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进展的重要组成部分。ECM的物理和机械性质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剂。在发育过程中以及在诸如癌症、纤维化和结缔组织异常的病理中，ECM的组成和物理状态可以显著变化。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分子相互作用在疾病中如何受到影响。该提案的总体目标是确定细胞周基质的组装如何支持和指导额外ECM组分的后续组装以构建组织合适的最终基质。纤连蛋白（FN）是一种普遍存在的ECM蛋白，其在细胞表面受体和其他ECM组分之间形成必要的连接。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，该基质可以通过在去污剂脱氧胆酸盐（DOC）中的不溶解性来识别。理解组织适当的ECM组装的关键问题是顺应性的影响，因为基质组装发生在硬度显著不同的组织中。在目的1中，我们将测试的假设，即硬度的细胞外基质管理FN矩阵组装通过调节一个或多个关键步骤中的组装过程。我们将分析的主要步骤的组装（整合素结合，FN构象变化，DOC不溶性）使用聚丙烯酰胺凝胶基板的定义的弹性模量，并将测试的想法，FN构象不同的矩阵组装在不同的刚度条件下使用构象敏感的抗体。不溶性FN基质形成了其他ECM蛋白（包括一些胶原蛋白）组装的基础，但我们对其结构的了解有限。在目标2中，我们将检验特定的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。突变重组FN将用于识别重要的相互作用残基和一种新的质谱筛选将应用于识别其他FN结构域参与DOC不溶性相互作用。为了理解FN组装、组织硬度和其他ECM蛋白的掺入之间的相互作用，在目的3中，我们将检验FN基质在组织分化模型（体外软骨形成）中控制细胞命运和胶原沉积的假设。我们将操纵不溶性FN基质的水平及其硬度，并确定在间充质干细胞的软骨分化过程中对分化标志物和胶原组装的影响。通过所提出的研究获得的信息将提供对正常基质组装机制的新见解，并将提出疾病可以改变组装以引起异常ECM积累的方式，如纤维化、瘢痕形成和癌症。

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.