Matrix Organization and Dimensionality

在胚胎发育和成人伤口修复的不同阶段，不同组织中的细胞与不同类型的细胞外基质相互作用。 该项目重点解决以下有关这些细胞与细胞外基质相互作用机制的主要问题： 1.细胞如何组装三维（3D）细胞外基质，特别是主要基于纤连蛋白原纤维的细胞外基质？ 2.在传统的二维（2D）细胞培养和体内微环境的各种3D基质特征中，信号传导和生物学之间有什么区别？ 3.什么独特的机制控制细胞在3D微环境中的行为？ 我们先前分析了介导基于整合素的3D纤连蛋白基质组装的分子机制，并发表了整合素活化和协同蛋白易位产生纤连蛋白原纤维的作用的证据。 我们正在继续使用一组新的整合素突变体，我们已经产生的纤连蛋白基质组装显示特定的缺陷的特点，基质组装，信号转导和细胞骨架机制的调节。 我们最近的初步研究结果表明，纤连蛋白基质组装依赖于整合素胞质结构域中的多个位点。 根据点突变，基质组装可以被破坏差异相比，整合素介导的粘附。 我们以前发表的证据的三维细胞外基质周围的成纤维细胞在各种细胞的生物学功能，包括迁移和增殖的重要性。 最近，我们完成了对组织形态发生和癌症的3D模型领域的回顾。 基于目前的文献，我们已经开始测试两个新的假设，细胞迁移的机制，在3D设置。 已知细胞在体内被生物化学和结构上不同的基质包围，在颅面神经嵴早期迁移过程中富含纤维连接蛋白原纤维的基质与成人结缔组织中具有不同交联的富含胶原的基质。 我们假设：（1）三维性和生化组成或交联的差异可以联合收割机确定细胞迁移所需的细胞骨架和蛋白水解反应的改变要求。 我们进一步假设（2）肌球蛋白IIA（可能还有IIB）的作用在3D和2D环境中以及不同类型的3D环境中可能不同。 我们的初步数据表明，根据3D基质的类型（细胞来源的胶原蛋白凝胶），肌球蛋白II亚型可能需要或可能不需要有效的细胞迁移;在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，其中其消融刺激迁移。 我们将测试3D环境中的进一步差异，比较其他类型的3D矩阵的要求，并确定它们是否可以通过使用不同的整合素和亚基而引起的不同相互作用和细胞内调节来解释，例如，胶原结合α-2亚基与纤连蛋白结合α-5整联蛋白。 我们还在开发在3D矩阵中可视化细胞表面和细胞骨架动态的方法。 虽然技术上很困难，但这种显微镜技术的进一步发展将是重要的，以便在2D与3D环境中直接比较细胞功能。 了解特定蛋白质在2D和3D中的需求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 different types of extracellular matrix in different tissues and at different stages of embryonic development and 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 primarily based on fibronectin fibrils? 2. What are the differences between signaling and biology in traditional two-dimensional (2D) cell culture and various 3D matrices characteristic of in vivo microenvironments? 3. What unique mechanisms control cell behavior in 3D microenvironments? 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 suggest that fibronectin matrix assembly depends on multiple sites in the integrin cytoplasmic domain. Depending on the point mutation, matrix assembly can be disrupted differentially compared to integrin-mediated adhesion. We previously published evidence for the importance of the three-dimensionality of extracellular matrix surrounding fibroblasts in a variety of cell biological functions including migration and proliferation. Very recently, we completed a review of the field of 3D models of tissue morphogenesis and cancer. Based on the current literature, we have initiated tests of two new 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 differences in 3D environments, compare requirements in other types of 3D matrices, and determine whether they can be explained by distinct interactions and intracellular regulation due to the use of different integrins and subunits, e.g., the collagen-binding alpha-2 subunit versus the fibronectin-binding alpha-5 integrin. We are also developing methods to visualize cell-surface and cytoskeletal dynamics in 3D matrices. Although technically difficult, this microscopy technology will be important to develop further to permit 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.