Cell-Matrix Interactions and Migration

整合素、细胞外基质分子和细胞骨架蛋白以复杂的方式促进细胞迁移和信号传递。我们正在解决以下问题： 1.什么亚细胞结构和信号通路对细胞高效迁移是重要的？ 2.整合素、细胞外基质和细胞骨架的功能是如何整合的，它们之间的调控串扰是如何协调以产生细胞迁移的？ 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、荧光嵌合体和活细胞相差或共聚焦时间推移显微镜。我们已经产生了细胞骨架蛋白的各种荧光分子嵌合体和突变体，作为长期计划的一部分，以分析它们在整合素介导的过程中的功能。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机制和空间调控中的功能。 局部微环境的地形对空间的调节可以改变多种细胞功能。为了深入分析细胞外基质分子的拓扑组织的作用，我们发展了一种称为微光消融的过程。这项技术的详细方案已经开发出来，并已标准化，供其他实验室使用。这种新的方法允许产生任何类型的基质蛋白。例如，它可以产生微米宽的图案线，作为细胞迁移的有效路径，这接近于模拟细胞沿三维(3D)细胞衍生的纤维的迁移。我们把这种细胞沿一条狭窄的基质蛋白质线迁移的过程称为“一维迁移”。我们以前证明了3D纤维基质中3D细胞迁移的许多方面，包括形态、迁移、细胞骨架组织和对基质密度的响应，与常规用于细胞培养的传统平面2D表面上的迁移模式相比，这种1D迁移过程要有效得多。 细胞可以利用细胞肌动蛋白和肌球蛋白产生细胞内张力，并在细胞力学转导过程中感受到微环境中的张力。在1D和3D与2D条件下的细胞迁移过程中，细胞对收缩抑制物的反应被鉴定为特别显著的差异。在2D纤维连接蛋白涂层表面用肌动球蛋白收缩抑制剂处理成纤维细胞，细胞迁移率略有增加，而相同处理的细胞在1D(纤维连接蛋白涂层)或3D纤维细胞衍生基质上的迁移显著抑制两倍以上。基于这些观察，加上我们的发现，在1D中与底层基质的粘连形成了一种独特的、长时间的粘连结构，而不是在2D表面上发现的，我们正在测试细胞-ECM粘连的地形和物理结构的改变是否会影响所提出的介导细胞迁移的基本形态和生化机制。为了量化可能包含细胞-基质相互作用中的离合器样机制的蛋白质的动力学，我们目前正在分析GFP连接的融合蛋白的光漂白后荧光恢复(FRAP)，以及其他活细胞成像技术(旋转圆盘和TIRF显微镜)，以跟踪假设的分子离合器中涉及的蛋白质的动力学，以确定1D ECM如何促进细胞迁移。更广泛地说，我们认为研究细胞在一维中的迁移将为分析细胞迁移的分子机制提供一个强大的新工具，因为分子机制的组件沿着稳定迁移的细胞的长度线性排列，而细胞保持单一方向定向。 非肌肉细胞肌球蛋白和肌动蛋白被认为在细胞迁移以及许多发育和创伤修复过程中发挥关键作用，但主要的肌球蛋白IIA基因的作用尚不清楚。我们和其他人最近发表了关于肌球蛋白II主要基因，肌球蛋白IIA和IIB的作用的研究。我们发现肌球蛋白IIA在成纤维细胞和胚胎干细胞的收缩、肌动蛋白细胞骨架的组织和细胞-基质粘连的组织中发挥核心作用。我们现在正在直接比较肌球蛋白IIA和IIB亚型在一维、二维和三维细胞迁移系统中的作用。我们以前也发现了肌球蛋白IIA和微管动力学之间强烈的交叉调节，调节RAC的定位和细胞迁移。我们正在探索2D和3D系统中这两个主要细胞骨架系统之间的串扰机制。 这些正在进行的关于整合素和相关的细胞内外分子在细胞迁移中的功能的研究，集中在我们对早期细胞突起和细胞内肌球蛋白和微管的活细胞分子动力学成像的能力上。所有这些过程都需要实时地并行分析，并在更具生理性的一维和三维基质环境中进行分析，才能了解体内细胞迁移的机制。这种结合的知识应该为理解、预防或改善细胞在异常发育和癌症中使用的迁移过程提供新的方法。深入了解细胞如何移动并与其基质环境相互作用也将有助于组织工程学研究。

Integrins, extracellular matrix molecules, and cytoskeletal proteins contribute in complex fashion to cell migration and signaling. We are addressing the following questions: 1. What subcellular structures and signaling pathways are important for efficient 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 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 functions of integrins and associated extracellular and intracellular molecules in the mechanisms and spatial regulation of cell migration. Spatial regulation by the topography of the local microenvironment can alter a wide variety of cellular functions. In order to analyze in depth the roles of topological organization of extracellular matrix molecules, we developed a procedure termed micro-photoablation. Detailed protocols for this technique have been developed and standardized for use by other laboratories. This new methodology permits the generation of any type of pattern of matrix proteins. For example, it can generate micron-wide patterned lines that serve as efficient paths of cell migration, which closely mimics cell migration along three-dimensional (3D) cell-derived fibrils. We term this process of cell migration along a narrow line of matrix proteins "1D migration." We previously showed that many aspects of 3D cell migration in a 3D fibrillar matrix, including morphology, migration, cytoskeletal organization, and responses to matrix density, are reproduced much more effectively by this process of 1D migration compared to the mode of migration on traditional flat, 2D surfaces routinely used for cell culture. Cells can