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视频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的机制和地形调节中的功能。 细胞和组织动力学发生在体内的三维(3D)环境中。我们已经确定了一维(1D)偏移对于理解3D偏移的重要性。使用一种新开发的名为微光消融的程序，我们证明了细胞通过排列的纤维3D细胞衍生基质的迁移很容易被简单的1.5微米宽的微图案线模拟，我们称之为1D迁移。我们发现3D纤维细胞迁移的许多方面，包括纺锤形细胞形态、迁移速度(1D和3D都比2D加快)、细胞骨架组织(基于肌动蛋白和微管)、中心体和高尔基体定向以及对收缩抑制物的反应，与传统的用于细胞培养的二维表面相比，这种具有1D纤维形态的系统可以更有效地再现。 一个特别有趣的发现涉及在一维、二维和三维条件下细胞迁移过程中细胞对收缩抑制物的不同反应。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，细胞迁移速度和细胞扩散名义上增加，而同样的处理在1D(纤维连接蛋白涂层)或3D纤维细胞衍生基质上的细胞基本上抑制两倍以上的迁移。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。出乎意料的是，与肌球蛋白II分子对细胞迁移必不可少的信念相反，我们发现肌球蛋白IIA并不是必需的，实际上它在2D细胞培养中起到了抑制迁移的作用。我们还发现，肌球蛋白IIA和微管动力学之间存在很强的交叉调节，调节RAC的定位和细胞迁移。由于技术原因，在我们最初的研究中，有必要使用标准的2D培养系统来可视化细胞骨架动力学和Rho GTP酶功能。在此之前，我们还确定了RAC在帮助调节这种2D环境中的定向细胞迁移方面的关键作用。两个新的项目正在测试肌球蛋白II串扰和Rho家族GTP酶在迁移中的这些原理在一维、二维和三维细胞迁移系统中是否正确。 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。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矩阵环境中进行分析，才能理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 video 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 topographical regulation of cell migration. Cell and tissue dynamics occur in three-dimensional (3D) environments in vivo. We have established the importance of one-dimensional (1D) migration for understanding 3D migration. Using a newly developed procedure termed micro photoablation, we demonstrated that cell migration through aligned fibrillar 3D cell-derived matrices is readily mimicked by simple 1.5 micron-wide micropatterned lines in a process we term 1D migration. We showed that many aspects of 3D fibrillar cell migration, including spindle cell morphology, migration velocity (both increased in 1D and 3D compared to 2D), cytoskeletal organization (both actin- and microtubule-based), centrosome and Golgi orientation, and responses to contractile inhibitors are reproduced much more effectively by this system with 1D fibrillar topography versus the traditional two-dimensional surfaces used for cell culture. A particularly interesting finding involves differences in the cellular responses to inhibitors of contractility during cell migration under 1D, 2D, and 3D conditions. Treatment of fibroblasts with inhibitors of actomyosin contractility on 2D fibronectin-coated surfaces leads to a nominal increase in cell migration velocity and cell spreading, whereas the same treatment of cells plated on 1D (fibronectin-coated) or 3D fibrillar cell-derived matrix substantially inhibits migration 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. Unexpectedly and in contradiction to the belief that myosin II molecules are essential for cell migration, we showed that myosin IIA is not required, and in fact it serves as a brake on migration in 2D cell culture. We also found strong cross-regulation between myosin IIA and microtubule dynamics that regulates Rac localization and cell migration. For technical reasons, it was necessary in our original study to use standard 2D culture systems for visualizing cytoskeletal dynamics and Rho GTPase functions. We had also previously established a key role for Rac in helping to regulate directional cell migration in such 2D settings. Two new projects are testing whether these principles of myosin II crosstalk and Rho family GTPase functions in migration are correct in 1D, 2D, and 3D cell migration 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 myosin and microtubules. All of these processes will 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.