Abstract

Junctional complexes between endothelial cells form a dynamic barrier that hinders passive diffusion of blood constituents into interstitial tissues. Remodelling of junctions is an essential process during leukocyte trafficking, vascular permeability, and angiogenesis. However, for many junctional proteins, the mechanisms of junctional remodelling have yet to be determined. Here, we used receptor mutagenesis, horseradish peroxidase (HRP), and ascorbate peroxidase 2 (APEX-2) proximity labelling, alongside light and electron microscopy (EM), to map the intracellular trafficking routes of junctional adhesion molecule-C (JAM-C). We found that JAM-C cotraffics with receptors associated with changes in permeability such as vascular endothelial cadherin (VE-Cadherin) and neuropilin (NRP)-1 and 2, but not with junctional proteins associated with the transmigration of leukocytes. Dynamic JAM-C trafficking and degradation are necessary for junctional remodelling during cell migration and angiogenesis. By identifying new potential trafficking machinery, we show that a key point of regulation is the ubiquitylation of JAM-C by the E3 ligase Casitas B-lineage lymphoma (CBL), which controls the rate of trafficking versus lysosomal degradation.

Highlights

  • Endothelial cells exist as a monolayer that lines the blood vasculature, and as such, they present a discrete barrier to the cellular and molecular components of blood

  • To characterise the amount and localisation of intracellular junctional adhesion molecule-C (JAM-C) present at steady state in cultured endothelial cells, we carried out immunofluorescence analysis in human umbilical vein endothelial cells (HUVECs) (Fig 1A–1D)

  • Incubating cells with the vacuolar-type H+-ATPase inhibitor bafilomycin (100 nM for 4 h) (Fig 1C), a reagent that blocks the acidification step essential for lysosomal degradation, markedly increased the number of junctional adhesion molecule (JAM)-C–positive intracellular punctae. These results indicate that JAM-C is constitutively trafficked into vesicles, and at least a proportion of the receptor in resting conditions is targeted for degradation in lysosomes

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Summary

Introduction

Endothelial cells exist as a monolayer that lines the blood vasculature, and as such, they present a discrete barrier to the cellular and molecular components of blood. This includes ions, small and large proteins, platelets, and leukocytes [1]. The endothelium plays a crucial role in responding to a pathogenic infection or tissue injury, both in the initial recruitment of leukocytes to the appropriate site [2] and in subsequent tissue repair and angiogenesis [3]. Endothelial dysfunction can predispose the vessel wall to leukocyte adhesion, platelet activation, oxidative stress, thrombosis, coagulation, and chronic inflammation, leading to the pathogenesis of numerous cardiovascular diseases [4].

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