Mechanisms of Delta-like 4 Endocytosis and Notch Activation During Blood Vessel Development

总结 内皮细胞（EC）是负责胚胎血管形成的细胞类型， 最终在成年期形成了大约10万英里长的脉管系统。在发展过程中，新的血液 血管从预先存在的脉管系统中出现，这一过程称为血管生成。Notch信号是基础 血管生成和成人血管内环境稳定。在没有Notch的情况下，血管表现出 以不受抑制的增殖和过度生长为特征的慢性发芽表型;这一证据和其他证据 压倒性地显示Notch是血管成熟和稳定所必需。当配体结合时， Notch胞内结构域被切割并易位到细胞核，作为转录因子发挥作用。δ样 配体4（DLL 4）是结合Notch的细胞外结构域的有效Notch配体。为了引出Notch 激活Notch的胞外域需要DLL 4的拉力以暴露S2切割位点。一旦 暴露后，Notch胞外结构域被切割，允许Notch胞内结构域的释放 （NICD）刺激信号传导激活。关于DLL 4的转录后调控知之甚少， 更不了解DLL 4施加持续拉力以激活 Notch介导的侧抑制。我们的初步数据首次描述了DLL 4内吞作用和 锚定到肌动蛋白细胞骨架产生暴露Notch的S2位点所需的机械力。 具体来说，这项建议将集中在两个基本上没有特征的蛋白质，我们认为是核心的力量- 产生DLL 4内吞作用、Eps 15同源结构域结合蛋白1（EHBP 1）和含有EH结构域的 蛋白2（EHD 2）。我们广泛的假设是EHBP 1将EHD 2锚定在f-肌动蛋白丝上，有助于EHD 2的表达。 DLL 4与Notch结合的转内吞作用。在Aim 1中，我们将详细介绍EHBP 1和EHD 2如何在 组合以促进DLL 4内吞作用，并且在这样做时，介导Notch信号传导。在AIM 2中，我们将 全面证明EHBP 1和EDH 2联合作用调节DLL 4内吞作用， 使用实时成像和CRISPR在斑马鱼血管形态发生过程中的下游Notch活性- 基于突变体生成。总的来说，这项建议将回答几个至关重要的问题， 血管发育，并为本科学者提供了一个强大的培训机会。

SUMMARY Endothelial cells (ECs) are the cell type responsible for the bulk of embryonic blood vessel formation, eventually leading to an estimated 100,000 miles of vasculature by adulthood. During development, new blood vessels emerge from pre-existing vasculature, a process termed angiogenesis. Notch signaling is fundamental to angiogenesis and adult blood vessel homeostasis. In the absence of Notch, blood vessels demonstrate a chronic sprouting phenotype marked by unchecked proliferation and overgrowth; this evidence and others overwhelmingly show Notch is required for blood vessel maturation and stability. When ligand-bound, the Notch intracellular domain is cleaved and translocates to the nucleus, acting as a transcription factor. Delta-like ligand 4 (DLL4) is a potent Notch ligand that binds to the extracellular domain of Notch. To elicit Notch activation the ectodomain of Notch requires a pulling force by DLL4 to expose a S2 cleavage site. Once exposed, the Notch extracellular domain is cleaved, allowing for release of the Notch intracellular domain (NICD) stimulating signaling activation. Very little is known about the post-transcriptional regulation of DLL4, and even less is understood about the mechanisms by which DLL4 exerts a sustained pulling force to activate Notch-mediated lateral inhibition. Our preliminary data describes, for the first time, how DLL4 endocytosis and anchoring to the actin cytoskeleton generates the mechanical force required to expose the S2 site of Notch. Specifically, this proposal will focus on two largely uncharacterized proteins we believe are central to force- generating DLL4 endocytosis, Eps15 homology domain binding protein 1 (EHBP1) and EH domain containing protein 2 (EHD2). Our broad hypothesis is that EHBP1 anchors EHD2 to f-actin filaments, aiding in the transendocytosis of DLL4 bound to Notch. In Aim1 we will detail how both EHBP1 and EHD2 work in combination to facilitate DLL4 endocytosis, and in doing so, mediate Notch signaling. In Aim2, we will comprehensively demonstrate that EHBP1 and EDH2 works in combination to regulate DLL4 endocytosis and downstream Notch activity during zebrafish blood vessel morphogenesis using live-imaging and CRISPR- based mutant generation. Overall, this proposal will answer several critically important questions pertaining to blood vessel development as well as provide a powerful training opportunity for undergraduate scholars.