Actin-related protein 6 facilitates proneural protein-induced gene activation for rapid neural differentiation
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PROVIDER: PRJNA926111 | ENA |
REPOSITORIES: ENA
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Project description:Actin-related protein 6 (Arp6), a core component of the H2A.Z exchange complex SWR1, interacts with proneural proteins and is crucial for efficient onset of proneural protein target gene expression.
2023-01-26 | GSE223416 | GEO
Project description:Changes in cell morphology require the dynamic remodeling of the actin cytoskeleton. Calcium fluxes have been suggested as an important signal to rapidly relay information to the actin cytoskeleton, but the underlying mechanisms remain poorly understood. Here, we identify the EF-hand domain containing protein EFhD2/Swip-1 as a conserved lamellipodial protein strongly upregulated in Drosophila macrophages at the onset of metamorphosis when macrophage behavior shifts from quiescent to migratory state. Loss- and gain-of-function analysis confirm a critical function of EFhD2/Swip-1 in lamellipodial cell migration in fly and mouse melanoma cells. Contrary to previous assumptions, TIRF-analyses unambiguously demonstrate that EFhD2/Swip-1 proteins efficiently cross-link actin filaments in a calcium-dependent manner. Using a single-cell wounding model, we show that EFhD2/Swip-1 promotes wound closure in a calcium-dependent manner. Mechanistically, our data suggest that transient calcium bursts reduce EFhD2/Swip-1 cross-linking activity and thereby promote rapid reorganization of existing actin networks to drive epithelial wound closure.
2022-04-19 | GSE200966 | GEO
Project description:Glioblastoma multiforme (GBM) is an aggressive, heterogeneous and highly vascularized brain tumor. GBM is thought to arise from glioblastoma stem-like cells (GSCs) which are characterized as being either proneural or mesenchymal. The former isolates of GSCs are tight sphere forming and slow growing while mesenchymal GSCs are lose sphere forming, fast growing, highly invasive and when dominant yield poorer patient prognosis. GSCs are known to be plastic in nature and can therefore evolve from a proneural to a mesenchymal state. Here, we observed that factors secreted by endothelial cells (which make up the brain vasculature) alter several properties of GSCs resulting in the acquisition of a more mesenchymal and invasive phenotype coupled with changes at the level of secretory and cellular proteome. Thus, using mass spectrometry, quantitative proteomic analysis and GO term filters, we identified several mesenchymal traits in proneural GSCs exposed to endothelial cell secretome. Specifically, proneural cells treated with the conditioned media derived from human umbilical vein endothelial cells (HUVEC) upregulated the expression of mesenchymal proteins such as CD44 and VIM, while downregulating the expression of the proneural proteins such as NOTCH1, activated NOTCH intracellular domain (NICD), SOX2 and NESTIN, which were validated using flow cytometry (FACS) and western blots (WB). Using DAVID analysis tool, we detected the features of cellular proteome indicative of the activation of NFkB, Wnt and several other pathways in the proneural cells treated with HUVEC conditioned media. Using conditioned media fractionation through several centrifugation steps we identified the extracellular vesicles (EV) sedimented at 100,000 x g using ultracentrifugation, as the source of activity in endothelial conditioned media capable of triggering mesenchymal shift in proneural GSCs. EVs are heterogeneous membrane structures containing multiple bioactive macromolecules, which have the ability to carry multiple bioactive proteins, transfer them to recipient cells and alter their function, signalling and biological programmes. We compared the effects of EVs, soluble fraction and unfractionated conditioned media in terms of their ability to trigger mesenchymal changes in the phenotype of proneural GSCs. Once the cultures were established, the culture medium was removed and replaced with HUVEC-derived material (conditioned media, supernatant or EV fraction) and responses evaluated over 7 days by microscopical analysis of sphere structures, and biochemically by following the aforementioned proneural or mesenchymal markers (WB, FACS). We observed an upregulation of mesenchymal proteins, as well as downregulation of the proneural proteins, mentioned above. These effects recapitulated those of unfractionated conditioned media and were absent from target cells exposed to EV-depleted conditioned media. The data analysis of EV proteome included canonical markers and pathways of cellular vesiculation as well as