Rab8 Trafficking of Apical Cargo is Required for Endothelial Lumen Formation and Sprouting Dynamics

Protein transport by way of intracellular vesicles, termed trafficking, is critically important for cell and tissue function. Rab GTPases are a family of proteins which function to orchestrate trafficking networks, facilitating cargo transport to various organelle compartments. In particular, Rab8 localizes to the Golgi Apparatus and previous reports indicate it is responsible for the trafficking to the apical membrane; however, these data have yet to be confirmed in blood vessel tissue. Accordingly, our investigation aimed to determine how Rab8 impacted trafficking of apically destined proteins in endothelial cells. Our results demonstrate that loss of Rab8 alters localization of vascular-endothelial cadherins, podocalyxin, and Tie-2. Additionally, basolateral beta 1-integrin was not impacted by Rab8 functionality, suggesting Rab8 specifically targets the apical membrane. In three-dimensional endothelial sprouts, Rab8 siRNA knockdown resulted in hyper-sprouting and long thin vessels lacking a defined lumen. These results suggest that Rab8 is required for endothelial lumen formation, an apical membrane-specific event, as well as regulation of sprouting dynamics. We also investigated Rab8’s necessity for organismal development. We performed a CRISPR knockout of Rab8 in larval zebrafish and observed that blood vessels lacked the normal lumen formation at 48 hours post-fertilization compared with controls; a finding in-line with our in vitro results. These results suggest that Rab8 serves an important function in apical membrane transport and vascular lumen formation.