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HEK293 CD63 CRISPR cell-derived small EVs

ABSTRACT: The tetraspanin protein CD63 has been recently described as a key factor in extracellular vesicle (EV) production and endosomal cargo sorting. In this study, we assemble a global scope of CD63-dependent protein trafficking into small secreted vesicles.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture, Early Embryonic Cell

SUBMITTER: Stephanie Hurwitz

LAB HEAD: David Meckes, Jr.

PROVIDER: PXD006514 | Pride | 2018-10-24

REPOSITORIES: pride

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			Action	DRS
	05202016_DM_930_Run1.raw	Raw
	05202016_DM_930_Run2.raw	Raw
	05202016_DM_930_Run3.raw	Raw
	05202016_DM_931_Run1.raw	Raw
	05202016_DM_931_Run2.raw	Raw

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Publications

Tetraspanin CD63 Bridges Autophagic and Endosomal Processes To Regulate Exosomal Secretion and Intracellular Signaling of Epstein-Barr Virus LMP1

Hurwitz Stephanie N SN Cheerathodi Mujeeb R MR Nkosi Dingani D York Sara B SB Meckes David G DG

Journal of virology 20180212 5

The tetraspanin protein CD63 has been recently described as a key factor in extracellular vesicle (EV) production and endosomal cargo sorting. In the context of Epstein-Barr virus (EBV) infection, CD63 is required for the efficient packaging of the major viral oncoprotein latent membrane protein 1 (LMP1) into exosomes and other EV populations and acts as a negative regulator of LMP1 intracellular signaling. Accumulating evidence has also pointed to intersections of the endosomal and autophagy pa ...[more]

PMID: 29212935

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Tetraspanin CD63 acts as a pro-metastatic factor via β-catenin stabilization

Project description:The tetraspanin CD63 is implicated in pro-metastatic signaling pathways, but so far, it is unclear how CD63 levels affect the tumor cell phenotype. Here, we investigated the effect of CD63 modulation in different metastatic tumor cell lines. In vitro, knock down of CD63 induced a more epithelial-like phenotype concomitant with increased E-cadherin expression, downregulation of its repressors Slug and Zeb1, and decreased N-cadherin. In addition, β-catenin protein was markedly reduced, negatively affecting expression of the target genes MMP-2 and PAI-1. β-catenin inhibitors mimicked the epithelial phenotype induced by CD63 knock down. Inhibition of β-catenin upstream regulators PI3K/AKT or GSK3β could rescue the mesenchymal phenotype underlining the importance of the β-catenin pathway in CD63-regulated cell plasticity. CD63 knock down-induced phenotypical changes correlated with a decrease of experimental metastasis, while CD63 overexpression enhanced the tumor cell-intrinsic metastatic potential. Taken together, our data show that CD63 is a crucial player in the regulation of the tumor cell-intrinsic metastatic potential by affecting cell plasticity. Stable knock down of CD63 was performed in SKOV3ipL ovarian carcinoma cell line using 2 shRNAs lentiviral constructs (sh49 and sh51), and as a negative control, a shNT construct. Parental cells, control shNT and the 2 shCD63 cell lines were seeded 24 hours prior to RNA isolation on a 10 cm dish, labelling and hybridization on microarrays. One color experiment with 2 biological replicates of the 4 experimental conditions: SKOV3ipL, SKOV3ipL_shNT, SKOV3ipL_sh49 and SKOV3ipL_sh51.

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Human - Cells and Extracellular vesicles of Hela and MNT-1 WT vs CD63 KO Proteomes 2

Project description:CD63 is a tetraspanin, which organize on cholesterol enriched microdomains, especially on late endosomes. Previous work of our team has shown that CD63 could regulate the sorting of cargoes into ILVs through an ESCRT-independent manner in melanocytes. However, CD63 has also been involved in other trafficking events in other cell type and so far, we have no clear idea of the general process that CD63 would control and would unify these disparate reports. In order to elucidate to general role of CD63 we compared two cell lines, Hela and MNT-1 that we Knock-out for CD63 by CRISPR in both cell lines and investigated the EV generation and the expression level of proteins involved in this process in cell lysates and the EV pellet obtained by SEC.

