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

Abstract

The adhesion receptor vascular endothelial (VE)-cadherin transduces an array of signals that modulate crucial lymphatic cell behaviors including permeability and cytoskeletal remodeling. Consequently, VE-cadherin must interact with a multitude of intracellular proteins to exert these functions. Yet, the full protein interactome of VE-cadherin in endothelial cells remains a mystery. Here, we use proximity proteomics to illuminate how the VE-cadherin interactome changes during junctional reorganization from dis-continuous to continuous junctions, triggered by the lymphangiogenic factor adrenomedullin. These analyses identified interactors that reveal roles for ADP ribosylation factor 6 (ARF6) and the exocyst complex in VE-cadherin trafficking and recycling. We also identify a requisite role for VE-cadherin in the in vitro and in vivo control of secretion of reelin-a lymphangiocrine glycoprotein with recently appreciated roles in governing heart development and injury repair. This VE-cadherin protein interactome shines light on mechanisms that control adherens junction remodeling and secretion from lymphatic endothelial cells.

Fig. 1. Validation of a functional VE-cadherin-V5-miniTurbo that recapitulates native VE-cadherin AM-induced junctional rearrangement in LECs.

A Schematic of adrenomedullin (AM)-mediated rearrangement of VE-cadherin; a central component on inter-endothelial adherens junctions. AM induces the rapid rearrangement of discontinuous junctions to continuous VE-cadherin junctions. B Experimental workflow used to obtain the VE-cadherin interactome during junctional rearrangements (predominantly discontinuous with 0 nM AM; predominantly continuous with 100 nM AM). C LECs were transduced with VE-cadherin-V5-miniTurbo (VE-cad-mT) lentivirus and treated with vehicle or 100 nM AM and 50 µM biotin for a 2 h labeling period to evaluate fusion protein expression and biotinylation by western blot after streptavidin affinity-purification. Anti-V5 evaluated fusion protein expression, streptavidin probed biotinylated proteins, ß-catenin probed a known VE-cadherin-interactor, and actin served as a load control. Shown is a representative experiment. A minimum of three independent experiments were performed. D Confocal microscopy to evaluate the cellular localization of VE-cadherin-mT fusion protein in transduced primary LECs treated with vehicle or 100 nM AM and 50 µM biotin for a 2 h labeling period. Cells were stained for VE-cadherin (magenta) to detect VE-cadherin-mT fusion proteins as well as native VE-cadherin, streptavidin (green) to stain for biotinylated proteins, and DAPI (cyan) to mark cell nuclei. Scale bar = 20 µm. A minimum of three independent experiments were performed. Source data are provided with this paper.

Fig. 2. Bioinformatic analysis of the AM-induced VE-cadherin interactome identifies enrichment of secretory and Rho family GTPase pathways.

A Gene Ontology (GO) Cell Component and Molecular Function annotation of top 20 most significant terms determined by enrichment false discovery rate (FDR), which indicates how likely the enrichment of the term is by chance and where smaller values correlate with a lower likelihood of random enrichment. Bar length correlates to enrichment FDR (displayed as -log10(FDR)) and the size of bar-terminating circles correlates to the number of genes found within that term. B Network analysis of top 20 enriched GO Cell Components and GO Molecular Functions annotations showing relationship between pathways. Node size correlates to size of gene sets, intensity and opacity of green shading refers to enrichment of gene sets (light/transparent indicates lower degree of enrichment, dark/opaque indicates higher degree of enrichment). Two pathways are considered connected if gene overlap between sets is >20% and is denoted by lines between nodes. Thickness of lines correlates to the degree of gene overlap between nodes. Pink outline indicates the ‘cell adhesion/actin organization super-node’.Blue outline indicates the ‘vesicular structures super-node’. Green outline indicates the ‘GTPase super-nodes’. C Top 10 enriched canonical pathways identified by Ingenuity Pathway Analysis (IPA) sorted by significance of number of proteins overlapping with the pathway (pathway overlap). Values displayed are -log(p-value) calculated by IPA (right-tailed Fisher’s exact test). D Top 10 enriched canonical pathways identified by IPA sorted by significance of activation of the pathway. Values displayed are z-scores calculated by IPA. E Top 10 Pathways with both high pathway overlap and activation. Both -log(p-value) and z-score are plotted for each pathway (right-tailed Fisher’s exact test). Source data are provided with this paper.

