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参与细胞迁移和信号传导。我们正在处理以下问题： 1.什么样的亚细胞结构和信号通路对有效的细胞迁移很重要？ 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，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。 局部微环境地形的空间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，我们开发了一种称为微光消融的程序。已经制定了这项技术的详细方案，并将其标准化，供其他实验室使用。这种新方法允许生成任何类型的基质蛋白质模式。例如，它可以产生微米宽的图案化线，其用作细胞迁移的有效路径，其紧密地模拟细胞沿沿着三维（3D）细胞衍生的原纤维的迁移。我们将这种细胞迁移过程称为基质蛋白质沿沿着的一维迁移。“我们以前表明，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反应，与传统的平面迁移模式相比，这种1D迁移过程更有效地再现，2D表面通常用于细胞培养。 细胞可以使用细胞肌动蛋白和肌球蛋白产生细胞内张力，并且它们可以在细胞机械转导过程中感受其微环境中的张力。一个特别显着的差异被确定在细胞迁移过程中的收缩性抑制剂在1D和3D与2D条件下的细胞反应。在2D纤连蛋白包被的表面上用肌动球蛋白收缩性抑制剂处理成纤维细胞导致细胞迁移速率略微增加，而在1D（纤连蛋白包被的）或3D纤维状细胞衍生基质上铺板的细胞的相同处理基本上抑制迁移超过两倍。基于这些观察结果，再加上我们的发现，即粘附到1D的底层形成了一个独特的，冗长的粘附结构，与2D表面上发现的不同，我们正在测试是否改变细胞-ECM粘附的地形和物理结构会影响基本的形态和生化机制，建议介导细胞迁移。为了量化的动态蛋白质可能包括一个离合器样机制牵连在细胞基质相互作用，我们目前正在分析荧光恢复后的GFP连接的融合蛋白的光漂白（FRAP）与其他活细胞成像技术（旋转磁盘和TIRF显微镜）跟踪的蛋白质的动态参与假设的分子离合器，以确定如何一维ECM增强细胞迁移。更一般地说，我们认为，研究一维迁移的细胞将为分析细胞迁移的分子机制提供一个强有力的新工具，因为分子机器的组成部分是沿沿着稳定迁移的细胞的长度线性排列的，该细胞保持单一方向取向。 非肌肉细胞肌球蛋白和肌动蛋白被认为在细胞迁移和许多发育和创伤修复过程中发挥关键作用，但主要肌球蛋白IIA基因的作用尚不清楚。我们和其他人最近发表了关于主要肌球蛋白II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性，肌动蛋白细胞骨架组织和组织的细胞基质粘附中起着重要作用。我们现在直接比较肌球蛋白IIA和IIB亚型在1D，2D和3D细胞迁移系统中的作用。我们以前也发现了肌球蛋白IIA和微管动力学之间的强交叉调节，调节Rac定位和细胞迁移。我们正在探索这两个主要的细胞骨架系统在2D和3D系统之间的串扰的机制。 这些正在进行的研究整合素和相关的细胞内和细胞外分子在细胞迁移的功能中心后，我们的能力，早期细胞突起和细胞内肌球蛋白和微管的图像活细胞分子动力学。所有这些过程都需要在真实的时间和更生理的1D和3D矩阵环境中并行分析，以便能够理解体内细胞迁移的机制。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。深入了解细胞如何移动并与其基质环境相互作用也将促进组织工程研究。

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.