develop intracellular tension using cellular actin and myosin, and they can sense tension in their microenvironment in the process of cellular mechanotransduction. A particularly striking discrepancy was identified in cellular responses to inhibitors of contractility during cell migration under 1D and 3D versus 2D conditions. Treatment of fibroblasts with inhibitors of actomyosin contractility on 2D fibronectin-coated surfaces leads to slight increases in rates of cell migration, whereas the same treatments of cells plated on 1D (fibronectin-coated) or 3D fibrillar cell-derived matrix substantially inhibits migration by greater than two-fold. Based on these observations, together with our finding that adhesions to the underlying substratum in 1D form a unique, lengthy adhesion structure unlike that found on 2D surfaces, we are testing whether changing the topography and physical structure of cell-ECM adhesions affects the basic morphological and biochemical mechanisms proposed to mediate cell migration. In order to quantify the dynamics of proteins potentially comprising a clutch-like mechanism implicated in cell-matrix interactions, we are currently analyzing fluorescence recovery after photobleaching (FRAP) of GFP-linked fusion proteins together with other live-cell imaging techniques (spinning disk and TIRF microscopy) to track the dynamics of the proteins involved in the postulated molecular clutch to determine how 1D ECM enhances cell migration. More generally, we feel that studying cells migrating in 1D will provide a powerful new tool for analyzing the molecular mechanisms of cell migration, because the components of the molecular machinery are arrayed linearly along the length of a steadily migrating cell that remains oriented in a single direction. Nonmuscle cellular myosins and actin are thought to play crucial roles in cell migration and in many developmental and wound repair processes, but the roles of the major myosin IIA gene were not clear. We and others recently published studies on the roles of the major myosin II genes, myosin IIA and IIB. We found that myosin IIA plays central roles in fibroblast and embryonic stem cell contractility, actin cytoskeletal organization, and organization of cell-matrix adhesions. We are now directly comparing the roles of myosin IIA and IIB isoforms in 1D, 2D, and 3D cell migration systems. We had also previously identified strong cross-regulation between myosin IIA and microtubule dynamics that regulates Rac localization and cell migration. We are exploring the mechanisms of this cross-talk between these two major cytoskeletal systems in 2D and 3D systems. These ongoing studies on the functions of integrins and associated intracellular and extracellular molecules in cell migration center upon our ability to image live-cell molecular dynamics of early cell protrusions and intracellular myosins and microtubules. All of these processes need to be analyzed in parallel in real time and in more physiological 1D and 3D matrix environments to be able to understand the mechanisms of in vivo cell migration. 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 exactly how cells move and interact with their matrix environment will also facilitate tissue engineering studies.