Fig. 3. Comparative analysis of VE-cadherin interactomes identified in whole cell or plasma membrane fractions reveals reproducibly captured proteins and pathways.

A Detection of VE-cad-mT, ß-catenin, and biotinylated protein isolated from LEC plasma membrane cell fractions. Membrane isolation was performed on LECs that were transduced with VE-cad-mT and treated with vehicle or 100 nM AM and 50 µM biotin for a 2 h labeling period, followed by streptavidin affinity purification and western blot analysis. INPUT and STREPT-AP fractions were profiled for V5 to evaluate fusion protein expression and pulldown, streptavidin to probe biotinylated proteins, ß-catenin to probe for a known VE-cadherin-interactor, and actin served as a load control. Shown is a representative experiment. A minimum of three independent experiments were performed. B Venn diagram of proteins uniquely or repeatedly captured across VE-cadherin interactomes generated from whole cell lysate (WCL1) or plasma membrane (PM) fractions. C Reactome analysis of proteins identified in the PM VE-cadherin interactome. Within clusters, branches and nodes with increasing orange color refers to increased significance of overlap with individual pathways comprising a cluster. Pink dashed box refers to (D). D Zoomed in view of the Cell-Cell Communication cluster showing expected enrichment of pathways associated with junction organization and adherens junctions. E List of proteins, with their gene names, identified in the PM VE-cadherin interactome which are sorted to the ‘Adherens Junction Interactions’ node of the Reactome Cell-Cell Communication Cluster. Source data are provided with this paper.

Fig. 4. Bioinformatic analysis of the plasma membrane-specific AM-induced VE-cadherin interactome identifies a role for secretory vesicles and GTPase activity in the remodeling of epithelial adherens junctions.

A Gene Ontology (GO) Cell Component and Molecular Function annotation of top 20 most significant terms determined by enrichment false discovery rate (FDR), which indicates how likely the enrichment of the term is by chance and where smaller values correlate with a lower likelihood of random enrichment. Bar length correlates to enrichment FDR (displayed as -log10(FDR)) and the size of bar-terminating circles correlates to the number of genes found within that term. B Network analysis of top 20 enriched GO Cell Components and GO Molecular Functions annotations showing relationship between pathways. Node size correlates to size of gene sets, intensity and opacity of green shading refers to enrichment of gene sets (light/transparent indicates lower degree of enrichment, dark/opaque indicates higher degree of enrichment). Two pathways are considered connected if gene overlap between sets is >20% and is denoted by lines between nodes. Thickness of lines correlates to the degree of gene overlap between nodes. Pink outline indicates the ‘cell adhesion/actin organization super-node’. Blue outline indicates the ‘vesicular structures super-node’. Green outline indicates the ‘GTPase super-node’. C–E Top 20 Pathways with high pathway overlap (significance of proteins overlapping with the pathway; displayed as -log(p-value)) and activation (displayed as z-score) when comparing samples that received (C) biotin versus a no biotin control, (D) biotin + AM versus a no biotin control, and (E) biotin + AM versus biotin only. Both -log(p-value) (right-tailed Fisher’s exact test) and z-score are plotted for each pathway. IPA analysis included all proteins having a log2 value > 0.5. n = 3 biological replicates of ‘biotin’ and ‘biotin + AM’. Source data are provided with this paper.

Fig. 5. AM-mediated VE-cadherin junction remodeling occurs through an endocytic-exocyst pathway.