2024-04-19 | PXD037147 | Pride

Human - Cells and Extracellular vesicles of Hela and MNT-1 WT vs CD63 KO Proteomes 1

2024-04-19 | PXD037146 | Pride

Human - Cells and Extracellular vesicles of Hela and MNT-1 WT vs CD63 KO Exosomes

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Tetraspanin CD63 acts as a pro-metastatic factor via β-catenin stabilization

2015-01-23 | GSE61957 | GEO

MS on EVs from the YSL of 3dpf zebrafish embryos

Project description:We set up a zebrafish model to study EVs in vivo by light microscopy, using CD63-pHluorin as a marker. To verify if exosomes from zebrafish have a comparable composition as human exosomes, and to validate the use of CD63 as a marker, we first analysed the compositon of EVs isolated from in vitro cultured fibroblasts (AB.9 ATCC), and could detect the presence of conventional exosome markers, including (zebrafish) CD63. In live embryos, we could track a population of exosomes from their source (the yolk syncytial layer (YSL)) to their final destination by live microscopy. To assess the composition of these exosomes, we dissociated YSL:CD63-pHluorin expressing 3dpf embryos and isolated exosomes from the supernatant and enriched for EVs from the YSL. This was compared to a background of total EVs isolated the control fish (non-expressing).

2019-04-10 | PXD011258 | Pride

CD63/CD9-GFP extracellular vesicles LC-MS/MS

Project description:This study aimed to identify subpopulations of extracellular vesicles (EVs) secreted by HeLa cells, and determine markers which enable to identify them, and which indicate their endosomal or plasma membrane origin. We used CD63 and CD9 as markers of EVs, and followed their intracellular trafficking using the RUSH assay. We used mass spectrometry to identify specific or common proteins in immunoprecipitated GFP+ EVs from HeLa transfected with the RUSH CD63-GFP or CD9-GFP, after a short (3h) or a long (24h) trafficking time from the endoplasmic reticulum.

2021-05-17 | PXD021515 | Pride

Gene expression changes upon CD63 knockdown in esophageal squamous cell carcinoma cell lines

Project description:The purpose of this analysis is to analyze the changes in gene expression that occur in cancer cells when CD63 is knocked down in esophageal squamous cell carcinoma (ESCC) cell lines. We performed a comprehensive analysis of mRNA in CD63 knockdown human ESCC cell lines (TE2, TE15) using microarrays.

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Jurkat cell exosomes bound by CD47, CD63 and MHC1 harbor different RNA content

Project description:Extracellular vesicles (EVs) mediate cell-cell communication including the intercellular transfer of RNAs, which alter gene expression in target cells. However, heterogeneity in the size, density, and composition of EVs has limited progress towards defining their physiological functions and utility as disease-specific biomarkers. CD63 and MHC-1 are widely used as markers to purify EVs. CD47 is also present on EVs and alters their effects on target cells, suggesting that specific surface markers define functionally distinct EVs. This hypothesis was addressed by comparing the properties of Jurkat T cell EVs captured using CD47, CD63, and MHC-1 antibodies. These EV subsets have similar sizes but divergent RNA contents.

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Jurkat cell exosomes bound by either CD47 or CD63 harbor different RNA content

Project description:xtracellular vesicles (EVs) mediate cell-cell communication including the intercellular transfer of RNAs, which alter gene expression in target cells. However, heterogeneity in the size, density, and composition of EVs has limited progress towards defining their physiological functions and utility as disease-specific biomarkers. CD63 and MHC-1 are widely used as markers to purify EVs. CD47 is also present on EVs and alters their effects on target cells, suggesting that specific surface markers define functionally distinct EVs. This hypothesis was addressed by comparing the properties of Jurkat T cell EVs captured using CD47, CD63, and MHC-1 antibodies. These EV subsets have similar sizes but divergent RNA contents.

2018-08-03 | GSE103310 | GEO

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