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

7.项目总结 这项拟议的工作试图利用细胞外基质(ECM)蛋白纤维连接蛋白中的一种新突变 (Fn)研究适当的细胞外基质组装在组织发育中的作用。细胞的组装能力和 操纵细胞外基质在组织的发育、维持和重塑过程中至关重要。适当的ECM生产 而功能是一系列令人震惊的生理过程的核心，而细胞外基质的失调 导致和促成许多疾病。已经对各种ECM成分的突变进行了研究 有助于理解这些蛋白质的加工、功能和在生理学和生物学上的意义 病理学。FN矩阵在早期ECM的形成中尤其关键，它是 细胞外基质的发展以及在指导其他细胞外基质蛋白的掺入和组装和生长方面的作用 在矩阵中加入各种因素。尽管它很重要，但我们缺乏关于FN矩阵如何发展以及如何发展的信息 矩阵组装指导电池和ECM的开发。这反过来又限制了我们理解正常组织的能力 并开发针对ECM组装和组织缺陷引起的疾病的治疗方法。我们的 有限的认识部分是由于缺乏自然发生的FN突变，作为FN的完全敲除 表达对发育中的胚胎是致命的。最近在一名患有黄斑狼疮的患者中发现了FN点突变 骨骼发育紊乱为研究FN的这种点突变提供了一个强有力的机会 组装域减少细胞外基质的数量(目标1)以及基质的减少如何影响细胞行为 (目标2)。由于FN基质组装是构建适合组织的ECM的第一步，我们 假设FN基质组装在发育早期的组织中的扰动将扰乱组织 细胞外基质以及细胞所需的细胞重排和基因表达的变化 差异化。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带有FN 装配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，并查看 FN突变对其他ECM组件组装的影响。在目标2中，我们将研究这些能力 突变干细胞利用软骨形成模型启动组织发育的早期阶段 分化，并调查不同ECM成分缺陷对这一发展的影响 进程。这项工作将对人类FN从头突变的影响提供新的理解。 基质组装，ECM组装如何引导组织发育和细胞分化，以及如何 ECM组装过程中的紊乱会导致发育缺陷和疾病。我们对此的描述 突变还将为深入研究与人类有关的任何其他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.