A Proposed model of AM-mediated adherens junction regulation, generated by aggregating elements from the WCL1 highly enriched pathways “Remodeling of Epithelial Adherens Junctions” and “CDC42 Signaling”. In accordance with IPA legend, pink outlines refer to proteins identified in the VE-cadherin interactome, orange fill refers to high confidence of predicted activation, orange lines with arrow heads refer to predicted activated relationships. Lines with flat heads refer to inhibition (purple refers to Rasarfin inhibition of ARF6, blue refers to shEXOC5 knockdown of EXOC5, brown refers to Endosidin-2 inhibition of Exocyst Complex (EX)). Western blot and densitometric analysis of plasma membrane fractions of LECs treated with +/− 100 nM AM as well as (B) 10 µM Rasarfin, (C) 100 µM Endosidin-2, or (D) shEXOC5 to determine AM, inhibitor, or shRNA time-dependent (4 or 24 h) changes on membrane-specific VE-cadherin and ß-catenin protein levels. Shown are representative blots. Densitometric quantification of VE-cadherin and ß-catenin levels were first normalized to their respective load control (actin or GAPDH) and then normalized relative control (DMSO or shScramble). Data represents n = 3-6 biological replicates for each inhibitor or shRNA (mean ± s.d). One-way ANOVA with Dunnett’s multiple comparisons test was used to calculate significance. Precise n numbers and p-values are found for each condition in Supplementary Data 10. Source data are provided with this paper. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Fig. 6. Inhibition of endocytic-exocyst pathways disrupts VE-cadherin junctional architecture in LECs.

A Representative images of VE-cadherin localization in LECs co-treated with vehicle or 100 nM AM for 4 h under the following additional conditions: DMSO (24 h), 10 µM Rasarfin (4 h), 100 µM Endosidin-2 (24 h), or transduction with shScramble, or shEXOC5 lentivirus (72 h). Cells were stained for VE-cadherin (magenta) and DAPI (cyan) to mark cell nuclei. Scale bar = 20 µm. B Quantification of the percentage of continuous junctions per field relative to total number of junctions for each condition shown in (A). n = 4 to 9 images analyzed for each condition. Unpaired two-tailed t-tests were used to calculate significance. Precise n numbers and p-values are found for each condition in Supplementary Data 10. Source data are provided with this paper. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Fig. 7. Lymphatic secretion of reelin requires VE-cadherin plasma membrane junctional organization in vitro and VE-cadherin expression in vivo.

A Time course of reelin levels (ng/mL) in conditioned media collected from LECs treated with +/− 100 nM AM and quantified by ELISA. n = 3-8 biological replicates (mean ± s.d). Significance was determined using a Mixed-effects model (REML) with Šídák’s multiple comparisons test. To examine the effect of VE-cadherin membrane levels on reelin levels (ng/mL) in LEC conditioned media, LECs were treated with +/− 100 nM AM as well as pretreated with (B) shCDH5 (48 h), (C) 10 µM Rasarfin (4 h), (D) 100 µM Endosidin-2 (24 h), or (E) shEXOC5 (48 h) and quantified by ELISA. n = 3-8 biological replicates for each condition (mean ± s.d). Significance was determined using a Mixed-effects model (REML) (panel B), a two-way ANOVA with Geisser-Greenhouse correction (C and E), or a one-way ANOVA (panel D) with Tukey’s multiple comparisons test. Statistics only shown for comparisons between samples within a given time point. All comparisons are shown in Supplementary Fig. 10. Shown are representative western blots of plasma membrane fractions from LECs to correlate time-dependent secretion of reelin with changes in plasma membrane levels of VE-cadherin. Actin served as a load control. F Serum reelin levels of Cdh5^ΔPROX1 and littermate Cdh5^fl/fl (WT) mice before tamoxifen treatment at 1–2 months old (D1, hollow symbols) and 3 weeks after tamoxifen treatment (D22, solid symbols). Significance was determined using a two-way ANOVA with Šídák’s multiple comparisons test. n = 8 mice per group (mean ± s.d). F Created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license. Precise n numbers and p-values are found for each condition in Supplementary Data 10. Source data are provided with this paper.*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.