Role of Extracellular Matrix Assembly in a Tissue Differentiation Model Probed with a Novel Fibronectin Mutant

细胞外基质组装在用新型纤连蛋白突变体探测的组织分化模型中的作用

• 批准号: 10090477
• 负责人: Eli Benjamin Cadoff
• 金额: $ 5.1万
• 依托单位: RBHS-ROBERT WOOD JOHNSON MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-02-01 至 2023-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10090477
• 关键词: Actins; Affect; Binding; Binding Sites; Biological Assay; CRISPR/Cas technology; Cell Culture Techniques; Cell Differentiation process; Cell physiology; Cells; Child; Chondrogenesis; Collagen; Collagen Type I; Cytoskeleton; Defect; Dependence; Deposition; Dermal; Development; Diabetes Mellitus; Differentiation Antigens; Disease; Embryo; Exposure to; Extracellular Matrix; Extracellular Matrix Proteins; FN1 gene; Fibroblasts; Fibronectins; Future; Gene Expression; Growth Factor; Heart; Human; Individual; Integrins; Knock-out; Lead; Link; Liver Failure; Lung diseases; Maintenance; Malignant Neoplasms; Mediating; Metaphyseal dysplasia Pyle type; Modeling; Molecular Conformation; Mutation; Normal tissue morphology; Osteogenesis Imperfecta; Pathogenesis; Pathology; Pathway interactions; Physiological Processes; Physiology; Point Mutation; Process; Production; Proteins; Recombinants; Research; Role; Skeletal Development; Spondyloepiphyseal Dysplasia; Structural Protein; Testing; Time; Tissue Differentiation; Tissues; Work; base; cell behavior; cell growth; de novo mutation; dimer; experimental study; human disease; insight; migration; mutant; novel; polymerization; receptor; skeletal; skeletal abnormality; skeletal disorder; skeletal dysplasia; stem cells; support network

7. Project Summary The proposed work seeks to use a novel mutation in the extracellular matrix (ECM) protein fibronectin (FN) to study the role of proper ECM assembly in tissue development. The ability of cells to assemble and manipulate the ECM is crucial in tissue development, maintenance, and remodeling. Proper ECM production and function is at the heart of a staggering range of physiologic processes, while dysregulation of the ECM causes and contributes to many diseases. The study of mutations in various ECM components has been instrumental in understanding these proteins' processing, function, and significance in physiology and pathology. The FN matrix specifically is critical in early ECM formation, serving as an essential framework for the development of the ECM and in guiding the incorporation and assembly of other ECM proteins and growth factors into the matrix. Despite its importance, we lack information about how the FN matrix develops and how matrix assembly directs cell and ECM development. This in turn limits our ability to understand normal tissue physiology and to develop treatments for diseases caused by defects in ECM assembly and organization. Our limited understanding is in part due to a lack of naturally occurring FN mutations, as complete knockout of FN expression is lethal to the developing embryo. The recent discovery of a FN point mutation in an individual with disordered skeletal development represents a powerful opportunity to study how this point mutation in FN's assembly domain reduces the amount of ECM (Aim 1) and how reductions in matrix affect cellular behavior (Aim 2). Because FN matrix assembly is the first step in construction of a tissue-appropriate ECM, we hypothesize that perturbations of FN matrix assembly early in a developing tissue will disrupt the organization of the ECM and the cell rearrangements and changes in gene expression that are required for cell differentiation. To test this hypothesis, in aim 1, we will perform an analysis of why fibroblasts with a FN assembly domain mutation form reduced FN and type I collagen matrices. We will assess the ability of mutant FN to bind to other FN molecules, track the secretion and fates of different ECM proteins, and look at the impact of a FN mutation on the assembly of other ECM components. In aim 2, we will study the ability of these mutant stem cells to initiate the early stages of tissue development using a model for chondrogenic differentiation, and investigate the impact of deficiency in different ECM components on this development process. This work will provide novel understanding of the effects of a de novo mutation in human FN on matrix assembly, how ECM assembly directs tissue development and cell differentiation, and how perturbations in ECM assembly can lead to developmental defects and disease. Our characterization of this mutation will also lay the groundwork for thoroughly studying any other FN mutations implicated in human disease.

7。项目摘要 拟议的工作试图在细胞外基质（ECM）蛋白质纤连蛋白中使用新型突变 （FN）研究适当的ECM组装在组织发育中的作用。细胞组装的能力和 操纵ECM对于组织发育，维护和重塑至关重要。适当的ECM生产 功能是惊人的生理过程范围的核心，而ECM失调 原因并导致许多疾病。各种ECM组件中突变的研究已经 有助于理解这些蛋白质在生理学中的加工，功能和意义 病理。 FN矩阵在早期ECM组中特别至关重要，它是一个基本框架 ECM的开发并指导其他ECM蛋白质和增长的结合和组装 矩阵中的因素。尽管它很重要，但我们缺乏有关FN矩阵如何发展的信息 矩阵组装指导细胞和ECM开发。反过来，这限制了我们理解正常组织的能力 生理学并开发由ECM组装和组织中缺陷引起的疾病的治疗方法。我们的 有限的理解部分是由于缺乏天然发生的FN突变，作为FN的完全敲除 表达对发育中的胚胎是致命的。最近发现的一个人中发现了一个fn点突变 骨骼发育无序是一个有力的机会，可以研究FN中的这一点突变 组装域减少ECM的数量（AIM 1）以及基质的减少如何影响细胞行为 （目标2）。因为FN矩阵组件是构建适合组织的ECM的第一步，我们 假设在发育组织的早期FN矩阵组装的扰动将破坏组织 ECM和细胞重排以及细胞所需的基因表达变化 分化。为了检验这一假设，在AIM 1中，我们将对为什么具有FN的成纤维细胞进行分析 组装结构域突变形成降低FN和I型胶原蛋白矩阵。我们将评估突变体的能力 FN与其他FN分子结合，跟踪不同ECM蛋白的分泌和命运，并查看 FN突变对其他ECM组件组装的影响。在AIM 2中，我们将研究这些能力 突变干细胞使用软骨的模型开始组织发育的早期阶段 分化，并研究不同ECM组件中不同ECM组件缺乏对这一发展的影响 过程。这项工作将提供对从头突变对人FN的影响的新了解 基质组件，ECM组装如何指导组织发育和细胞分化，以及如何 ECM组装中的扰动会导致发育缺陷和疾病。我们对此的特征 突变还将为彻底研究与人类有关的任何其他FN突变奠定基础 疾病。