Advanced Methods for the Investigation of Cell Contact Dynamics in Endothelial Cells Using Florescence-Based Live Cell Imaging
| Autor: | Mohammed Aldirawi, Quentin Leidl, Christine Schimp, Jiahui Cao, Irene Hofer, Jochen Seebach, Muna Taha, Annelie Ahle, Hans J. Schnittler |
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| Rok vydání: | 2018 |
| Předmět: |
0301 basic medicine
Gibson assembly Endothelium Physiology Genetic Vectors Green Fluorescent Proteins Immunoblotting Gene Expression Cell junction Antibodies Viral vector Mice 03 medical and health sciences Live cell imaging Human Umbilical Vein Endothelial Cells medicine Animals Humans Cloning Molecular beta Catenin Fluorescent Dyes Chemistry Goats Antibodies Monoclonal Endothelial Cells Cadherins Cell biology Endothelial stem cell Intercellular Junctions 030104 developmental biology medicine.anatomical_structure Rabbits gamma Catenin Lamellipodium Cardiology and Cardiovascular Medicine mCherry |
| Zdroj: | Journal of Vascular Research. 55:350-364 |
| ISSN: | 1423-0135 1018-1172 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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