Molecular Analysis of Extracellular Matrix Assembly

细胞外基质组装的分子分析

• 批准号: 8827275
• 负责人: Jean E Schwarzbauer
• 金额: $ 32.55万
• 依托单位: PRINCETON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-05-01 至 2016-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8827275
• 关键词: Actins; Address; Affect; Aging; Antibodies; Binding; Biological Assay; Cell Surface Receptors; Cell physiology; Cells; Chondrogenesis; Cicatrix; Collagen; Collagen Type II; Connective Tissue; Connective Tissue Diseases; Cues; Cytoskeleton; Defect; Deposition; Detergents; Development; Differentiation Antigens; Disease; Disease Outcome; Disease Progression; Environment; Extracellular Matrix; Extracellular Matrix Proteins; Fibronectins; Fibrosis; Foundations; Goals; Heparin; Homeostasis; Individual; Integrin Binding; Integrins; Lead; Malignant Neoplasms; Maps; Mass Spectrum Analysis; Measures; Mechanics; Mediating; Mesenchymal Stem Cells; Molecular Analysis; Molecular Conformation; Molecular Probes; Morphogenesis; Pathology; Process; Property; Proteins; Reaction; Recombinant Proteins; Recombinants; Role; Signal Transduction; Small Interfering RNA; Structure; Testing; Tissue Differentiation; Tissues; Wound Healing; base; cell growth; crosslink; deoxycholate; design; dimer; human disease; improved; in vitro Model; insight; mutant; novel; physical state; polyacrylamide gels; polymerization; prevent; receptor; research study; treatment strategy; tumorigenesis

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.

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 的物理和机械特性通过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癌症、纤维化和结缔组织异常等病理过程中，ECM 的成分和物理状态可能会发生巨大变化。为了开发治疗方法来预防或逆转异常 ECM 的影响，我们需要了解组装适合组织的基质的分子相互作用以及它们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其他 ECM 组件的后续组装，以构建适合组织的最终基质。纤连蛋白 (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，该基质可通过去污剂脱氧胆酸盐 (DOC) 中的不溶性来识别。了解适合组织的 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硬度显着不同的组织中。在目标 1 中，我们将测试细胞周 ECM 的刚度通过调节组装过程中的一个或多个关键步骤来控制 FN 矩阵组装的假设。我们将使用具有确定弹性模量的聚丙烯酰胺凝胶基质分析组装的主要步骤（整合素结合、FN 构象变化和 DOC 不溶性），并使用构象敏感抗体测试在不同硬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，我们将检验以下假设：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。突变重组 FN 将用于鉴定重要的相互作用残基，并且将应用新型质谱筛选来鉴定参与 DOC 不溶性相互作用的其他 FN 结构域。为了了解 FN 组装、组织硬度和其他 ECM 蛋白掺入之间的相互作用，在目标 3 中，我们将测试 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蛋白沉积的假设。我们将操纵不溶性 FN 基质的水平及其硬度，并确定间充质干细胞软骨分化过程中对分化标记物和胶原蛋白组装的影响。通过拟议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，并将提出疾病改变组装导致异常 ECM 积累（如纤维化、疤痕形成和癌症）的方法。