Angiogenesis is a crucial process under both physiological and pathological conditions. Vascular endothelial growth factor (VEGF) A and its cognate receptor, vascular endothelial growth factor receptor 2 (VEGFR2) are key regulators of angiogenesis. Plasma membrane (PM) levels of VEGFR2 are regulated by de novo synthesis, and by both exocytic and endocytic trafficking. VEGF-binding to VEGFR2 induces phosphorylation of key tyrosine residues located in the cytosolic domain of the receptor, followed by clathrin-mediated endocytosis and signal transduction leading to vascular morphogenesis. Disabled protein 2 (Dab2) is a cytosolic, clathrin-adaptor protein that is known to regulate endocytosis of certain cell surface receptors. Studies of Dab2 function have revealed its role in the development of embryonic vasculature. However, the mechanism of Dab2 function, particularly in conjunction with endosomal VEGFR2, remains poorly understood. Our results show that Dab2 interacts with VEGFR2 and that upon VEGF stimulation the two proteins co-localize within Rab5-positive early endosomes. Knockdown of Dab2 reduces levels of VEGF-induced phosphorylation of VEGFR2 at residue Y1175. This is significant because phosphorylation of VEGFR2-Y1175 is crucial for pro-angiogenic signal transduction. Moreover, knockdown of Dab2 causes an increased trafficking of VEGFR2 to late endosomes (LE). Finally, this altered VEGFR2 trafficking following Dab2 knockdown has major functional consequences for endothelial cells, as they are unable to undergo morphogenesis into tube-like structures in an in vitro assay of angiogenesis. Collectively, our data show that Dab2 plays a crucial role in VEGFR2 trafficking in the endocytic system and this impacts receptor signaling and endothelial cell morphogenesis during angiogenesis.

RNA interference (RNAi) is a gene regulation mechanism conserved among eukaryotes. To silence gene expression, RNAi relies on a short single-stranded guide RNA to steer the RNA-induced Silencing Complex (RISC) to mRNAs with guide strand-complementary sequences. RNAi is a highly membrane-associated process. The RISC complex is likely loaded at the rough Endoplasmic Reticulum, where it can bind to and degrade mRNAs. Components of the RISC complex also colocalize to late endosomes, and the efficiency of RNAi-mediated silencing is affected by changes in late endosome to lysosome fusion. RNAi can be systemic and inherited, effecting gene silencing in distal tissues and in the offspring. In this thesis, the model organism Caenorhabditis elegans was used to identify and characterize factors connecting systemic and inherited RNAi to the endomembrane system. We identify two SNARE proteins, SEC-22 and SYX-6, that both act as negative regulators of RNAi. SNAREs are necessary for vesicle fusion. Both SEC-22 and SYX-6 localize to late endosomes, and both interact with systemic RNAi protein SID-5 in a yeast two-hybrid (Y2H) screen. We find that in addition to its function in systemic RNAi, SID-5 is required for proper maturation of late endosomes. Furthermore, we identify the putative RNA-binding protein C12D8.1 as a novel regulator of RNAi inheritance. Mutant C12D8.1 animals will have enhanced inheritance of RNAi silencing, which negatively affects the ability of the progeny to silence new targets using RNAi. Finally, we describe a novel, object-based method for estimating significance in colocalization studies. This method helped us describe and quantify spatial relations between fluorophore-labeled proteins in situations where such analyses would otherwise be impossible. In conclusion, the work presented here further elucidates the connection between cellular RNAi, the endomembrane system, and the outside world.