Matrix Organization and Dimensionality

在胚胎发育的不同阶段和成人伤口修复过程中，细胞与不同组织中结构和生物化学类型的细胞外基质相互作用。该项目的重点是解决以下有关这些细胞-细胞外基质相互作用机制的主要问题： 1.哺乳动物细胞使用什么独特的机制来迁移通过三维（3D）细胞外基质环境，而不是在平坦的培养表面？ 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。 我们一直在比较在不同的三维基质环境中迁移的原代人成纤维细胞的细胞骨架和信号系统的定位。我们最近的研究表明，尽管活性Rac 1，Cdc 42和PIP 3在3D胶原蛋白中基于板状伪足的迁移过程中朝向前沿极化，但在基于叶状伪足的迁移过程中，Cdc 42，Rac 1和PIP 3信号的这种极化丢失。相反，信号集中在领先的突起后面，沿着两侧，并在细胞的后部的焦点簇。降低肌动球蛋白收缩性将细胞切换到基于板层脂质的3D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，我们正在研究基于板状伪足和叶伪足的迁移过程中细胞内细胞骨架机制的功能。这些研究比较了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。我们目前特别集中在细胞内压力在叶足细胞迁移中的潜在作用。 已知响应于与不同细胞外基质分子的相互作用的细胞迁移的可塑性对于有效的组织发育和伤口修复是必需的，并且其在癌症中通常是失调的。Rho家族GTPases，Cdc 42，Rac 1和RhoA的差异激活，已被牵连在管理不同的形态和迁移表型与不同的基质蛋白的相互作用所产生的。然而，一个基本的未回答的问题是如何特定的GTdR信号转导途径管理细胞迁移的调节不同的细胞外基质蛋白。我们的假设是，粘附到不同的基质分子，如胶原蛋白和纤连蛋白，将触发差异调节鸟嘌呤核苷酸交换因子（GEFs），以调节细胞迁移反应。虽然已知GEF激活Rho GTP酶，但通过细胞与不同类型基质的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种基质特异性GEF调节剂及其调节Rho家族GTPases的机制，以及它们在决定细胞迁移模式和速度方面的调节串扰。

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.