markers and pathways of interest with regards to the biological effects associated with treatment of GSC recipient cells. In this regard we observed several EV related tetraspanin markers, which were validated using western blot including CD9, CD63, CD81 and a purity control, BIP. Although we identified several potential effectors associated with endothelial cell EVs that could impact proneural cell phenotype, we focused on MMPs for at least three reasons: (i) evidence in the literature (see text) indicated that MMPs may induce differentiation programs in neural stem cells; (ii) MMPs in EV cargo were relatively abundant and have been implicated in various biological processes; (iii) MMPs released from endothelial cells could be functionally involved in disrupting proneural cell sphere structures that we observed in the presence of endothelial cell secretome. We noted the expressions of MMP1, MMP2, MMP11 and MP14 in our HUVEC-EV mass spectrometry dataset, the activity of which was validated using the MMP activity assay kit from abcam (ab112146). Using GO terms we also detected a signal for NFkB pathway activation in the proteome of endothelial (HUVEC) conditioned media-treated proneural GSCs. NFkB activation is regarded as hallmark of mesenchymal phenotype in GSCs and GBM cells. We validated that the upregulation of NFkB, was also true for the proneural cells treated with HUVEC derived EVs. Moreover, upon blocking MMP expression in proneural cells treated with endothelial cell EVs, we inhibited the activation of NFkB activity thereby documenting that the initial effects of MMPs trigger a shift in cellular phenotype toward NfkB activation and mesenchymal reprogramming. Briefly, we compared GO terms of GSC157 cells treated with their own or HUVEC-derived EVs. Validation of the NFkB pathway activation was analysed using WB and immunofluorescence for levels of NFkB and phosphor-NFkB.
2022-10-14 | PXD034819 | Pride
Project description:Nuclear actin has been demonstrated to be essential for optimal transcription, but the molecular mechanisms and direct binding partner for actin in the RNA polymerase complex have remained unknown. By using purified proteins in several biochemical assays, we demonstrate a direct and specific interaction between monomeric actin and Cdk9, the kinase subunit of the positive transcription elongation factor b (P-TEFb) required for RNA polymerase II (Pol II) pause-release. This interaction is not dependent on kinase activity of Cdk9 and is not involved with releasing P-TEFb from its inhibitor 7SK snRNP complex. Supporting the specific role for actin in the elongation phase of transcription, chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) reveals that actin interacts with genes only upon their active transcription elongation. This study therefore provides novel insights into the mechanisms by which actin facilitates the transcription process.
2023-09-01 | GSE239771 | GEO
Project description:As many other tumors, a subset of gliobastoma is thought to be maintained by a restricted population of cancer cells, stem-like cells that express CD133 transmembrane protein. Expression levels of CD133 gene has been linked to a poor prognostic molecular subgroup and is not overexpressed by the PDGF-driven proneural group. Thus, the significance of CD133+ cells for gliomagenesis of the proneural group is undetermined. In addition, the role of the CD133 protein remains elusive and controversial, which results from the difficult isolation of CD133+ cells that has largely relied on the use of antibodies to ill-defined glycosylated epitopes of CD133. Here, we used a knockin lacZ reporter mouse, Prom1lacZ/+, to track Prom1+ cells in the brain and found that Prom1 (prominin1, murine CD133 homologue) is expressed by cells that co-express markers characteristic of neuronal, glial and vascular lineage phenotype. In proneural tumors derived from injection of RCAS-PDGF into the brain of tv-a;Ink4a-Arf-/- Prom1lacZ/+ mice, Prom1+ cells co-express markers for astrocytes and endothelial cells. Therefore, we characterize the tumor propagation in a murine model and found that the mice co-transplanted with Prom1 endothelium and proneural tumor spheres cells had significant tumor burden and vascular proliferation (angiogenesis). Specific genes in Prom1 endothelium are identified that code for endothelial signaling modulators that most likely support proneural tumor progression and can be potential targets for anti-angiogenic therapy. Cells were sorted via FACS to obtain a population of CD31+CD133- cells and a population of CD31+CD133+ cells. Total RNA was extracted from each population and gene expression was assayed on Affymetrix Mouse 430 2.0 arrays with one array per cell population.
