Matrix Organization and Dimensionality

在不同的组织中，在胚胎发育的不同阶段，以及在成人伤口修复期间，细胞与结构和生化上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.与平板细胞培养底物相比，哺乳动物细胞使用什么独特的机制在不同的三维(3D)细胞外基质环境中迁移？ 2.在不同的3D微环境中，有哪些不同的信号转导机制控制着细胞的行为？ 我们正在探索使用常规2D细胞培养建立的细胞运动和信号的经典模型是否适用于组织中发现的结构复杂的3D环境。我们发现2D和3D环境之间以及不同3D环境之间的细胞迁移和信号传递存在差异，例如，与富含纤维连接蛋白的细胞基质相比，基于胶原的基质在结构上有所不同。在这些基质中，原代人皮肤成纤维细胞以不同的速度和不同的迁移方式迁移，一个主要的区别是使用了基于肌动蛋白聚合的片状脂膜迁移和基于细胞内压力的叶状迁移。对于叶轴迁移，我们以前发现了中间细丝蛋白vientin、肌动蛋白、肌球蛋白II和核骨架-细胞骨架连接蛋白Nesprin 3之间的收缩依赖蛋白质复合体。我们与Michael Davidson合作开发了基于FRET的生物传感器，以可视化这些连接细胞外基质和细胞核的连接。 尽管平面(2D)细胞外基质的物理性质可以调节细胞黏附动力学和整体细胞迁移，但对于不同类型3D基质之间的局部微环境差异对活细胞动态行为的影响却知之甚少。我们制备了不同基质微结构的3D胶原凝胶，以寻找3D黏附动力学和细胞迁移的差异调节。胶原水凝胶在相同的胶原蛋白浓度下聚合，但在不同的温度下聚合，显示出显著的结构差异，从高度网状的非常短的纤维到大束平行的胶原纤维。尽管基于整体流变学的经典测量显示硬度只有很小的差异，但使用微米级的原子力显微镜探针对这些基质进行更仔细的分析后发现，这些以不同结构组织的纤维在硬度上相差10倍。不同的胶原蛋白结构经常在体内紧密地存在，因此这些体外模型可以提供容易处理的系统来比较每种不同类型的结构的细胞反应。 原子力显微镜下，在细胞粘连的大小范围内，捆绑的胶原纤维被发现局部僵硬，而网状纤维则柔软。这些3D微环境随后被用来比较局部僵硬对细胞黏附稳定性和细胞迁移率的各种参数的影响。总体而言，细胞在3D胶原蛋白中的迁移似乎需要通过整合素受体来稳定细胞黏附，以达到最有效的细胞迁移速度，从而局部平衡收缩能力和特定的基质硬度水平。有趣的是，这些人细胞在不同的3D胶原环境中显示出类似的整合素激活和聚集水平，与细胞培养中培养在扁平胶原基质上的细胞相比显著升高。这种增强的整合素激活伴随着对细胞收缩的要求，显然是为了使这些细胞能够有效地从基于增强的整合素与3D纤维的附着中分离出来，以介导有效的细胞迁移。这项研究的下一阶段将直接比较恶性肿瘤细胞与3D纤维基质的相互作用，以及有效细胞迁移所需的信号。

Cells interact with structurally and biochemically distinct types of extracellular matrix in different tissues, 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. What unique mechanisms do mammalian cells use to migrate through different three-dimensional (3D) extracellular matrix environments compared to flat cell culture substrates? 2. What distinct signal transduction mechanisms control cell behavior in different 3D microenvironments? We are exploring whether classical models of cell motility and signaling established using regular 2D cell culture are valid in the structurally complex 3D environments found in tissues. We find differences in cell migration and signaling between 2D and 3D environments, but also between different 3D environments, such as collagen-based matrices that differ in architecture compared to fibronectin-rich cell-derived matrices. In these matrices, primary human dermal fibroblasts migrate at different speeds and with distinct modes of migration, with one major difference being the use of actin polymerization-based lamellipodial migration versus intracellular pressure-based lobopodial migration. For lobopodial migration, we had previously identified a contractility-dependent protein complex between the intermediate filament protein vimentin, actin, myosin II, and the nucleoskeleton-cytoskeleton linker protein nesprin 3. We collaborated with Michael Davidson to develop FRET-based biosensors to visualize these connections linking the extracellular matrix to the nucleus. Although the physical properties of extracellular matrices on flat (2D) extracellular matrices are known to modulate cell adhesion dynamics and overall cell migration, little is known about the roles of local micro-environmental differences between various types of 3D matrix on the dynamic behavior of living cells. We have generated 3D collagen gels of different matrix micro-architectures to search for differential regulation of 3D adhesion dynamics and cell migration. Collagen hydrogels polymerized at identical collagen concentrations but at different temperatures display dramatic differences in architecture, ranging from highly reticular with very short fibrils to large bundles of parallel collagen fibrils. Although classical measurements based on bulk rheology showed only minor differences in stiffness, more careful analyses of these matrices using atomic force microscopy probes at the micron-size scale of cell adhesions reveals that these fibrils organized in distinct architectures differ 10-fold in stiffness. Different collagen architectures frequently exist in close proximity in vivo, so these in vitro models can provide tractable systems for comparing cellular responses to each distinct type of architecture. Using atomic force microscopy, bundled collagen fibrils were found to be locally stiff at the size scale of cell adhesions, whereas reticular fibrils were soft. These 3D microenvironments were then used to compare effects of local stiffness on a variety of parameters of cell adhesion stability and rates of cell migration. Overall, cells migrating in 3D collagen appeared to require a local balancing of contractility with the specific local level of matrix stiffness through integrin receptors to stabilize cell adhesions in order to achieve the most efficient rate of cell migration. Interestingly, these human cells in the various 3D collagen environments displayed similar levels of integrin activation and clustering that were markedly elevated compared to the levels for the cells cultured on flat collagen matrices in cell culture. This enhanced integrin activation was accompanied by a requirement for cellular contractility, apparently in order for these cells to be able to detach effectively from their enhanced integrin-based attachments to 3D fibrils to mediate efficient cell migration. The next phase of this study will directly compare the interactions of malignant tumor cells with 3D fibrillar matrices, as well as the signaling required for efficient cell migration.