted therapeutic interventions. However, insufficient knowledge of the fundamental biology of GSCs is a significant bottleneck hindering these efforts. Here, we show that patient-derived GSCs recruit an elevated level of proteins that ensure the temporal cilium disassembly, leading to suppressed ciliogenesis. Depleting the cilia disassembly complex components at the ciliary base is sufficient to induce ciliogenesis in a subset of GSCs via sequestering PDGFR-α from its original location to newly induced cilium. Importantly, restoring ciliogenesis caused GSCs to behave like healthy NPCs switching from self-renewal to differentiation. Finally, using an organoid-based glioma invasion assay and brain xenografts in mice, we establish that ciliogenesis-induced differentiation can prevent the infiltration of GSCs into the brain. Our findings illustrate a crucial role for cilium as a molecular switch in determining GSCs' fate and suggest that cilium induction is an attractive strategy to intervene in GSCs proliferation.
2021-07-08 | GSE179439 | GEO
Project description:Human melanomas frequently harbor amplifications of EZH2. However, the oncogenic contribution of this methyltransferase to melanoma formation has remained elusive. Taking advantage of murine melanoma models, we now show that EZH2 drives tumorigenesis from benign BrafV600E or NrasQ61K-expressing melanocytes. EZH2 oncogenicity results from silencing of genes relevant for the integrity of the primary cilium, a signaling organelle projecting from the surface of vertebrate cells. Consequently, gain of EZH2 function promotes loss of primary cilia in benign melanocytic lesions. In contrast, blockade of EZH2 activity evokes ciliogenesis and cilia-dependent growth inhibition in malignant melanoma. Finally, we demonstrate that loss of cilia enhances pro-tumorigenic WNT/β-catenin signaling and is itself sufficient to drive metastatic melanoma in benign cells. Thus, primary cilia deconstruction is a key process in EZH2-driven melanomagenesis.
2019-04-04 | GSE112677 | GEO
Project description:Primary cilia are tiny membrane protrusions emanating from the surface of almost all mammalian cell types. Recently, a picture has emerged of the primary cilium functioning as a cellular antenna that senses extracellular stimuli via receptors, locally processes the signal using cilia-specific signalling pathways, and transduces this information into a cellular response. Components of the cyclic AMP (cAMP) signalling cascade have been proposed to be part of the ciliary signalling pathways. We aimed to shed light on whether ciliary cAMP signaling controls gene expression, and if yes, which gene expression program is particularly targeted by ciliary cAMP signaling. To this end, we targeted an optogenetic tool to increase cAMP levels (bPAC) to the primary cilium in murine, kidney-derived inner medullary collecting duct (mIMCD-3) cells (mIMCD-3 cilia-bPAC cells). As controls, we used cells were bPAC is targeted to the cytosol (mIMCD-3 cyto-bPAC cells) and wild-type cells (mIMCD-3 WT cells). We increased cAMP levels by light stimulation and next analysed gene expression using bulk RNA-sequencing. As an additional control, we also included WT cells treated with the compound Forskolin, which targets endogeneous adenylyl cyclases and thus, in principle, is believed to increase cAMP levels in both compartments, cilium and cell soma.
2022-05-11 | GSE182339 | GEO
Project description:The neuronal primary cilium and centriolar satellites have established roles in neurogenesis. However, the role of these regions in the postnatal brain is not clear. Here we show that ablation of the pericentriolar material 1 gene (Pcm1) in the mouse leads to enlarged lateral ventricles and behavioral abnormalities in adult but not juvenile animals. Adults have concomitant reductions in cortical mass and altered hippocampal morphology. Cytoanatomical analyses revealed progressive ciliary shortening, most prominently in the CA1 hippocampus, prelimbic cortex, and amygdala, as psychomotor and cognitive function declined. RNAseq of affected regions revealed significant transcriptional changes in amine- and G-protein coupled receptor activity pathways. The physiological relevance of this phenotype was supported by decreased dopamine D2 receptor (D2R) protein levels, and the failure to rescue adult behavioral defects with antipsychotic drugs. Immunoprecipitations showed an association with Pcm1 and D2Rs. Finally, we sequenced PCM1 in two cohorts of humans with severe schizophrenia. Systematic modeling of all discovered rare alleles by in vivo complementation in zebrafish revealed a significant enrichment for pathogenic alleles. Taken together, our data highlight a role for the pericentriolar material in the postnatal brain; they suggest that a progressive degenerative ciliary/centriolar phenotype can induce behavioral defects in mice; and support a contributory role for PCM1 in individuals with severe psychosis.
