Matrix Organization and Dimensionality

矩阵组织和维度

• 批准号: 7733931
• 负责人: Kenneth Yamada
• 金额: $ 72.3万
• 依托单位: NATIONAL INSTITUTE OF DENTAL & CRANIOFACIAL RESEARCH
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7733931
• 关键词: Ablation; Address; Adhesives; Adult; Amino Acids; Biochemical; Biological; Biological Process; Cell Adhesion; Cell Communication; Cell physiology; Cell surface; Cells; Characteristics; Collagen; Complex; Condition; Connective Tissue; Cues; Cultured Cells; Cytoplasmic Tail; Data; Defect; Embryonic Development; Environment; Extracellular Matrix; Fibrin; Fibroblasts; Fibronectins; Gel; Goals; Homeostasis; Human; Integrins; Mediating; Methods; Microscopy; Molecular; Mus; Mutate; Myosin Type II; Nature; Neural Crest; Nonmuscle Myosin Type IIA; Play; Process; Protein Isoforms; Protein translocation; Proteins; Publishing; Regulation; Role; Signal Transduction; Site; Staging; Structure; System; Technology; Testing; Tissues; Wound Healing; base; cell behavior; cell motility; craniofacial; crosslink; in vivo; migration; mutant; novel; physical property; response; tissue/cell culture; two-dimensional

Cells interact with distinct types of extracellular matrix in different tissues and at different stages of embryonic development and during adult wound repair. This project focuses on addressing the following major questions concerning the mechanisms of these cell-extracellular matrix interactions: 1. How do cells assemble a three-dimensional (3D) extracellular matrix, particularly one that is based on fibronectin? 2. What are the differences in cell adhesive structures and biological responses between 2D and 3D matrices, as well as between different types of 3D matrices characteristic of different in vivo microenvironments? 3. What unique mechanisms control cell behavior in 3D microenvironments, especially those without oriented topological cues? We previously analyzed the molecular machinery mediating integrin-based assembly of a 3D fibronectin matrix and published evidence for the role of integrin activation and concerted protein translocation to generate fibronectin fibrils. We are continuing to characterize the regulation of matrix assembly, signal transduction, and cytoskeletal mechanisms using a set of novel integrin mutants we have generated that display specific defects in fibronectin matrix assembly. Our recent preliminary findings using these mutants suggest that fibronectin matrix assembly depends on multiple sites in the integrin cytoplasmic domain. Depending on the amino acid residue that is mutated, we also find that the process of matrix assembly can be disrupted independently from the process of integrin-mediated cell spreading. We are exploring the effects of these mutants on integrin activation state and on multimolecular cell adhesion complexes. We previously published evidence for the importance of the three-dimensionality of the extracellular matrix surrounding fibroblasts in a variety of cell biological functions including migration and proliferation. We have initiated tests of two hypotheses concerning the mechanisms of cell migration in 3D settings. Cells are known to be surrounded by biochemically and structurally distinct matrices in vivo, e.g., a matrix rich in fibronectin fibrils during early craniofacial neural crest migration versus a collagen-rich matrix with varying crosslinking in adult connective tissue. We hypothesize that (1) three-dimensionality and differences in biochemical composition or crosslinking can combine to determine altered requirements for cytoskeletal and proteolytic responses needed for cell migration. We further hypothesize that (2) the role of myosin IIA (and possibly IIB) may differ in 3D versus 2D settings, as well as in different types of 3D environments. Our preliminary data suggest that depending on the type of 3D matrix (cell-derived versus collagen gels), myosin II isoforms may or may not be required for effective cell migration; this dichotomy in 3D contrasts with its migratory down-regulatory function in 2D cell culture, where its ablation stimulates migration. We will test for further biological differences in 3D environments, comparing requirements in other types of 3D matrices such as collagen, fibrin, and complex cell-derived matrices. We are also developing methods to visualize cell-surface and cytoskeletal molecular complexes and dynamics in 3D matrices. Although technically difficult, this microscopy technology will be important to develop further in order to allow direct comparisons of cellular functions in 2D versus 3D environments. Understanding whether the extent and nature of requirements for a specific protein differ in 2D and 3D appears important, since initial conclusions from in 2D vitro studies may differ under 3D or in vivo conditions.

细胞与不同组织中的不同类型的细胞外基质相互作用，在胚胎发育的不同阶段和成人伤口修复期间相互作用。该项目侧重于解决以下有关这些细胞 - 托管矩阵相互作用的机制的主要问题： 1。细胞如何组装三维（3D）细胞外基质，尤其是基于纤连蛋白的基质？ 2. 2D和3D矩阵之间的细胞粘合剂结构和生物学反应的差异是什么，以及不同类型的3D矩阵在体内微环境中具有不同类型的3D矩阵特征？ 3。哪些独特的机制控制3D微环境中的细胞行为，尤其是那些没有定向拓扑的线索？ 我们先前先前分析了3D纤连蛋白基质的基于整联蛋白的组装的分子机械，并发表了证据，证明了整联蛋白激活和协同蛋白易位以产生纤连蛋白质原纤维的作用。我们继续表征使用一组新型整合素突变体的基质组件，信号转导和细胞骨架机制的调节，我们已经生成了在纤连蛋白基质组件中显示特定缺陷的。我们最近使用这些突变体的初步发现表明，纤连蛋白基质组件取决于整联蛋白细胞质结构域中的多个位点。根据突变的氨基酸残基，我们还发现基质组件的过程可以独立于整联蛋白介导的细胞扩散过程中破坏。 我们正在探索这些突变体对整联蛋白激活状态和多分子细胞粘附复合物的影响。 我们先前发表了证据证明，在各种细胞生物学功能（包括迁移和增殖）中，围绕成纤维细胞的细胞外基质的三维重要性的重要性。我们已经对3D设置中细胞迁移的机制进行了两个假设的测试。已知细胞在体内被生物化学和结构上不同的基质包围，例如，在早期颅面神经rest迁移期间，富含纤连蛋白原纤维的基质与富含胶原蛋白的基质在成人结合组织中具有不同的交联。我们假设（1）生化组成或交联的三维性和差异可以结合起来，以确定细胞迁移所需的细胞骨架和蛋白水解反应的改变。我们进一步假设（2）肌球蛋白IIA（以及可能的IIB）的作用在3D与2D设置以及不同类型的3D环境中可能有所不同。我们的初步数据表明，取决于3D矩阵（细胞衍生的胶原凝胶与胶原蛋白）的类型，肌球蛋白II同工型可能需要或可能不需要有效的细胞迁移。在3D中，这种二分法与其在2D细胞培养中的迁移下调功能形成对比，在2D细胞培养中，其消融刺激了迁移。我们将在3D环境中测试进一步的生物学差异，以比较其他类型的3D矩阵（例如胶原蛋白，纤维蛋白和复杂细胞衍生的矩阵）的需求。 我们还在开发可视化细胞表面和细胞骨架分子复合物以及3D矩阵中的动力学的方法。尽管在技术上很困难，但这种显微镜技术对于进一步发展至关重要，以便直接比较2D与3D环境中的细胞功能。了解特定蛋白质需求的程度和性质在2D和3D中是否有所不同，因为在2D体外研究中的初步结论可能在3D或体内条件下可能有所不同。