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

7.项目摘要 拟议的工作旨在利用一种新的突变细胞外基质（ECM）蛋白纤连蛋白 (FN)研究适当的ECM组装在组织发育中的作用。细胞的组装能力， 操纵ECM在组织发育、维持和重塑中至关重要。正确的ECM生产 和功能是一系列惊人的生理过程的核心，而ECM的失调 导致并促成了许多疾病。各种ECM成分中突变的研究已经被广泛应用。 有助于了解这些蛋白质的加工，功能和生理学意义， 病理FN基质在早期ECM形成中特别重要，作为ECM形成的基本框架。 ECM的发展和指导其他ECM蛋白的掺入和组装以及生长 因素进入矩阵。尽管新军矩阵很重要，但我们缺乏关于新军矩阵如何发展以及 基质组装指导细胞和ECM发育。这反过来又限制了我们理解正常组织的能力 本发明的目的是提供一种新的治疗方法，用于治疗由ECM组装和组织缺陷引起的疾病。我们 有限的理解部分是由于缺乏自然发生的FN突变，因为FN的完全敲除 表达对发育中的胚胎是致命的。最近发现的FN点突变的个人与 骨骼发育紊乱是研究FN中的点突变 组装域减少ECM的量（目的1）以及基质减少如何影响细胞行为 (Aim 2）。由于FN基质组装是构建组织合适ECM的第一步，我们 假设发育组织早期FN基质组装的扰动将破坏组织 ECM和细胞重排以及细胞生长所需的基因表达的变化， 分化为了验证这一假设，在目标1中，我们将分析为什么成纤维细胞与FN 组装结构域突变形成还原的FN和I型胶原基质。我们将评估变种人的能力 FN与其他FN分子结合，跟踪不同ECM蛋白的分泌和命运，并观察 FN突变对其他ECM组分组装的影响。在目标2中，我们将研究这些 突变干细胞启动组织发育的早期阶段，使用软骨形成模型， 分化，并研究不同ECM成分的缺乏对这种发展的影响 过程这项工作将提供新的理解的影响，从头突变的人FN的， 基质组装，ECM组装如何指导组织发育和细胞分化， ECM组装中的扰动可导致发育缺陷和疾病。我们对此的描述 这种突变也将为彻底研究人类中涉及的任何其他FN突变奠定基础。 疾病

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.