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.哺乳动物细胞使用什么独特的机制在三维细胞外基质环境中迁移，而不是在平坦的培养表面上迁移？ 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。我们发现了一种独特的3D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3D细胞外基质体外模型。在真皮组织外植体和细胞基质中迁移的原代真皮成纤维细胞被发现使用钝的圆柱形突起，称为叶脚，以迁徙的阿米巴使用的一种细胞内压力驱动的突起命名。相反，在3D胶原凝胶中迁移的细胞表现出类似于2D细胞培养的基于板脂的迁移，在前沿有富含F-肌动蛋白的小的扇形突起。 我们一直在比较细胞骨架和信号系统在原代人成纤维细胞中的定位，这些细胞在不同的3D基质环境中迁移。我们最近的研究表明，尽管在基于片脂的3D胶原迁移过程中，活性的rac1、cc42和PIP3被极化到前沿，但在基于叶脚的迁移过程中，这种极化的CDc42、rac1和PIP3信号会丢失。相反，信号集中在前导突起后面、细胞两侧和细胞后部的焦点簇中。降低肌动球蛋白的收缩能力会将细胞切换到基于板脂蛋白的3D迁移。为了探索3D细胞外基质如何调节机械转导来控制前沿突起的模式，我们正在研究细胞内细胞骨架机制在基于板脂和叶脚的迁移过程中的功能。这些研究正在比较纤维系统的定位和功能，包括基于肌球蛋白II和中间丝的细胞骨架系统。我们目前特别关注细胞内压力在小叶细胞迁移中的潜在作用。 细胞迁移的可塑性与不同的细胞外基质分子相互作用被认为是有效的组织发育和伤口修复所必需的，而且在癌症中经常被解除调控。Rho家族GTP酶，CDC42，rac1和RhoA的不同激活，被认为控制了与不同基质蛋白相互作用所产生的不同的形态和迁移表型。然而，一个尚未解答的基本问题是，不同的细胞外基质蛋白是如何调控特定的GTPase信号通路来调控细胞迁移的。我们的假设是，黏附于不同的基质分子，如胶原和纤维连接蛋白，将触发鸟嘌呤核苷酸交换因子(GEF)的不同调节，以调节细胞的迁移反应。虽然已知GEF可以激活Rho GTP酶，但细胞与不同类型的基质相互作用对其生化活性的可能调节尚不清楚。我们最近的研究集中在确定一个基质特异性的全球环境基金调节因子及其对Rho家族GTP酶的调节机制，以及它们在决定细胞迁移模式和速度方面的调控串扰。

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 three-dimensional (3D) extracellular matrix environments rather than on flat culture surfaces? 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 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. We have been comparing the localization of cytoskeletal and signaling systems in primary human fibroblasts migrating in different 3D matrix environments. Our studies recently demonstrated that even though active Rac1, Cdc42, and PIP3 are polarized towards the leading edge during lamellipodia-based migration in 3D collagen, during lobopodia-based migration, this polarization of Cdc42, Rac1, and PIP3 signaling is lost. Instead, signaling is concentrated in focal clusters behind leading protrusions, along the sides, and at the rear of the cell. Reducing actomyosin contractility switches the cells to lamellipodia-based 3D 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 the intracellular cytoskeletal machinery during lamellipodia- and lobopodia-based migration. These studies are comparing the localization and function of fibrillar systems, including cytoskeletal systems based on myosin II and intermediate filaments. We are currently focusing particularly intensely on the potential role of intracellular pressure in lobopodial cell migration. Plasticity of cell migration in response to interaction with different extracellular matrix molecules is known to be necessary for efficient tissue development and wound repair, and it is often deregulated in cancer. Differential activation of the Rho family GTPases, Cdc42, Rac1, and RhoA, has been implicated in governing the distinct morphological and migratory phenotypes resulting from interaction with different matrix proteins. However, a fundamental unanswered question is how specific GTPase signaling pathways governing cell migration are regulated by different extracellular matrix proteins. Our hypothesis is that adhesion to different matrix molecules, such as collagen and fibronectin, will trigger differential regulation of guanine nucleotide exchange factors (GEFs) to regulate the cell migratory response. Although GEFs are known to activate the Rho GTPases, the possible regulation of their biochemical activity by cell interactions with different types of matrix is not known. Our recent studies have focused on identifying a matrix-specific GEF regulator and the mechanisms of its regulation of Rho family GTPases and their regulatory crosstalk in dictating the mode and speed of cell migration.