Control of epithelial morphology and bioenergetics by Toll receptors during dynamic tissue remodeling

动态组织重塑过程中 Toll 受体对上皮形态和生物能的控制

基本信息

批准号：
10357749
负责人：
Adam Christopher Pare
金额：
$ 21.27万
依托单位：
UNIVERSITY OF ARKANSAS AT FAYETTEVILLE
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：
2021-04-01 至 2026-02-28
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10357749
关键词：
Adhesions Affect Antibodies Architecture Arkansas Attention Bioenergetics Biological Biomechanics Birth CRISPR/Cas technology Cell Adhesion Inhibition Cell Polarity Cell Shape Cell-Cell Adhesion Cells Cellular Morphology Complex Congenital Abnormality Consumption Data Analyses Data Science Defect Development Disease Drosophila genus Embryo Epithelial Epithelial Cells Family Fluorescence G Protein-Coupled Receptor Signaling G-Protein-Coupled Receptors GPCR Signaling Pathway Genetic Genus Hippocampus Glycolysis Goals Human Human Pathology Image Immune signaling Individual Lead Light Link Measures Mentors Metabolic Metabolism Mitochondria Molecular Molecular Probes Morphogenesis Morphology NADH Natural Immunity Nature Neural Tube Defects Oxidation-Reduction Oxidative Phosphorylation Oxygen Consumption Pathway interactions Pattern Play Positioning Attribute Process Receptor Signaling Research Rho-associated kinase Role Shapes Signal Pathway Signal Transduction Structure Study models System Techniques Tertiary Protein Structure Testing Tissues Work base cell behavior cofactor convergent extension epithelial wound experimental study functional loss in vivo evaluation insight intercalation loss of function member multiphoton microscopy mutant novel overexpression receptor receptor expression receptor function repaired rho rho GTP-Binding Proteins wound closure

项目摘要

Project Summary – Project Leader Adam Paré Epithelial cells undergo significant changes in shape and relative position during development to build proper tissue architecture. Errors in epithelial remodeling directly contribute to some of the most common types of congenital abnormalities––neural tube defects––which affect approximately 1 in 2000 births. However, the upstream signals that control epithelial morphology remain poorly understood. A paradigm for studying epithelial remodeling is cell intercalation in the Drosophila neurectoderm, and it was shown that three members of the highly conserved Toll receptor family are expressed in overlapping striped patterns to organize rapid cell rearrangements in this tissue. Toll receptors are widely expressed throughout human epithelia, and they have been extensively studied in the context of innate immune signaling. However, the control of cell morphology by Toll receptors has received very little attention. The focus of this proposal is to understand how non-uniform Toll receptor expression affects cortical tension, cell-cell adhesion, and mitochondrial dynamics to control cell shape and behavior during epithelial remodeling. We will use newly developed CRISPR/Cas9-derived genetic backgrounds and antibodies to characterize how Toll receptors control cell polarity to trigger intercalation; we will apply non-destructive techniques to characterize the bioenergetics of epithelial reorganization in intact living embryos; and we will investigate unaddressed links between Toll receptor, Rho, and G protein-coupled receptor signaling. Our first hypothesis is that neighboring cells sense differences in the expression of individual Toll receptor types to increase cortical tension and decrease cell-cell adhesion. We have developed a genetic system for expressing individual receptors in a single stripe that we will use to systematically characterize and compare the effects of each Toll receptor type on cell morphology and to identify the protein domains necessary for modulating cell shape. Our second hypothesis is that rapid cellular rearrangements during neurectoderm elongation require specific changes in mitochondrial architecture to fuel cytoskeletal and junctional reorganization. To test this, we will use the Agilent Seahorse flux analyzer to measure oxygen consumption rates and multiphoton microscopy to visualize cellular redox state in live embryos during epithelial remodeling, and then use gain- and loss-of-function techniques to determine what role mitochondrial fusion and fission play in epithelial reorganization. Our third hypothesis is that Toll receptor and GPCR signaling converge to activate Rho Kinase to trigger cell intercalation specifically in the neurectoderm. We will use gain- and loss-of-functional analyses to determine how these two signaling pathways intersect to control cortical tension, cell-cell adhesion, mitochondrial dynamics during epithelial remodeling. Successful completion of these experiments will give us an understanding of how Toll receptors function at a molecular level to control cell morphology and bioenergetics during dynamic tissue remodeling, which could shed light on the cell biological underpinning of neural tube defects and other epithelia-based diseases.

项目摘要 - 项目负责人亚当·帕雷（AdamParé）上皮细胞在发育过程中的形状和相对位置发生重大变化以构建正确组织结构。上皮重塑的错误直接导致了一些最常见的类型先天性异常 - 神经管缺陷 - 在2000年出生中大约有1个。但是，上游信号表明，控制上皮形态仍然知之甚少。研究上皮的范式重塑是果蝇神经毒性中的细胞插入，结果表明三个成员高度保守的收费受体家族以重叠的条纹图案表示，以组织快速细胞该组织中的重排。收费接收器在整个人类上皮中得到广泛表达，并且在先天免疫信号传导的背景下进行了广泛研究。但是，通过收费接收器几乎没有受到关注。该提议的重点是了解不均匀的损失受体表达会影响皮质张力，细胞细胞粘合剂和线粒体动力学以控制细胞形状和上皮重塑期间的行为。我们将使用新开发的CRISPR/CAS9衍生的通用背景和抗体表征通行器接收器如何控制细胞极性触发插入；将采用非破坏性技术来表征完整生活中上皮重组的生物能学胚胎；我们将研究收费受体，RHO和G蛋白偶联受体之间的未解决的联系信号。我们的第一个假设是，相邻细胞在单个通行的表达中感觉差异受体类型增加皮质张力并降低细胞细胞粘合剂。我们已经开发了一个遗传系统用于在单个条纹中表达单个接收器，我们将使用该条纹来系统地表征和比较每种收费受体类型对细胞形态的影响，并确定所需的蛋白质结构域调节细胞形状。我们的第二个假设是神经诊所中的快速细胞重排伸长需要线粒体结构的特定变化以燃料细胞骨架和交界处重组。为了测试这一点，我们将使用安捷伦的海马通量分析仪来测量氧气消耗率和多光子显微镜，可在上皮重塑期间可视化活胚中的细胞氧化还原状态，并且然后使用功能丧失技术来确定线粒体融合和裂变在上皮重组。我们的第三个假设是Toll受体和GPCR信号转化以激活Rho 激酶在神经涂片性中特别触发细胞插入。我们将使用功能障碍分析以确定这两个信号通路如何相交以控制皮层张力，细胞 - 细胞粘合剂，上皮重塑过程中的线粒体动力学。这些实验的成功完成将为我们提供了解通行器受体如何在分子水平上发挥作用来控制细胞形态和生物能学在动态组织重塑期间，可以揭示神经管的细胞生物学基础缺陷和其他基于上皮的疾病。