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

血管发育过程中 Delta-like 4 内吞作用和 Notch 激活的机制

基本信息

批准号：
10202195
负责人：
Erich J Kushner
金额：
$ 43.9万
依托单位：
UNIVERSITY OF DENVER (COLORADO SEMINARY)
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-06-01 至 2024-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10202195
关键词：
3-Dimensional Actins Adult Affect Alleles Behavior Binding Biochemical Biochemistry Blood Vessels CRISPR/Cas technology Caveolins Cell Culture System Cell Nucleus Chemosensitization Chronic Clathrin Cleaved cell Cloning Clustered Regularly Interspaced Short Palindromic Repeats Communication Cytoskeleton Data Data Analyses Data Collection Development Embryo Endocytosis Endothelial Cells Endothelium Environment Exposure to Extracellular Domain F-Actin Generations Genetic Genotype Homeostasis Image Imaging Techniques Impairment In Vitro Knock-out Label Laboratories Lateral Ligand Binding Ligands Mediating Microfilaments Microscopy Modeling Molecular Molecular Biology Morphogenesis Notch Signaling Pathway Nutrient Organism Oxygen Phenotype Post-Transcriptional Regulation Primary Cell Cultures Process Protein Binding Domain Proteins Reporter Resolution Scientist Signal Transduction Site Students System Testing Time Training Transgenic Organisms Work Zebrafish angiogenesis base blood vessel development cell type experience experimental study gain of function genome editing in vivo in vivo imaging live cell imaging mechanical force mutant notch protein prevent programs protein function receptor training opportunity transcription factor undergraduate student

项目摘要

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.

概括内皮细胞（EC）是负责大部分胚胎血管形成的细胞类型，最终导致成年后估计达到100,000英里的脉管系统。在发育期间，新的血液血管从现有的脉管系统中出现，这是一种称为血管生成的过程。 Notch信号是基本的血管生成和成人血管稳态。在没有缺口的情况下，血管证明了慢性发芽表型以未经检查的增殖和过度生长为标志；这个证据和其他人绝大多数表明血管成熟和稳定性是必需的。当配体结合时，切割凹槽内结构域并易位到核，充当转录因子。类似三角洲配体4（DLL4）是一种有效的Notch配体，与Notch的细胞外结构域结合。引起缺口激活NOTCH的外域域需要DLL4的拉力来暴露S2裂解位点。一次暴露于凹口外域裂解，从而释放了凹口内结构域（NICD）刺激信号传导激活。关于DLL4的转录后调节知之甚少，关于DLL4施加持续的拉力激活的机制的理解甚至更少。缺口介导的侧向抑制。我们的初步数据首次描述了DLL4内吞作用和锚定在肌动蛋白细胞骨架上会产生暴露Notch S2位点所需的机械力。具体而言，该提案将集中于两种在很大程度上没有特征的蛋白质上，我们认为是武力的核心生成DLL4内吞作用，EPS15同源结构域结合蛋白1（EHBP1）和EH结构域含有蛋白2（EHD2）。我们广泛的假设是，EHBP1将EHD2锚定在F-肌动蛋白丝上，以帮助 DLL4与Notch结合的跨核细胞增多。在AIM1中，我们将详细说明EHBP1和EHD2如何工作促进DLL4内吞作用的组合，并在其中介导缺口信号传导。在AIM2中，我们将全面证明EHBP1和EDH2可以组合起来调节DLL4内吞作用和斑马鱼血管形态发生过程中的下游槽口活性，使用现场成像和crispr- 基于突变的产生。总体而言，该提案将回答几个至关重要的问题血管发展，并为本科学者提供强大的培训机会。

项目成果

期刊论文数量（8）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

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

DOI：
10.1038/s41467-022-32853-5
发表时间：
2022-09-08
期刊：
Nature communications
影响因子：
16.6
作者：
通讯作者：

Lipidure-based micropattern fabrication for stereotyping cell geometry.

DOI：
10.1038/s41598-023-47516-8
发表时间：
2023-11-22
期刊：
Scientific reports
影响因子：
4.6
作者：
通讯作者：

Trafficking in blood vessel development.

DOI：
10.1007/s10456-022-09838-5
发表时间：
2022-08
期刊：
Angiogenesis
影响因子：
9.8
作者：
Francis CR;Kushner EJ
通讯作者：
Kushner EJ

Correction to: Notch regulates vascular collagen IV basement membrane through modulation of lysyl hydroxylase 3 trafficking.

DOI：
10.1007/s10456-021-09801-w
发表时间：
2021-11
期刊：
Angiogenesis
影响因子：
9.8
作者：
Gross SJ;Webb AM;Peterlin AD;Durrant JR;Judson RJ;Raza Q;Kitajewski JK;Kushner EJ
通讯作者：
Kushner EJ

EHD2 modulates Dll4 endocytosis during blood vessel development.

DOI：
10.1111/micc.12740
发表时间：
2022-01
期刊：
MICROCIRCULATION
影响因子：
2.4
作者：
Webb, Amelia M.;Francis, Caitlin R.;Judson, Rachael J.;Kincross, Hayle;Lundy, Keanna M.;Westhoff, Dawn E.;Meadows, Stryder M.;Kushner, Erich J.
通讯作者：
Kushner, Erich J.