Criado Santos Nina, Chehab Tarek, Holthenrich Anna, Gerke Volker
Centre for Molecular Biology of Inflammation, Institute of Medical Biochemistry, University of Münster, Münster, Germany.
Department of Cell Physiology and Metabolism, University of Geneva, Geneva, Switzerland.
Methods Mol Biol. 2019;1929:437-445. doi: 10.1007/978-1-4939-9030-6_27.
Endothelial cells respond to blood vessel injury by the acute release of the procoagulant von Willebrand factor, which is stored in unique secretory granules called Weibel-Palade bodies (WPBs). Stimulated, Ca-dependent exocytosis of WPBs critically depends on their proper targeting to the plasma membrane, but the mechanism of WPB-plasma membrane tethering prior to fusion is not well characterized. Here we describe a method to visualize and analyze WPB tethering and fusion in living human umbilical vein endothelial cells (HUVEC) by total internal reflection fluorescence (TIRF) microscopy. This method is based on automated object detection and allowed us to identify components of the tethering complex of WPBs and to monitor their dynamics in space and time. An important tethering factor identified by this means was Munc13-4 that was shown to interact with S100A10 residing in a complex with plasma membrane-bound annexin A2.
内皮细胞通过急性释放促凝血性血管性血友病因子来应对血管损伤，该因子存储在称为魏尔-帕拉德小体（WPB）的独特分泌颗粒中。受刺激后，WPB依赖于钙的胞吐作用关键取决于它们正确靶向质膜，但融合前WPB与质膜的拴系机制尚未得到充分表征。在这里，我们描述了一种通过全内反射荧光（TIRF）显微镜观察和分析人脐静脉内皮细胞（HUVEC）中WPB拴系和融合的方法。该方法基于自动目标检测，使我们能够识别WPB拴系复合体的成分，并监测它们在空间和时间上的动态变化。通过这种方法鉴定出的一个重要拴系因子是Munc13-4，它被证明与位于与质膜结合的膜联蛋白A2形成的复合物中的S100A10相互作用。
