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：
10574572
负责人：
Adam Christopher Pare
金额：
$ 20.63万
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 2023-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10574572
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 Clustered Regularly Interspaced Short Palindromic Repeats Complex Congenital Abnormality Consumption Cytoskeleton Data Analyses Data Science Defect Development Disease Drosophila genus Embryo Epithelial Cells Epithelium 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 Intercalated Cell 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 Visualization Work cell behavior cofactor convergent extension data integration epithelial wound experimental study functional loss gain of function in vivo evaluation insight intercalation loss of function member multiphoton microscopy mutant novel overexpression receptor receptor expression receptor function repaired rho 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 Adam Paré 上皮细胞在发育过程中形状和相对位置发生显着变化，以构建适当的结构。组织结构的错误直接导致一些最常见的类型。先天性畸形——神经管缺陷——影响大约 2000 名新生儿中的 1 名。控制上皮形态的上游信号仍然知之甚少。重塑是果蝇神经外胚层中的细胞嵌入，研究表明，重塑是果蝇神经外胚层中的三个成员高度保守的 Toll 受体家族以重叠的条纹模式表达以组织快速细胞 Toll 受体在该组织中广泛表达，并且它们具有然而，细胞形态的控制在先天免疫信号传导的背景下得到了广泛的研究。 Toll 受体受到的关注很少，该提案的重点是了解 Toll 的非均匀性。受体表达影响皮质张力、细胞间粘附和线粒体动力学以控制细胞形状我们将使用新开发的 CRISPR/Cas9 衍生的基因。背景和抗体来表征 Toll 受体如何控制细胞极性以触发嵌入；将应用非破坏性技术来表征完整活体中上皮重组的生物能学胚胎；我们将研究 Toll 受体、Rho 和 G 蛋白偶联受体之间未解决的联系我们的第一个假设是邻近细胞感知个体 Toll 表达的差异。增加皮质张力和减少细胞间粘附的受体类型我们开发了一个遗传系统。用于在单个条带中表达单个受体，我们将用它来系统地表征和比较每种 Toll 受体类型对细胞形态的影响，并鉴定所需的蛋白质结构域我们的第二个假设是神经外胚层期间细胞的快速重排。伸长需要线粒体结构的特定变化来促进细胞骨架和连接为了测试这一点，我们将使用安捷伦海马通量分析仪来测量耗氧率。和多光子显微镜，以可视化上皮重塑过程中活胚胎中的细胞氧化还原状态，以及然后使用功能获得和丧失技术来确定线粒体融合和裂变在其中发挥什么作用我们的第三个假设是 Toll 受体和 GPCR 信号汇聚以激活 Rho。激酶专门在神经外胚层中触发细胞嵌入，我们将使用功能的获得和丧失。分析以确定这两个信号通路如何交叉来控制皮质张力、细胞间粘附、成功完成这些实验将为我们提供上皮重塑过程中的线粒体动力学。了解 Toll 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过程中，这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。