Matrix Organization and Dimensionality

在不同的组织中，在胚胎发育的不同阶段和成人伤口修复期间，细胞与不同类型的细胞外基质相互作用。本项目重点解决与这些细胞-细胞外基质相互作用的机制有关的以下主要问题： 1.细胞如何组装三维(3D)细胞外基质，特别是基于纤维连接蛋白的基质？ 2.2D和3D基质之间以及不同类型3D基质之间的细胞黏附结构和生物学反应有何差异？ 3.在三维微环境中，特别是在那些没有定向拓扑线索的微环境中，什么独特的机制控制着细胞的行为？ 我们之前分析了基于整合素的3D纤维连接蛋白基质组装的分子机制，并发表了整合素激活和协同蛋白转位在产生纤维连接蛋白纤维中的作用的证据。我们正在继续利用我们产生的一组新的整合素突变体来表征基质组装、信号转导和细胞骨架机制的调节，这些突变体显示出纤维连接蛋白基质组装中的特定缺陷。我们最近使用这些突变体的初步发现表明，纤维连接蛋白基质的组装依赖于整合素细胞质区域中的多个位置。根据突变的氨基酸残基，我们还发现基质组装过程可以独立于整合素介导的细胞扩散过程而被破坏。我们正在探索这些突变体对整合素激活状态和多分子细胞黏附复合体的影响。 我们之前发表的证据表明，成纤维细胞周围的细胞外基质的三维特性在包括迁移和增殖在内的各种细胞生物学功能中具有重要作用。我们已经启动了关于3D环境中细胞迁移机制的两个假设的测试。已知在体内细胞被不同的生物化学和结构上不同的基质所包围，例如，在早期颅面神经脊迁移过程中，富含纤维连接蛋白的基质，而在成人结缔组织中，富含胶原的基质具有不同的交联性。我们假设(1)三维和生化成分或交联的差异可以结合在一起来确定细胞迁移所需的细胞骨架和蛋白分解反应的变化要求。我们进一步假设(2)肌球蛋白IIA(以及可能的IIB)在3D和2D环境中的作用可能不同，以及在不同类型的3D环境中也可能不同。我们的初步数据表明，根据3D基质的类型(细胞衍生和胶原凝胶)，肌球蛋白II亚型可能是有效的细胞迁移所必需的，也可能不是；3D中的这种二分法与其在2D细胞培养中的迁移下调功能形成对比，在2D细胞培养中，其消融刺激迁移。我们将在3D环境中测试进一步的生物学差异，比较其他类型的3D基质的要求，如胶原、纤维蛋白和复杂细胞衍生基质。 我们还在开发在3D矩阵中可视化细胞表面和细胞骨架分子络合物和动力学的方法。尽管技术上有困难，但这项显微技术的进一步发展将是重要的，以便能够直接比较2D和3D环境中的细胞功能。了解2D和3D对特定蛋白质的需求的程度和性质是否不同似乎很重要，因为2D体外研究的初步结论在3D或体内条件下可能不同。

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.