Cervero Pasquale, Vrenken Kirsten, Klose Matthias, Rehm Kerstin, Linder Stefan
Institut für medizinische Mikrobiologie, Virologie und Hygiene, Universitätsklinikum Eppendorf, Martinistr. 52, 20246 Hamburg, Germany.
Institut für medizinische Mikrobiologie, Virologie und Hygiene, Universitätsklinikum Eppendorf, Martinistr. 52, 20246 Hamburg, Germany; Laboratory of Pediatric Oncology, Radboud Institute for Molecular Life Sciences, Radboud university medical center, Nijmegen, P.O.Box 9101, 6500 HB Nijmegen, The Netherlands.
Eur J Cell Biol. 2021 Sep-Nov;100(7-8):151184. doi: 10.1016/j.ejcb.2021.151184. Epub 2021 Nov 20.
Cell-cell junctions undergo constant remodeling, which is crucial for the control of vascular integrity. Indeed, transport of junctional components such as cadherins is understood in increasing depth. However, little is known about the respective pathways regulating localization of nectin at cell-cell junctions. Here, we performed an siRNA-based screen of vesicle regulators of the RabGTPase family, leading to the identification of a novel role for Rab5a in the endocytosis nectin-2 at adherens junctions of primary human endothelial cells (HUVEC). Confocal microscopy experiments revealed disordered nectin-2 localization at adherens junctions upon Rab5a depletion. In addition, internalized nectin-2 was shown to prominently localize to Rab5a-positive vesicles in both fixed and living cells. As shown previously, nectin-2 stabilization at junctions is achieved via drebrin-dependent coupling to the subcortical actin cytoskeleton. Consistently, depletion of drebrin in this study leads to enhanced internalization of nectin-2 from junctions. Strikingly, simultaneous silencing of Rab5a and drebrin restored the junctional localization of nectin-2, pointing to Rab5a as counteracting the drebrin-dependent stabilization of nectin-2 at adherens junctions. This mechanism could be further validated by transendothelial resistance measurements. Collectively, our results identify Rab5a as a key player in the endocytosis of nectin-2 and thus in the regulation of adherens junction integrity in primary human endothelial cells.
细胞间连接不断进行重塑，这对于控制血管完整性至关重要。实际上，对诸如钙黏蛋白等连接成分的运输理解得越来越深入。然而，对于调节nectin在细胞间连接定位的各自途径却知之甚少。在这里，我们对RabGTPase家族的囊泡调节因子进行了基于siRNA的筛选，从而确定了Rab5a在原代人内皮细胞（HUVEC）黏附连接处对nectin-2内吞作用中的新作用。共聚焦显微镜实验显示，Rab5a缺失后，nectin-2在黏附连接处的定位紊乱。此外，在固定细胞和活细胞中，内化的nectin-2都显著定位于Rab5a阳性囊泡。如先前所示，通过drebrin依赖的与皮质下肌动蛋白细胞骨架的偶联，可实现nectin-2在连接处的稳定。与此一致的是，本研究中drebrin的缺失导致nectin-2从连接处的内化增强。令人惊讶的是，同时沉默Rab5a和drebrin可恢复nectin-2的连接处定位，表明Rab5a抵消了drebrin依赖的nectin-2在黏附连接处的稳定作用。这种机制可通过跨内皮电阻测量进一步验证。总的来说，我们的结果确定Rab5a是nectin-2内吞作用的关键参与者，从而也是原代人内皮细胞中黏附连接完整性调节的关键参与者。
