Cell-Matrix Interactions and Migration

整合素、细胞外基质分子和细胞骨架蛋白通过复杂的整合机制促进细胞迁移和信号传导。我们正在处理以下具体问题： 1.什么样的亚细胞结构和信号通路对细胞快速迁移很重要？ 2.整合素、细胞外基质和细胞骨架的功能是如何整合的，它们之间的调节串扰是如何协调的以产生正常的细胞迁移？ 我们正在使用各种细胞和分子生物学方法来解决这些问题，包括生化分析，荧光嵌合体，活细胞相衬或共聚焦延时显微镜。我们已经产生了各种荧光分子嵌合体和突变体的细胞骨架蛋白作为一个长期计划的一部分，以分析其功能的整合素介导的过程。我们一直特别关注整合素和相关的细胞外和细胞内分子在细胞迁移的机制和空间调节中的功能。 我们以前建立的细胞外基质（ECM）的拓扑结构在调节细胞骨架组织，细胞形态和细胞迁移中起着至关重要的作用，通过证明一维（1D）微图案线模拟三维细胞衍生基质中发现的纤维状ECM结构的功能。我们扩展了这些研究，以建立机制，其中纤维状地形引起快速，有效的细胞迁移成纤维细胞，以及这种迁移模式如何不同于迁移研究以前使用常规的二维（2D）组织培养基质。我们发现，间充质细胞迁移的两个关键过程，突出的前缘和粘连内形成的板层，在一维迁移过程中得到增强，并间接控制细胞收缩。这些研究还确定了与细胞源性基质原纤维的1D粘附和3D粘附与基质更稳定地结合，与延长的细胞粘附一致。 我们正在继续探索ECM的拓扑结构和物理特性，如硬度在调节成纤维细胞粘附，迁移和机械转导的作用。这些正在进行的研究比较了成纤维细胞对不同结构的3D胶原水凝胶的反应，以表征细胞粘附于不同厚度和顺应性的胶原纤维的动力学，以及确定对应于体内不同类型细胞外基质的不同类型胶原基质中的细胞迁移模式。 我们还评估了肌动球蛋白收缩性和微管翻译后修饰在细胞粘附，迁移，基质组装和器官分支发育中的调节和功能串扰。我们的研究正在确定肌动球蛋白介导的收缩和微管乙酰化水平之间的稳态平衡。这种平衡影响纤连蛋白基质组装、细胞迁移和胚胎器官分支的有效性。 这些综合知识应该提供新的方法来理解，预防或改善细胞在异常发育和癌症中使用的迁移过程。深入了解细胞移动和与其基质环境相互作用的精确方式也将促进组织工程研究。

Integrins, extracellular matrix molecules, and cytoskeletal proteins contribute to cell migration and signaling by complex, integrated mechanisms. We are addressing the following specific questions: 1. What subcellular structures and signaling pathways are important for rapid cell migration? 2. How are the functions of integrins, the extracellular matrix, and the cytoskeleton integrated, and how is the regulatory crosstalk between them coordinated to produce normal cell migration? We are using a variety of cell and molecular biology approaches to address these questions, including biochemical analyses, fluorescent chimeras, and live-cell phase-contrast or confocal time-lapse microscopy. We have generated a variety of fluorescent molecular chimeras and mutants of cytoskeletal proteins as part of a long-term program to analyze their functions in integrin-mediated processes. We have been focusing particularly on the functions of integrins and associated extracellular and intracellular molecules in the mechanisms and spatial regulation of cell migration. We previously established that the topography of the extracellular matrix (ECM) plays a vital role in regulating cytoskeletal organization, cell morphology, and cell migration by demonstrating that one-dimensional (1D) micropatterned lines mimic the functions of the fibrillar ECM structures found in three-dimensional cell-derived matrix. We extended these studies to establish the mechanism by which fibrillar topography evokes rapid, efficient cell migration in fibroblasts and how this mode of migration differs from migration studied previously using regular two-dimensional (2D) tissue culture substrates. We found that two key processes of mesenchymal cell migration, protrusion of the leading edge and adhesions formed within the lamella, are enhanced during 1D migration and are controlled indirectly by cellular contractility. These studies also established that 1D adhesions and 3D adhesions to cell-derived matrix fibrils are more stably associated with the matrix, consistent with prolonged cell adhesiveness. We are continuing to explore the role of ECM topography and physical properties such as stiffness in regulating fibroblast adhesion, migration, and mechanotransduction. These ongoing studies are comparing fibroblast responses to 3D collagen hydrogels of differing architecture in order to characterize the dynamics of cell adhesions to collagen fibrils of differing thickness and compliance, as well as determining the mode of cell migration in different types of collagen matrix that correspond to different types of extracellular matrix in vivo. We are also evaluating the regulatory and functional crosstalk between actomyosin contractility and microtubule post-translational modification in cell adhesion, migration, matrix assembly, and organ branching in development. Our studies are identifying a homeostatic balance between actomyosin-mediated contraction and the level of microtubule acetylation. This balance affects fibronectin matrix assembly, cell migration, and the effectiveness of embryonic organ branching. This combined knowledge should provide novel approaches to understanding, preventing, or ameliorating migratory processes that cells use in abnormal development and cancer. An in-depth understanding of the precise manner in which cells move and interact with their matrix environment will also facilitate tissue engineering studies.