Matrix Organization and Dimensionality

矩阵组织和维度

基本信息

批准号：
8553345
负责人：
Kenneth Yamada
金额：
$ 57.48万
依托单位：
NATIONAL INSTITUTE OF DENTAL & CRANIOFACIAL RESEARCH
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/8553345
关键词：
1-Phosphatidylinositol 3-Kinase Actins Actomyosin Address Adhesives Adult Amoeba genus Behavior Biochemical Biological Biosensor Cell Communication Cell Culture Techniques Cells Characteristics Collaborations Collagen Complex Dermal Embryonic Development Environment Exhibits Extracellular Matrix F-Actin Family Fibroblasts Fluorescence Gel Goals Guanosine Triphosphate Phosphohydrolases Homeostasis Human Immigration Life Lobopodia Measures Mediating Modeling Mus Names National Institute on Deafness and Other Communication Disorders Play Proteolysis Relative (related person)Resolution Role Shapes Side Signal Transduction Staging Structure System Tissues Wound Healing base cell behavior cell motility cellular imaging crosslink in vitro Model in vivo migration novel physical property polymerization pressure response rho tissue culture two-dimensional

项目摘要

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.

细胞与不同组织中的结构不同类型的细胞外基质相互作用，在胚胎发育的不同阶段以及成人伤口修复期间。该项目侧重于解决以下有关这些细胞 - 托管矩阵相互作用的机制的主要问题： 1。细胞粘合剂结构和对2D矩阵的生物响应的差异以及不同类型的3D矩阵在体内微环境中的特征的差异是什么？ 2。哪些信号转导机制控制不同3D微环境中的细胞行为？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是否在组织中发现的结构复杂的3D环境中有效。我们使用高分辨率活细胞成像发现了一种独特的3D迁移模式，使用3D细胞外基质的不同体外模型可视化细胞内信号传导。发现原发性皮肤成纤维细胞在皮肤组织外植体中迁移和细胞衍生的基质使用钝的，圆柱形突起，称为小叶植物，以通过迁移的动物群使用的细胞内压力驱动的突出命名。相反，在3D胶原蛋白凝胶中迁移的细胞表现出类似于2D细胞培养的基于层状型的迁移，而小型，扇形的突起富含前缘的F-肌动蛋白。我们方法的一个主要特征是测量RHO家族GTPase信号传导和PI 3-激酶产物PIP3的极化。从经典上讲，Rac1，cdc42和pip3在2D底物上迁移到细胞的前缘强烈极化。这些信号被认为可以调节运动细胞前缘层状脂蛋白的肌动蛋白聚合，并有助于确定迁移的方向性。使用基于荧光的生物传感器的活细胞成像，我们比较了这些各种信号系统在不同3D矩阵环境中迁移的原代人成纤维细胞中的定位。在3D胶原蛋白的基于薄片的迁移期间，活性Rac1，cdc42和pip3都朝向前缘偏振。然而，在基于小叶的迁移过程中，丢失了cdc42，rac1和pip3信号的极化。取而代之的是，信号传导集中在前导突起，侧面和细胞后部后面的焦点簇中。降低肌动球蛋白的收缩力将细胞切换为基于层状的3D迁移。与NIDCD IRP的Nria Gavara和Richard Chadwick进行了富有成果的合作，有助于揭示这些3D迁移模式受矩阵的物理性质调节。通过有限的蛋白水解修饰细胞来源的基质改变了其弹性行为，使其非线性弹性并过渡了3D细胞运动的模式，向基于Lamellipodia的迁移。相反，通过交联的3D胶原蛋白或蛋白水解的细胞衍生的基质触发了基于小叶斑的迁移来诱导矩阵中的线性弹性行为。因此，细胞内信号传导的相对极化确定了由肌动蛋白收缩性本质上控制的3D细胞迁移的两个不同模式，并由3D细胞外基质的弹性行为外在。这些新颖的发现还表明，与教条，极化Rac1，cdc42和PIP3信号相反，对于持久的成纤维细胞迁移，不需要lamellipodia。为了探索3D细胞外基质如何调节机械转移来控制前沿突出的模式，我们正在研究基于薄片和基于叶虫的迁移期间细胞内肌球蛋白机械的功能。我们还正在探索人成纤维细胞对不同生化组成的3D细胞外基质的特定信号反应的机械基础。