2020-07-16 | GSE145915 | GEO
Project description:Primary cilia are microtubule based sensory organelles important for receiving and processing cellular signals. Recent studies have shown that cilia also release extracellular vesicles (EVs) but little is known about their composition and function. Because EVs have been shown to exert various functions in physiology and pathology, these findings have the potential to fundamentally alter our understanding of how the primary cilium is able to regulate specific signalling pathways in development and disease. Compared to control, ciliary mutant mammalian cells demonstrated increased secretion of small EVs (smEVs) and a change in EV composition. Small RNA sequencing and mass spectrometry identified an enrichment for WNT signalling-associated miRNAs and proteins loaded into ciliary mutant EVs. Furthermore, we show that smEVs secreted from mammalian cells are biologically active and modulate the WNT response in recipient cells. These results highlight a possible new smEV-dependent ciliary signalling mechanism which could provide us with new insights into paracrine ciliary signalling as well as ciliopathy disease pathogenesis.
2021-07-19 | PXD020466 | Pride
Project description:The primary cilium is a signaling organelle with a unique membrane composition maintained by a diffusional barrier residing at the transition zone. Many transition zone proteins, such as the tectonic complex, are linked to preserving ciliary composition; however, whether these proteins regulate active transport or physically impede membrane diffusion remains unknown. To understand tectonic’s role, we generated a photoreceptor specific Tctn1 knockout mouse. Loss of Tctn1 resulted in an absence of the entire tectonic complex yet had minimal effects on transition zone structure. Interestingly, we found that protein composition of the photoreceptor cilium was disrupted as non-resident membrane proteins accumulated in the cilium over time, ultimately resulting in photoreceptor degeneration. We further show that membrane proteins moved faster through the transition zone illustrating that the tectonic complex acts as a physical barrier to slow diffusion of membrane proteins so they can be properly sorted by ciliary transport carriers.
2023-10-24 | PXD042270 | Pride
Project description:Centrosomes are orbited by centriolar satellites, dynamic multiprotein assemblies nucleated by PCM1. To study the requirement for centriolar satellites, we generated mice lacking PCM1. Pcm1-/- mice display partially expressive perinatal lethality with survivors exhibiting hydrocephalus, oligospermia and cerebellar hypoplasia. Pcm1-/- multiciliated ependymal cells and PCM1-/- retinal pigmented epithelial 1 (RPE1) cells showed reduced ciliogenesis. PCM1-/- RPE1 cells displayed reduced docking of the mother centriole to the ciliary vesicle and removal of CP110 and CEP97 from the distal mother centriole, indicating compromised early ciliogenesis. We show these molecular cascades are maintained in vivo, although we propose that the cellular threshold for ciliogenesis initiation varies between cell types. We propose that PCM1 and centriolar satellites facilitate efficient trafficking of proteins to and from centrioles, including the departure of CP110 and CEP97 to initiate ciliogenesis.
2023-05-10 | PXD031920 | Pride
Project description:EZH2-mediated primary cilium deconstruction drives metastatic melanoma formation
| PRJNA448706 | ENA
Project description:Primary cilia are microtubule based sensory organelles that protrude from almost every cell type. Their membrane contains highly specialized receptors important for receiving and processing extracellular signals, which enables them to regulate several signalling pathways. A recently discovered characteristic is that cilia are also able to release extracellular vesicles (EVs) from the ciliary membrane. Since EVs have been shown to exert numerous functions in physiology and pathology, these findings have the potential to dramatically alter our understanding of how the primary cilium is able to regulate various signalling pathways in development and disease. In our study we focused on the release of EVs from a ciliated kidney cell line. Using control and mutant cell lines, in which ciliary trafficking was disrupted, we observed that loss of primary cilia function leads to altered EV secretion and composition in NTA (Nanoparticle Tracking Analysis) and Western blot. Cilia mutant cells released more small EVs compared to control, and their composition was also changed between mutant and control. Protein identification via mass spectrometry identified both cilia- as well as WNT signalling-associated proteins and miRNA sequencing determined WNT-related miRNAs, which were differentially expressed in small EVs isolated from the cilia mutant cell line. Because of the presence of differentially expressed WNT related molecules in these small EVs, we tested whether this would have an effect on the WNT activity of recipient cells. We observed that small EVs secreted from cilia mutant cells differentially modulated the WNT response in recipient cells compared to control. Our results highlight a possible new small EV-dependent ciliary signalling mechanism, since deficient primary cilia lead to a change in EV secretion, resulting in an altered signal transduction. These results provide us with new insights into ciliopathy disease pathogenesis.
2021-07-19 | GSE153227 | GEO