2013-12-31 | E-GEOD-43931 | biostudies-arrayexpress
Project description:Active segregation of DNA in bacteria is catalyzed by cytomotive structures. Mediators of subcellular plasmid positioning are Walker-type ATPases (ParA), actin-like proteins, or tubulin homologs. These motor proteins are coupled to the DNA via adaptor proteins that recognize specific DNA motifs. Here, we describe that a temperate phage, CGP3, integrated into the genome of Corynebacterium glutamicum ATCC 13032 encodes an actin-like protein, AlpC. Biochemical characterization confirms that AlpC is a bona fide actin-like protein and cell biological analysis shows that AlpC forms dynamic filamentous structures upon phage induction. The co-transcribed AlpA protein binds to a specific region of the phage DNA, possibly functioning as an adaptor protein that connects circular phage DNA to the tips of the AlpC filaments. The AlpC filaments transport phage DNA to the cell membrane of the host cell. Furthermore, both AlpA and AlpC are required for efficient phage replication. This is remarkably similar to actin-assisted membrane localization of eukaryotic viruses that use the actin cytoskeleton to concentrate virus particles at the egress sites.
2015-05-01 | GSE45906 | GEO
Project description:Active segregation of DNA in bacteria is catalyzed by cytomotive structures. Mediators of subcellular plasmid positioning are Walker-type ATPases (ParA), actin-like proteins, or tubulin homologs. These motor proteins are coupled to the DNA via adaptor proteins that recognize specific DNA motifs. Here, we describe that a temperate phage, CGP3, integrated into the genome of Corynebacterium glutamicum ATCC 13032 encodes an actin-like protein, AlpC. Biochemical characterization confirms that AlpC is a bona fide actin-like protein and cell biological analysis shows that AlpC forms dynamic filamentous structures upon phage induction. The co-transcribed AlpA protein binds to a specific region of the phage DNA, possibly functioning as an adaptor protein that connects circular phage DNA to the tips of the AlpC filaments. The AlpC filaments transport phage DNA to the cell membrane of the host cell. Furthermore, both AlpA and AlpC are required for efficient phage replication. This is remarkably similar to actin-assisted membrane localization of eukaryotic viruses that use the actin cytoskeleton to concentrate virus particles at the egress sites.
2015-05-01 | GSE45905 | GEO
Project description:Polycomb protein RYBP facilitates super-enhancer activity
| PRJNA1183812 | ENA
Project description:As many other tumors, a subset of gliobastoma is thought to be maintained by a restricted population of cancer cells, stem-like cells that express CD133 transmembrane protein. Expression levels of CD133 gene has been linked to a poor prognostic molecular subgroup and is not overexpressed by the PDGF-driven proneural group. Thus, the significance of CD133+ cells for gliomagenesis of the proneural group is undetermined. In addition, the role of the CD133 protein remains elusive and controversial, which results from the difficult isolation of CD133+ cells that has largely relied on the use of antibodies to ill-defined glycosylated epitopes of CD133. Here, we used a knockin lacZ reporter mouse, Prom1lacZ/+, to track Prom1+ cells in the brain and found that Prom1 (prominin1, murine CD133 homologue) is expressed by cells that co-express markers characteristic of neuronal, glial and vascular lineage phenotype. In proneural tumors derived from injection of RCAS-PDGF into the brain of tv-a;Ink4a-Arf-/- Prom1lacZ/+ mice, Prom1+ cells co-express markers for astrocytes and endothelial cells. Therefore, we characterize the tumor propagation in a murine model and found that the mice co-transplanted with Prom1 endothelium and proneural tumor spheres cells had significant tumor burden and vascular proliferation (angiogenesis). Specific genes in Prom1 endothelium are identified that code for endothelial signaling modulators that most likely support proneural tumor progression and can be potential targets for anti-angiogenic therapy.
2013-12-31 | GSE43931 | GEO
Project description:Fluorescence-activated cell sorting of M4-GFP wing imaginal disc cells was used to recover a purified population of the cells that comprise the “proneural clusters” from which sensory organ precursors of the peripheral nervous system (PNS) arise. Whole-genome microarray analysis and in situ hybridization was then used to identify and verify a set of genes that are preferentially expressed in proneural cluster cells. Genes in this set encode proteins with a diverse array of implied functions, and loss-of-function analysis of two candidate genes shows that they are indeed required for normal PNS development. Keywords = Proneural Keywords = PNS Keywords = PNC Keywords = SOP Keywords: parallel sample
2005-03-05 | GSE2359 | GEO