Defining the molecular and cellular bases of tissue compartmentalization

定义组织区室化的分子和细胞基础

• 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
• 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

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.

项目摘要 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，隔室边界是多细胞肌动蛋白丰富的结构， 相邻的细胞combustion边界的典型代表是高度稳定的细胞-细胞界面的对齐“电缆”， 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 在脊椎动物的脑、肠、肢芽和体节的不同区域之间被鉴定。虽然研究 表明边界完整性的丧失会导致出生缺陷，如颅额鼻综合征， 由于癌症转移，表征这些结构的分子基础的努力受到了阻碍 缺乏专门针对边界细胞的遗传工具。最近有报道称，两种细胞表面 富含亮氨酸的重复蛋白质Tartan和端神经元Ten-m是直接的空间线索， 果蝇神经外胚层的边界形成。这些上游触发器的识别最终使得 它可以回答有关区划边界的性质和功能的长期存在的问题。在 根据这项建议，我们将使用各种遗传技术来改变Tartan和Ten-m在 神经外胚层，以解决该领域的三个重大知识差距。第一，确定 膜张力和粘附导致边界形成，我们将使用基因工程技术， 破坏隔室边界并使活胚胎中的细胞骨架和连接标记可视化。我们还将 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 组织结构其次，为了确定Tartan和Ten-m如何在分子水平上相互作用以触发边界， 我们将进行体内结构-功能分析，以确定Tartan如何控制定位 以及哪些十m细胞外结构域是边界形成所必需的。第三，定性 Ten-m下游的效应蛋白在边界处给予细胞-细胞界面其独特的物理性质， 我们将进行互补的生化和高分辨率成像分析。以识别 假定的Ten-m相互作用伙伴，我们将比较免疫沉淀/质谱分析之间 已经富集或耗尽隔室边界细胞的胚胎。为了直接可视化 纳米结构的分隔边界，我们将使用扩展显微镜物理放大 果蝇胚胎中的细胞骨架蛋白和连接蛋白的分布， 形态学这项工作的成功完成将大大提高我们的知识如何分室 边界形成并发挥作用。我们的发现也将作为一个范例，了解这两个 广泛表达和发育重要的家族--富含亮氨酸的重复蛋白和端蛋白--可能 在其他发展背景下互动。