Molecular Analysis of Extracellular Matrix Assembly

描述（由申请人提供）：细胞外基质（ECM）组装是发育、组织稳态、衰老、伤口愈合以及疾病发生和发展的重要组成部分。ECM的物理和机械特性通过其对细胞组织和信号传导的影响而成为这些过程的关键调节因子。在发育过程中，在癌症、纤维化和结缔组织异常等病理中，ECM的组成和物理状态可能会发生巨大变化。为了开发预防或逆转异常ECM影响的治疗方法，我们需要了解组装组织适当基质的分子相互作用以及这些相互作用在疾病中如何受到影响。本提案的总体目标是确定细胞周围基质的组装如何支持和指导后续额外ECM成分的组装，以构建适合组织的最终基质。纤维连接蛋白（FN）是一种普遍存在的ECM蛋白，它在细胞表面受体和其他ECM成分之间建立了重要的联系。FN通过细胞介导的过程组装成纤维状ECM，该过程涉及FN构象变化以促进FN-FN相互作用，并最终形成最终的FN基质，该基质可通过在洗涤剂脱氧胆酸盐（DOC）中的不溶性来识别。理解适合组织的ECM组装的一个关键问题是顺应性的影响，因为基质组装发生在刚度差异很大的组织中。在Aim 1中，我们将通过调节装配过程中的一个或多个关键步骤来检验细胞外ECM的刚度控制FN矩阵组装的假设。我们将使用弹性模量确定的聚丙烯酰胺凝胶底物分析组装的主要步骤（整合素结合、FN构象变化和DOC不溶性），并将使用构象敏感抗体测试在不同刚度条件下组装的基质中FN构象不同的想法。不溶性FN基质构成了其他ECM蛋白（包括一些胶原）组装的基础，但我们对其结构的了解有限。在目标2中，我们将检验特定FN-FN相互作用介导最终DOC-不溶性基质组装的最后一步的假设。突变型重组FN将用于鉴定重要的相互作用残基，新型质谱筛选将用于鉴定doc不溶性相互作用中涉及的其他FN结构域。为了了解FN组装、组织刚度和其他ECM蛋白结合之间的相互作用，在Aim 3中，我们将在组织分化模型（体外软骨形成）中测试FN基质控制细胞命运和胶原沉积的假设。我们将操纵不溶性FN基质的水平及其刚度，并确定在间充质干细胞软骨分化过程中对分化标志物和胶原组装的影响。通过提出的研究获得的信息将为正常基质组装的机制提供新的见解，并将提出疾病可以改变组装导致异常ECM积累的方法，如纤维化，疤痕形成和癌症。

DESCRIPTION (provided by applicant): Extracellular matrix (ECM) assembly is an essential part of development, tissue homeostasis, aging, wound healing, and initiation and progression of disease. The physical and mechanical properties of the ECM are emerging as critical regulators of these processes through their effects on cell organization and signaling. During development and in pathologies such as cancer, fibrosis, and connective tissue abnormalities, the composition and the physical state of the ECM can vary dramatically. To develop treatments to prevent or reverse the effects of abnormal ECM, we need to understand the molecular interactions that assemble a tissue-appropriate matrix and how these are affected in disease. The overall goal of this proposal is to determine how the assembly of the pericellular matrix supports and directs the subsequent assembly of additional ECM components to build a tissue-appropriate definitive matrix. Fibronectin (FN) is a ubiquitous ECM protein that makes an essential connection between cell surface receptors and other ECM components. FN is assembled into a fibrillar ECM via a cell-mediated process that involves FN conformational changes to promote FN-FN interactions and culminates with the definitive FN matrix that can be identified by insolubility in the detergent deoxycholate (DOC). A critical question for understanding tissue-appropriate ECM assembly is the effect of compliance since matrix assembly occurs in tissues that differ significantly in stiffness. In Aim 1 we will test the hypothesis that stiffness of the pericellular ECM governs FN matrix assembly by regulating one or more key steps in the assembly process. We will analyze the major steps of assembly (integrin binding, FN conformational changes, and DOC insolubility) using polyacrylamide gel substrates of defined elastic modulus and will test the idea that FN conformation differs in matrices assembled under different stiffness conditions using a conformation-sensitive antibody. The insoluble FN matrix forms the foundation for assembly of other ECM proteins, including some collagens, yet we have limited understanding of its structure. In Aim 2, we will test the hypothesis that specific FN-FN interactions mediate the final step in assembly of the definitive DOC- insoluble matrix. Mutant recombinant FNs will be used to identify important interacting residues and a novel mass spectrometry screen will be applied to identify other FN domains involved in DOC-insoluble interactions. To understand the interplay between FN assembly, tissue stiffness, and incorporation of other ECM proteins, in Aim 3 we will test the hypothesis that FN matrix controls both cell fate and deposition of collagens in a tissue differentiation model, in vitro chondrogenesis. We will manipulate the level of insoluble FN matrix and its stiffness and determine the effects on differentiation markers and assembly of collagens during chondrogenic differentiation of mesenchymal stem cells. The information obtained through the proposed studies will provide novel insights into the mechanisms of normal matrix assembly and will suggest ways in which disease can change assembly to cause abnormal ECM accumulation as in fibrosis, scar formation, and cancer.