Defining the molecular and cellular bases of tissue compartmentalization

定义组织区室化的分子和细胞基础

基本信息

批准号：
10292120
负责人：
Adam Christopher Pare
金额：
$ 43.71万
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：
2021-09-01 至 2024-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10292120
关键词：
Actins Address Adhesions Affect Animals Architecture Binding Biochemical Biotin Brain region CRISPR/Cas technology Cell Surface Proteins Cell membrane Cells Cellular Morphology Complex Congenital Abnormality Cues Cytoskeletal Proteins Data Set Development Disease Drosophila genus Embryo Epithelial Event Excision Extracellular Domain Family Genes Genetic Genetic Engineering Genetic Techniques Genome engineering Growth Imaging Device Immunoprecipitation Insecta Integral Membrane Protein Invertebrates Knowledge Lead Length Light Limb Bud Link Location Mass Spectrum Analysis Mediating Membrane Messenger RNA Microscopy Molecular Morphology Mutation Nature Neoplasm Metastasis Neuroectoderm Nose Pattern Process Property Proteins RNA Interference Reporting Research Personnel Role Shapes Signal Transduction Somites Spatial Distribution Specimen Structure Study models Syndrome System Techniques Testing Tissues Transgenes Vertebrates Visualization Work base cell behavior cell motility convergent extension experimental study extracellular genetic manipulation high resolution imaging imaging approach in vivo insight intercalation knock-down leucine-rich repeat protein multiple myeloma M Protein nanobodies nanoscale overexpression physical property recruit response tool

项目摘要

Project Summary A conserved mechanism for keeping complex tissues organized during growth and remodeling is to separate groups of cells using compartment boundaries, which are multicellular actin-rich structures formed between adjacent cells. Compartment boundaries are typified by aligned “cables” of highly stable cell-cell interfaces, and they were first described in insect embryos nearly 40 years ago. Since then, similar boundary structures have been identified in vertebrates between different regions of the brain, gut, limb buds, and somites. While studies indicate that the loss of boundary integrity contributes to birth defects such as cranio-fronto-nasal syndrome and cancer metastasis, efforts to characterize the molecular underpinnings of these structures have been stymied by a lack of genetic tools for specifically targeting boundary cells. It was recently reported that two cell-surface proteins––the leucine-rich repeat protein Tartan and the teneurin Ten-m––are the direct spatial cues that initiate boundary formation in the Drosophila neuroectoderm. The identification of these upstream triggers finally makes it possible to answer long-standing questions concerning the nature and function of compartment boundaries. In this proposal, we will use a variety of genetic techniques to alter the expression patterns of Tartan and Ten-m in the neuroectoderm to address three significant knowledge gaps in the field. First, to identify the changes in membrane tension and adhesion that lead to boundary formation, we will use genetic engineering techniques to disrupt compartment boundaries and visualize cytoskeletal and junctional markers in live embryos. We will also use gene-swapping techniques to alter the location of boundaries to determine how their presence affects overall tissue architecture. Second, to determine how Tartan and Ten-m interact at a molecular level to trigger boundary formation, we will perform in vivo structure-function analyses to determine how Tartan controls the localization of Ten-m and which Ten-m extracellular domains are necessary for boundary formation. Third, to characterize the effector proteins downstream of Ten-m that give cell-cell interfaces at boundaries their unique physical properties, we will perform complementary biochemical and high-resolution imaging analyses. To identify putative Ten-m interaction partners, we will compare immunoprecipitation/mass-spectrometry analyses between embryos that have been enriched or depleted for compartment boundary cells. To directly visualize the nanoscale structure of compartment boundaries, we will use expansion microscopy to physically enlarge Drosophila embryos and analyze the distribution of cytoskeletal and junctional proteins that mediate cell morphology. Successful completion of this work will greatly enhance our knowledge of how compartment boundaries are formed and function. Our findings will also serve as a paradigm for understanding how these two widely expressed and developmentally important families––leucine-rich repeat proteins and teneurins––might interact in other developmental contexts.

项目概要在生长和重塑过程中保持复杂组织有序的保守机制是分离使用隔室边界的细胞群，隔室边界是在细胞之间形成的富含肌动蛋白的结构相邻细胞的边界以高度稳定的细胞-细胞界面的对齐“电缆”为代表，并且大约 40 年前，它们首次在昆虫胚胎中被描述，此后，类似的边界结构出现了。研究中已在脊椎动物的大脑、肠道、肢芽和体节的不同区域中发现了这种现象。表明边界完整性的丧失会导致出生缺陷，例如颅额鼻综合征和由于癌症转移，表征这些结构的分子基础的努力受到阻碍最近有报道称，由于缺乏专门针对边界细胞的遗传工具，有两种细胞表面。蛋白质——富含亮氨酸的重复蛋白 Tartan 和 tenurin Ten-m——是启动的直接空间线索果蝇神经外胚层中边界的形成最终使得这些上游触发器的识别成为可能。有可能回答有关隔间边界的性质和功能的长期存在的问题。在这个提案中，我们将使用多种遗传技术来改变Tartan和Ten-m的表达模式神经外胚层解决该领域的三个重大知识空白首先，确定神经外胚层的变化。导致边界形成的膜张力和粘附力，我们将利用基因工程技术来我们还将破坏活胚胎中的隔室边界并可视化细胞骨架和连接标记。使用基因交换技术来改变边界的位置，以确定它们的存在如何影响整体其次，确定 Tartan 和 Ten-m 如何在分子水平上相互作用以触发边界。形成后，我们将进行体内结构功能分析，以确定 Tartan 如何控制定位十米以及哪些十米细胞外结构域对于边界形成是必需的第三，表征。 Ten-m 下游的效应蛋白赋予边界处的细胞-细胞界面独特的物理特性性质，我们将进行补充生化和高分辨率成像分析。假定的十米相互作用伙伴，我们将比较免疫沉淀/质谱分析已富集或耗尽隔室边界细胞的胚胎直接可视化。隔室边界的纳米级结构，我们将使用膨胀显微镜进行物理放大果蝇胚胎并分析介导细胞的细胞骨架和连接蛋白的分布这项工作的成功完成将极大地增强我们对隔室结构的了解。我们的研究结果也将作为理解这两者如何形成和发挥作用的范例。广泛表达且对发育重要的家族——富含亮氨酸的重复蛋白和 tenurins——可能在其他发展环境中相互作用。