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Proximity interactome of lymphatic VE-cadherin reveals mechanisms of junctional remodeling and reelin secretion.

ABSTRACT: In this study, we are the first to use the MiniTurboID generation of the proximity-dependent biotin identification (BioID) technology to illuminate how the lymphatic endothelial cell (LEC) VE-cadherin interactome changes during junctional remodeling in response to the lymphangiocrine factor adrenomedullin. Here, we created a UBC-driven lentiviral expression vector containing the coding sequence for a Ve-Cadehrin-V5-MiniTurboID fusion-protein. After transduction of LECs with Ve-Cadehrin-MiniTurboID lentivirus, we treated these cells with 100 nM adrenomedullin (AM) or vehicle and labeled proximal proteins with 50 µM for 2 hours to capture dynamic changes in the Ve-Cadehrin interactome during AM-induced junctional rearrangement. Biotin-labeled proximity interactors were isolated via streptavidin-affinity purification (AP) and identified using LC-MS/MS mass spectrometry. Our dataset consists of distinct biological replicates consisting of AP enriched proteins from whole cell lysates (WCL, n = 2) or plasma membrane (PM, n = 3) fractions. WCL fractions allow us to capture proximity networks involved in Ve-Cadehrin trafficking and recycling to the PM, while PM fractions allows us to examine membrane-specific changes in AM-induced adherens junctions assembly.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell

SUBMITTER: Christine Mills

LAB HEAD: Kathleen Caron

PROVIDER: PXD040452 | Pride | 2024-09-05

REPOSITORIES: Pride

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			Action	DRS
	20211129_PHCOQEHF_PC913-Caron_2_R1.raw	Raw
	20211129_PHCOQEHF_PC913-Caron_2_R2.raw	Raw
	20211129_PHCOQEHF_PC913-Caron_3_R1.raw	Raw
	20211129_PHCOQEHF_PC913-Caron_3_R2.raw	Raw
	20220902_PHCOQEHF_PC1018-Caron_10.raw	Raw

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Project description:The formation of a zygote by the fusion of egg and sperm involves the two gametic transcriptomes. In flowering plants, the embryo sac embedded within the ovule contains the egg cell, while the pollen grain contains two sperm cells inside a supporting vegetative cell. The difficulties of collecting isolated gametes and consequent low recovery of RNA have restricted in-depth analysis of gametic transcriptomes in flowering plants. We isolated living egg cells, sperm cells, and pollen vegetative cells from rice, and identified transcripts for ~36,000 genes by deep sequencing. The three transcriptomes are highly divergent, with about three quarters of those genes differentially expressed in the different cell types. Distinctive expression profiles were observed for genes involved in chromatin conformation, including an unexpected expression in the sperm cell of genes associated with active chromatin. Furthermore, both the sperm cell and the pollen vegetative cell were deficient in expression of key RNAi components. Differences in gene expression were also observed for genes for hormonal signaling and cell cycle regulation. The egg cell and sperm cell transcriptomes reveal major differences in gene expression to be resolved in the zygote, including pathways affecting chromatin configuration, hormones and cell cycle. The sex-specific differences in expression of RNAi components suggest that epigenetic silencing in the zygote might act predominantly through female-dependent pathways. More generally, this study provides a detailed gene expression landscape for flowering plant gametes, enabling the identification of specific gametic functions and their contributions to zygote and seed development. The gene expression profiles of the three gametic cells, egg cell (EC), sperm cell (Sp) and vegetative cell (Ve) were compared via RNA-seq using three biological replicates for each. Differential expression (DE) analysis was performed using edgeR and the resulting DE genes were assessed for Gene Ontology term enrichment.

Project description:Background: We have previously used the rat 4 day Complete Freund's Adjuvant (CFA) model to screen compounds with potential to reduce osteoarthritic pain. The aim of this study was to identify genes altered in this model of osteoarthritic pain and use this information to infer analgesic potential of compounds based on their own gene expression profiles using the Connectivity Map approach. Results: Using microarrays, we identified differentially expressed genes in L4 and L5 dorsal root ganglia (DRG) from rats that had received intraplantar CFA for 4 days compared to matched, untreated control animals. Analysis of these data indicated that the two groups were distinguishable by differences in genes important in immune responses, nerve growth and regeneration. This list of differentially expressed genes defined a âCFA signatureâ. We used the Connectivity Map approach to identify pharmacologic agents in the Broad Institute Build02 database that had gene expression signatures that were inversely related (ânegatively connectedâ) with our CFA signature. To test the predictive nature of the Connectivity Map methodology, we tested phenoxybenzamine (an alpha adrenergic receptor antagonist) â one of the most negatively connected compounds identified in this database - for analgesic activity in the CFA model. Our results indicate that at 10mg/kg, phenoxybenzamine demonstrated analgesia comparable to that of Naproxen in this model. Conclusion: Evaluation of phenoxybenzamine-induced analgesia in the current study lends support to the utility of the Connectivity Map approach for identifying compounds with analgesic properties in the CFA model. A naive (control) group of rats (n = 6) and a group of rats injected with CFA (n = 6) were used for gene expression profiling experiments. One animal from the control group did not yield sufficient amount of RNA for microarray and thus was omitted from further processing. In total, 5 microarrays each from the 5 animals in the control group, and 6 microarrays each from the 6 animals in the CFA group were analyzed.

Project description:Vascular endothelial protein tyrosine phosphatase (VE-PTP, PTPRB) is a receptor type phosphatase that is crucial for the regulation of endothelial junctions and blood vessel development. VE-PTP regulates vascular integrity by dephosphorylating substrates which are key players in endothelial junction stability, such as the angiopoietin receptor TIE2, the endothelial adherens junction protein VE-cadherin and the vascular endothelial growth factor receptor VEGFR2. Here, we have systematically searched for novel substrates of VE-PTP in endothelial cells by utilizing two approaches. First, we studied changes in the endothelial phosphoproteome upon exposing cells to a highly VE-PTP-specific phosphatase inhibitor followed by affinity isolation and mass-spectrometric analysis of phosphorylated proteins by phosphotyrosine-specific antibodies. Second, we used a substrate trapping mutant of VE-PTP to pull down phosphorylated substrates in combination with SILAC-based quantitative mass spectrometry measurements. We identified a set of substrate candidates of VE-PTP, of which a remarkably large fraction is related to cell junctions (48/165; 29.1%). Several of those were found in both screens and displayed very high connectivity in predicted functional interaction networks. The receptor protein tyrosine kinase EPHB4 was the most prominently phosphorylated protein upon VE-PTP inhibition among those VE-PTP targets that were identified by both proteomic approaches. Further analysis revealed that EPHB4 forms a ternary complex with VE-PTP and TIE2 in endothelial cells. VE-PTP controls the phosphorylation of each of these two tyrosine kinase receptors. Despite of their simultaneous presence in a ternary complex, stimulating each of the receptors with their own specific ligand did not cross-activate the respective partner receptor. Our systematic approach has led to the identification of novel substrates of VE-PTP, of which many are relevant for the control of cellular junctions further promoting the importance of VE-PTP as a key player of junctional signalling.

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Proximity interactome of lymphatic VE-cadherin reveals mechanisms of junctional remodeling and reelin secretion.

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