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.
Highlights
Critical steps during early vasculogenesis and endothelial cell (EC) lumen formation include both the establishment of asymmetric cytoskeletal polarization and the trafficking of pinocytic intracellular vacuoles or other intracellular vesicles to create an apical membrane surface[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16]
We show that intracellular vacuoles are highly enriched in Caveolin-1, Src family kinases and RalA which proceed to target to the apical membrane surface while the small GTPases Cdc42, Rab11A and Rab27A are localized in a polarized, subapical region
Using combinations of siRNAs, we demonstrated that combined knockdown of Rab8A with Rab11A or Rab27A had a greater blocking effect compared to knockdown of Rab8A alone and that combined knockdown of Rab27A and Caveolin-1 had a greater blocking influence than knockdown of Rab27A or Caveolin-1 alone
Summary
Critical steps during early vasculogenesis and endothelial cell (EC) lumen formation include both the establishment of asymmetric cytoskeletal polarization (i.e. modified tubulins subapically and F-actin basally) and the trafficking of pinocytic intracellular vacuoles or other intracellular vesicles to create an apical membrane surface[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16]. A central question is how intracellular vacuolization, directed trafficking of vacuoles and vesicles along an asymmetrically polarized cytoskeleton, and coalescence of vacuoles/vesicles to form the lumen are spatially and temporally controlled by molecular regulators of membrane trafficking events, including Rab, Ral, and Rho GTPases
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