Matrix Organization and Dimensionality

在胚胎发育的不同阶段和成人伤口修复期间，细胞与不同组织中结构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. 2D与3D基质的细胞粘附结构和生物学反应有何差异，以及不同体内微环境的不同类型3D基质之间有何差异？ 2.在不同的3D微环境中，什么样的信号转导机制控制着细胞的行为？ 我们探讨了使用常规2D细胞培养建立的细胞运动和信号传导的经典模型在组织中发现的结构复杂的3D环境中是否有效。我们发现了一种独特的三维迁移模式，使用高分辨率活细胞成像来可视化细胞内信号，使用不同的体外模型的三维细胞外基质。发现在真皮组织外植体和细胞衍生基质中迁移的原代真皮成纤维细胞使用钝的圆柱形突起，称为lobopodia，以迁移阿米巴使用的细胞内压力驱动突起命名。相比之下，细胞迁移在3D胶原凝胶表现出类似于2D细胞培养的基于板状伪足的迁移，在前缘具有富含F-肌动蛋白的小的扇形突起。 我们的方法的一个中心特征是测量Rho家族GT3信号传导和PI 3-激酶产物PIP 3的极化。经典的，Rac 1，Cdc 42，和PIP 3强烈极化的前沿细胞迁移的二维基板上，这些信号被认为是调节肌动蛋白聚合在片状伪足的运动细胞的前沿，并帮助确定方向性的迁移。使用基于荧光的生物传感器的活细胞成像，我们比较了这些不同信号系统在不同3D基质环境中迁移的原代人成纤维细胞中的定位。活性Rac 1，Cdc 42，和PIP 3都被极化的前沿在三维胶原基于板状伪足的迁移。然而，在基于叶足的迁移过程中，Cdc 42、Rac 1和PIP 3信号的极化丢失。相反，信号集中在领先的突起后面的焦点簇中，沿着两侧，以及在细胞的后部。降低肌动球蛋白收缩性将细胞转换为基于板层脂质的3D迁移。 与Nria Gavara和Richard Chadwick在NIDCD IRP中进行了富有成效的合作，帮助揭示了这些3D迁移模式受到基质物理性质的调节。通过有限的蛋白水解修饰细胞衍生的基质改变了其弹性行为，使其具有非线性弹性，并将3D细胞运动模式转变为基于板状伪足的迁移。相反，通过交联3D胶原蛋白或蛋白水解的细胞衍生基质诱导基质中的线性弹性行为触发了基于叶足的迁移。因此，细胞内信号的相对极化识别了两种不同的3D细胞迁移模式，其内在地由肌动球蛋白收缩性支配，并且外在地由3D细胞外基质的弹性行为支配。这些新的发现还表明，板状伪足是不必要的有效的3D运动，相反的教条，极化Rac 1，Cdc 42，和PIP 3信号是不需要定向持久的成纤维细胞迁移。 为了探索3D细胞外基质如何调节机械转导以控制前缘突起的模式，我们正在研究细胞内肌动球蛋白机械在基于板状伪足和叶伪足的迁移过程中的功能。我们还在探索人类成纤维细胞对不同生化成分的3D细胞外基质的特异性信号传导反应的机制基础。

Cells interact with structurally 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 are the differences in cell adhesive structures and biological responses to 2D versus 3D matrices, as well as between different types of 3D matrices characteristic of different in vivo microenvironments? 2. What signal transduction mechanisms control cell behavior in different 3D microenvironments? We explored 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 discovered a unique mode of 3D migration using high-resolution live-cell imaging to visualize intracellular signaling using different in vitro models of 3D extracellular matrix. Primary dermal fibroblasts migrating in dermal tissue explants and cell-derived matrix were found to use blunt, cylindrical protrusions termed lobopodia, named after an intracellular pressure-driven protrusion used by migrating amoeba. In contrast, cells migrating in 3D collagen gels exhibited lamellipodia-based migration similar to 2D cell culture, with small, fan-shaped protrusions enriched in F-actin at the leading edge. A central feature of our approach was to measure polarization of Rho family GTPase signaling and the PI 3-kinase product PIP3. Classically, Rac1, Cdc42, and PIP3 are strongly polarized to the leading edge of cells migrating on 2D substrates; these signals are thought to regulate actin polymerization in lamellipodia at the leading edge of motile cells and help to determine directionality of migration. Using live-cell imaging with fluorescence-based biosensors, we compared the localization of these various signaling systems in primary human fibroblasts migrating in different 3D matrix environments. Active Rac1, Cdc42, and PIP3 were all polarized towards the leading edge during lamellipodia-based migration in 3D collagen. During lobopodia-based migration, however, polarization of Cdc42, Rac1, and PIP3 signaling was lost. Instead, signaling was concentrated in focal clusters behind leading protrusions, along the sides, and at the rear of the cell. Reducing actomyosin contractility switched the cells to lamellipodia-based 3D migration. A fruitful collaboration with Nria Gavara and Richard Chadwick in the NIDCD IRP helped to reveal that these modes of 3D migration were regulated by physical properties of the matrix. Modifying cell-derived matrix by limited proteolysis changed its elastic behavior, rendering it non-linearly elastic and transitioning the mode of 3D cell motility to lamellipodia-based migration. Conversely, inducing linear elastic behavior in the matrices by crosslinking 3D collagen or proteolyzed cell-derived matrix triggered lobopodia-based migration. Thus, the relative polarization of intracellular signaling identifies two distinct modes of 3D cell migration governed intrinsically by actomyosin contractility, and extrinsically by the elastic behavior of the 3D extracellular matrix. These novel findings also indicate that lamellipodia are not necessary for efficient 3D motility and that, contrary to dogma, polarized Rac1, Cdc42, and PIP3 signaling is not required for directionally persistent fibroblast migration. In order to explore how a 3D extracellular matrix regulates mechanotransduction to control the mode of leading-edge protrusion, we are investigating the function of intracellular actomyosin machinery during lamellipodia- and lobopodia-based migration. We are also exploring the mechanistic basis of the specific signaling responses of human fibroblasts to 3D extracellular matrix of differing biochemical compositions.