Supplementary Material for: Advanced Methods for the Investigation of Cell Contact Dynamics in Endothelial Cells Using Florescence-Based Live Cell Imaging

Endothelial cells of the vascular system are dynamic cells whose molecular adaptability is decisive for the adjustment of homeostasis and organ perfusion. Advanced microscopic techniques, automation processing, and image analysis software was shown to improve the understanding of vascular biology. In this work, we describe advanced methods that allow investigating the dynamics of endothelial cell contacts. The development of viral vectors has contributed significantly to the genetic manipulation of endothelial cells. We used the Gibson assembly as a quick and cheap cloning system for introducing sequences into the lentiviral-based pFUGW vector. Furthermore, classical fluorescence tags such as mCherry and EGFP were compared with self-labeling tags such as Halo and SNAP for their suitability to study junction dynamics in cultured endothelium, and found the self-labeling tags as useful tools. Using such combinations, we found maintained cell junction integrity during shear stress-induced junction remodeling using VE-cadherin-EGFP. Remodeling was accompanied by VE-cadherin plaque formation, indicating that this process is mediated by the for­mation of the actin-driven junction-associated intermittent lamellipodia, JAIL. The combined methods including the Gibson assembly, lentiviral mediated gene transfer, spinning disk-based live cell imaging, and software for quantification allow analyses of the endothelial cell junction dynamics under static and under shear stress conditions.

血管系统内皮细胞属于一类动态细胞，其分子适应性对于机体内稳态（homeostasis）的调节与器官灌注（organ perfusion）的调控至关重要。先进的显微成像技术、自动化处理流程与图像分析软件，已被证实可有效加深人们对血管生物学（vascular biology）的理解。本研究阐述了可用于探究内皮细胞接触动态变化的先进实验方法。
病毒载体（viral vector）技术的发展，极大地推动了内皮细胞的遗传操控研究。我们采用吉布森组装（Gibson assembly）作为快速且经济的克隆系统，将目标序列插入基于慢病毒的pFUGW载体中。此外，本研究对比了mCherry、EGFP等经典荧光标签与Halo、SNAP等自标记标签（self-labeling tag）在培养内皮细胞中研究连接动态变化的适用性，结果证实自标记标签为极具实用价值的研究工具。
通过上述组合方法，我们借助VE-钙粘蛋白-EGFP（VE-cadherin-EGFP）标记观察到，在剪切应力（shear stress）诱导的细胞连接重塑过程中，细胞连接完整性得以维持。该重塑过程伴随VE-钙粘蛋白斑的形成，提示此过程由肌动蛋白（actin）驱动的连接相关间歇性片状伪足（junction-associated intermittent lamellipodia，JAIL）形成所介导。本研究所整合的方法包括吉布森组装、慢病毒介导的基因转染、基于转盘式的活细胞成像技术以及定量分析软件，可用于分析静态与剪切应力条件下的内皮细胞连接动态变化。