Molecular Analysis of Extracellular Matrix Assembly

细胞外基质组装的分子分析

• 批准号: 9519235
• 负责人: Jean E Schwarzbauer
• 金额: $ 3.24万
• 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 2018-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9519235
• 关键词: Actins; Address; Affect; Aging; Alpha Cell; 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; Dimerization; Disease; Disease Outcome; Disease Progression; ECM receptor; Environment; Extracellular Matrix; Extracellular Matrix Proteins; Fibronectins; Fibrosis; Foundations; Goals; Heparin; Homeostasis; In Vitro; Individual; Instruction; Integrin Binding; Integrins; Lead; Malignant Neoplasms; Maps; Mass Spectrum Analysis; Measures; Mediating; Mesenchymal Differentiation; Mesenchymal Stem Cells; Modeling; Modulus; Molecular Analysis; Molecular Conformation; Molecular Probes; Morphogenesis; Pathology; Polymers; Process; 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; experimental study; human disease; improved; insight; knock-down; mechanical properties; mutant; novel; physical property; physical state; polyacrylamide gels; polymerization; prevent; receptor; 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，伤口愈合以及疾病的起步和进展的重要组成部分。 ECM的物理和机械性能通过这些过程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。在发育和癌症，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大大变化。为了开发治疗以防止或扭转异常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在疾病中如何影响这些基质的分子相互作用。该提案的总体目标是确定周围细胞基质的组装如何支持并指导随后组装其他ECM组件以构建适合组织的确定性基质。纤连蛋白（FN）是一种普遍存在的ECM蛋白，在细胞表面受体和其他ECM成分之间建立了必不可少的联系。 FN通过细胞介导的工艺组装到纤维状ECM中，该过程涉及FN构象变化，以促进FN-FN相互作用，并与确定的FN矩阵达到顶点，这可以通过对去污剂脱氧酸（DOC）中的不溶性识别。理解适合组织的ECM组装的一个关键问题是依从性的效果，因为基质组装发生在刚度显着不同的组织中。在AIM 1中，我们将检验以下假设：细胞周围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基质组件。我们将使用定义的弹性模量的聚丙烯酰胺凝胶底物分析组装（整联蛋白结合，FN构象变化和DOC不溶性）的主要步骤。不溶性FN基质为组装其他ECM蛋白（包括一些胶原蛋白）的基础构成了基础，但我们对其结构的理解有限。在AIM 2中，我们将检验以下假设：特定的FN-FN相互作用介导了确定的文档不溶性矩阵的最后一步。突变重组FN将用于识别重要的相互作用残基，并将应用新颖的质谱屏幕来识别与DOC-Insoluble相互作用有关的其他FN域。为了了解FN组装，组织刚度和其他ECM蛋白的掺入之间的相互作用，在AIM 3中，我们将测试以下假设：FN矩阵控制组织分化模型中胶原蛋白的细胞命运和沉积，体外软骨发生。我们将操纵不溶性FN基质及其刚度的水平，并确定在间充质干细胞软骨分化过程中对分化标记和胶原蛋白组装的影响。通过拟议的研究获得的信息将提供有关正常基质组件机制的新见解，并将提出疾病可以改变组装以引起异常ECM积累的方法，例如纤维化，疤痕形成和癌症。