Rab35 governs apicobasal polarity through regulation of actin dynamics during sprouting angiogenesis.

In early blood vessel development, trafficking programs, such as those using Rab GTPases, are tasked with delivering vesicular cargo with high spatiotemporal accuracy. However, the function of many Rab trafficking proteins remain ill-defined in endothelial tissue; therefore, their relevance to blood vessel development is unknown. Rab35 has been shown to play an enigmatic role in cellular behaviors which differs greatly between tissue-type and organism. Importantly, Rab35 has never been characterized for its potential contribution in sprouting angiogenesis; thus, our goal was to map Rab35's primary function in angiogenesis. Our results demonstrate that Rab35 is critical for sprout formation; in its absence, apicobasal polarity is entirely lost in vitro and in vivo. To determine mechanism, we systematically explored established Rab35 effectors and show that none are operative in endothelial cells. However, we find that Rab35 partners with DENNd1c, an evolutionarily divergent guanine exchange factor, to localize to actin. Here, Rab35 regulates actin polymerization through limiting Rac1 and RhoA activity, which is required to set up proper apicobasal polarity during sprout formation. Our findings establish that Rab35 is a potent brake of actin remodeling during blood vessel development.

EHD2 modulates Dll4 endocytosis during blood vessel development.

OBJECTIVE: Despite the absolute requirement of Delta/Notch signaling to activate lateral inhibition during early blood vessel development, many mechanisms remain unclear about how this system is regulated. Our objective was to determine the involvement of Epsin 15 Homology Domain Containing 2 (EHD2) in delta-like ligand 4 (Dll4) endocytosis during Notch activation. APPROACH AND RESULTS: Using both in vivo and in vitro models, we demonstrate that EHD2 is a novel modulator of Notch activation in endothelial cells through controlling endocytosis of Dll4. In vitro, EHD2 localized to plasma membrane-bound Dll4 and caveolae. Chemical disruption of caveolae complexes resulted in EHD2 failing to organize around Dll4 as well as loss of Dll4 internalization. Reduced Dll4 internalization blunted Notch activation in endothelial cells. In vivo, EHD2 is primarily expressed in the vasculature, colocalizing with junctional marker VE-cadherin and Dll4. Knockout of EHD2 in zebrafish produced a significant increase in dysmorphic sprouts in zebrafish intersomitic vessels during development and a reduction in downstream Notch signaling. CONCLUSIONS: Overall, we demonstrate that EHD2 is necessary for Dll4 transcytosis and downstream Notch activation.