Dynamic trafficking and turnover of JAM-C is essential for endothelial cell migration

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.内皮细胞间的连接复合物构成动态屏障，能够阻断血液成分经被动扩散进入间质组织。连接复合物的重塑是白细胞转运、血管通透性调节及血管生成过程中的核心生物学过程。然而，诸多连接蛋白的连接重塑具体分子机制仍有待阐明。本研究联合运用受体诱变技术、辣根过氧化物酶（horseradish peroxidase, HRP）邻近标记法、抗坏血酸过氧化物酶2（ascorbate peroxidase 2, APEX-2）邻近标记法，以及光学与电子显微镜（EM）技术，对黏着连接分子-C（junctional adhesion molecule-C, JAM-C）的细胞内转运通路开展了系统定位与分析。研究结果显示，JAM-C可与参与血管通透性调控的受体（如血管内皮钙粘蛋白（vascular endothelial cadherin, VE-Cadherin）、神经纤毛蛋白（neuropilin, NRP）-1与NRP-2）发生共转运，但不会与介导白细胞跨内皮迁移的连接蛋白产生共转运。动态的JAM-C转运与降解过程，是细胞迁移及血管生成阶段连接复合物重塑的必要前提。本研究通过鉴定全新的潜在转运调控机制，证实E3泛素连接酶Cbl（Casitas B-lineage lymphoma, CBL）对JAM-C的泛素化修饰是核心调控节点，该修饰可精准调控JAM-C的转运速率与溶酶体降解通路的选择。