1. Control of endothelial tubulogenesis by Rab and Ral GTPases, and apical targeting of caveolin-1-labeled vacuoles
- Author
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George E. Davis, Zheying Sun, and Pieter R. Norden
- Subjects
Hydrolases ,Cell Membranes ,Caveolin 1 ,GTPase ,Vacuole ,Biochemistry ,Small interfering RNAs ,RNA, Small Interfering ,Cytoskeleton ,Tube formation ,0303 health sciences ,RALB ,Multidisciplinary ,Chemistry ,030302 biochemistry & molecular biology ,RALA ,Cell biology ,Enzymes ,rac GTP-Binding Proteins ,Nucleic acids ,Protein Transport ,src-Family Kinases ,Medicine ,Collagen ,Cellular Structures and Organelles ,Research Article ,Cell Physiology ,Science ,Green Fluorescent Proteins ,Models, Biological ,Exocytosis ,03 medical and health sciences ,Genetics ,Human Umbilical Vein Endothelial Cells ,Humans ,Vesicles ,Non-coding RNA ,030304 developmental biology ,Cell Membrane ,Biology and Life Sciences ,Proteins ,Cell Biology ,Intracellular Membranes ,Apical membrane ,Gene regulation ,Guanosine Triphosphatase ,Membrane Trafficking ,rab GTP-Binding Proteins ,Vacuoles ,Enzymology ,RNA ,ral GTP-Binding Proteins ,Rab ,Gene expression ,Collagens - Abstract
Here, we examine known GTPase regulators of vesicle trafficking events to assess whether they affect endothelial cell (EC) lumen and tube formation. We identify novel roles for the small GTPases Rab3A, Rab3B, Rab8A, Rab11A, Rab27A, RalA, RalB and caveolin-1 in co-regulating membrane trafficking events that control EC lumen and tube formation. siRNA suppression of individual GTPases such as Rab3A, Rab8A, and RalB markedly inhibit tubulogenesis, while greater blockade is observed with combinations of siRNAs such as Rab3A and Rab3B, Rab8A and Rab11A, and RalA and RalB. These combinations of siRNAs also disrupt very early events in lumen formation including the formation of intracellular vacuoles. In contrast, knockdown of the endocytosis regulator, Rab5A, fails to inhibit EC tube formation. Confocal microscopy and real-time videos reveal that caveolin-1 strongly labels intracellular vacuoles and localizes to the EC apical surface as they fuse to form the luminal membrane. In contrast, Cdc42 and Rab11A localize to a perinuclear, subapical region where intracellular vacuoles accumulate and fuse during lumen formation. Our new data demonstrates that EC tubulogenesis is coordinated by a series of small GTPases to control polarized membrane trafficking events to generate, deliver, and fuse caveolin-1-labeled vacuoles to create the apical membrane surface.
- Published
- 2020