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：
10737093
负责人：
Adam Christopher Pare
金额：
$ 31.15万
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：
2023
资助国家：
美国
起止时间：
2023-09-01 至 2028-07-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10737093
关键词：
Actomyosin Address Adhesions Affect Animals Antibodies Architecture Area Atlases Attention Bioenergetics Biomechanics CRISPR/Cas technology Cell Adhesion Inhibition Cell Polarity Cell Shape Cell membrane Cell-Cell Adhesion Cells Cellular Metabolic Process Cellular Morphology Chronic Clustered Regularly Interspaced Short Palindromic Repeats Complex Congenital Abnormality Cytoskeleton Data Defect Development Developmental Biology Disease Drosophila genus Embryo Embryonic Development Embryonic Structures Engineering Epithelial Cells Epithelium Family Fluorescence G Protein-Coupled Receptor Signaling G-Protein-Coupled Receptors GPCR Signaling Pathway Genetic Genetic Techniques Genome engineering Genomics Health Homeostasis Human Human Pathology Immune signaling Impaired wound healing Individual Inflammation Intercalated Cell Label Light Link Malignant Neoplasms Measures Mediator Metabolic Metabolism Microscopy Mitochondria Modeling Molecular Morphology Multiphoton Fluorescence Microscopy Mutation Myosin ATPase NADH Nature Neoplasm Metastasis Neuroectoderm Oxidation-Reduction Pathway interactions Pattern Play Positioning Attribute Process Production Proteins Receptor Activation Receptor Gene Receptor Signaling Regulation Rho-associated kinase Role Shapes Signal Pathway Signal Transduction Stereotyping Structure Study models System Techniques Tertiary Protein Structure Testing Tissues Toll-like receptors Transgenic Organisms Visualization cell behavior cofactor convergent extension epithelial wound experimental study fluorescence lifetime imaging functional loss gain of function in vivo intercalation loss of function member model organism multiphoton microscopy receptor receptor expression receptor function rho seal tumor wound healing

项目摘要

Project Summary During development, epithelial cells undergo programmed changes in morphology and position to create complex tissues. Studies in model organisms have identified a conserved set of effector proteins that directly alter cell shape, although the upstream pathways that coordinate these processes across large groups of cells 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 changes in mitochondrial signaling to drive cytoskeletal and junctional reorganization. To test this, we will use multiphoton microscopy to visualize the endogenous autofluorescence of metabolic cofactors to quantify 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 and trigger cell intercalation 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, and mitochondrial dynamics during epithelial remodeling. Successful completion of these experiments will give us a more comprehensive understanding of how Toll receptors function at a molecular level to control cellular biomechanics and bioenergetics during dynamic tissue remodeling.

项目摘要在开发过程中，上皮细胞经历了形态和位置的编程变化以创建复杂的组织。模型生物的研究已经确定了一组保守的效应蛋白，直接改变细胞形状，尽管上游途径是在大量细胞上协调这些过程的上游途径保持不当理解。用于研究上皮重塑的范式是果蝇中的细胞插入神经抑制剂，并且表明表达高度保守的收费受体家族的三个成员在重叠的条纹图案中，以组织该组织中的快速细胞重排。收费受体广泛在整个人类上皮中表达，它们在先天免疫的背景下进行了广泛的研究信号。但是，收费受体对细胞形态的控制很少受到关注。重点该建议的是了解不均匀的通行器受体表达如何影响皮层张力，细胞细胞粘附和线粒体动力学，以控制上皮重塑过程中细胞形状和行为。我们将使用新开发的CRISPR/CAS9衍生的遗传背景和抗体来表征损失受体控制细胞极性以触发插入；我们将采用非破坏性技术来表征完整活胚胎中上皮重组的生物能学；我们将调查未解决的链接在收费受体，RHO和G蛋白偶联受体信号传导之间。我们的第一个假设是细胞在单个收费受体类型的表达中感知差异，以增加皮质张力和减少细胞细胞粘附。我们已经开发了一个遗传系统，用于在一个单一的中表达单个受体我们将使用的条纹系统地表征和比较每种收费受体类型对细胞的影响形态并确定调节细胞形状所需的蛋白质结构域。我们的第二个假设是神经外胚层伸长过程中的快速细胞重排需要改变线粒体信号转导至驱动细胞骨架和连接重组。为了测试这一点，我们将使用多光子显微镜可视化代谢辅助因子的内源性自动荧光，以量化活细胞氧化还原状态在活胚中上皮重塑，然后使用功能丧失技术来确定线粒体的作用上皮重组中的融合和裂变发挥。我们的第三个假设是Toll受体和GPCR信号传导收敛以激活Rho激酶并触发神经外胚层中的细胞插入。我们将使用增益和损失 - 官能分析以确定这两种信号通路如何相交以控制皮质张力，细胞 - 细胞粘附和上皮重塑过程中的线粒体动力学。这些成功完成实验将使我们对收费受体如何在分子水平的功能方面有更全面的了解控制动态组织重塑期间的细胞生物力学和生物能力。