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、整合的机制促进细胞迁移和信号传递。我们正在解决以下具体问题： 1.哪些亚细胞结构和信号通路对细胞的快速迁移是重要的？ 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正常的细胞迁移的？ 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。 我们先前证实，细胞外基质(ECM)的形态在调节细胞骨架组织、细胞形态和细胞迁移方面起着至关重要的作用，因为我们证明了一维(1D)微图案线模拟了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，以建立成纤维细胞中纤维形态引起快速、有效细胞迁移的机制，以及这种迁移模式与以前使用常规二维(2D)组织培养底物研究的迁移有何不同。我们发现，间充质细胞迁移的两个关键过程，前缘的突出和板层内形成的粘连，在一维迁移过程中得到加强，并间接受到细胞收缩的控制。 利用高速成像，我们发现在一维迁移过程中，迁移细胞前沿的细胞膜突起形成得更快、更频繁。这种增强依赖于将侧向粘连面积减少到非常窄的基板(1.5微米宽)。量化显示，前缘突出率和净突出率分别增加了2.8倍和10倍，迁移率增加了50%。相反，2D迁移的特征是在前沿有一个宽阔的片层，沿片层-板层边界横向分布着多个细胞粘连。这些发现表明，物理ECM约束可以调节前沿动态、粘连和细胞迁移的速度。 出乎意料的是，与细胞来源的基质纤维的1D和3D黏附被发现在相当程度上与基质保持更稳定的联系(例如，比2D长6倍)。为了检测粘连中的蛋白质动力学，我们对表达GFP标记或KikGR标记的粘连分子的细胞进行了光漂白后荧光恢复(FRAP)和光转换后荧光损失(FLAP)实验。动力学分析表明，与2D底物上形成的粘连相比，1D粘连中的paxlin、vinculin和actin与基础基质保持着更稳定的联系，这与延长细胞粘连的时间一致。有趣的是，虽然2D底物上的整合素显示逆行流向细胞中心，但1D粘附物中的整合素相对于细胞外基质保持稳定，这表明细胞外基质和肌动蛋白细胞骨架之间的物理联系增加。用Blebbistatin抑制肌球蛋白II的功能或条件性敲除肌球蛋白IIA后，肌球蛋白II的收缩能力丧失，破坏了一维黏附稳定性，降低了前沿突起效率。我们认为，肌球蛋白IIA通过稳定细胞和底物之间的分子离合器中的黏附蛋白，进而调节迁移效率，增加了细胞对纤维底物的粘附性。 我们使用3D胶原水凝胶和3D细胞衍生基质，继续探索细胞外基质在调节成纤维细胞黏附、迁移和机械转导中的作用。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。深入了解细胞移动和与其基质环境相互作用的精确方式也将有助于组织工程学研究。

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 have 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. Using high-speed imaging, we discovered that protrusions of the cell membrane at the leading edge of migrating cells form more rapidly and frequently during 1D migration. This enhancement depends on reducing lateral adhesion area to a very narrow substrate (1.5 micrometers wide). Quantification showed 2.8- and 10-fold increases in leading edge protrusion rate and net protrusion, respectively, with a 50% increase in migration rate. In contrast, 2D migration is characterized by a wide lamellipodium at the leading edge with multiple cell adhesions spread laterally along the lamellipodium-lamellum border. These findings indicate that physical ECM constraints can regulate leading edge dynamics, adhesions, and the speed of cell migration. Unexpectedly, both 1D adhesions and 3D adhesions to cell-derived matrix fibrils were found to remain more stably associated with the matrix to a substantial degree (e.g., 6-fold longer than in 2D). To examine protein dynamics within adhesions, we used fluorescence recovery after photobleaching (FRAP) and fluorescence loss after photoconversion (FLAP) experiments with cells expressing GFP-tagged or KikGR-tagged adhesion molecules. Kinetic analysis revealed that paxillin, vinculin, and actin within 1D adhesions remain more stably associated with the underlying matrix compared to adhesions formed on 2D substrates, consistent with prolonged cell adhesiveness. Interestingly, while integrins on 2D substrates demonstrated retrograde flow towards the cell center, integrins within 1D adhesions remained stable with respect to the ECM, suggesting increased engagement of the physical link between the ECM and the actin cytoskeleton. Loss of contractility after inhibiting myosin II function with blebbistatin or conditional knockout of myosin IIA disrupted 1D adhesion stability and reduced leading edge protrusion efficiency. We suggest that myosin IIA increases cell adhesiveness to fibrillar substrates by stabilizing adhesion proteins found within a molecular clutch between the cell and the substrate, which in turn regulates migration efficiency. We are continuing to explore the role of ECM topography in regulating fibroblast adhesion, migration, and mechanotransduction using 3D collagen hydrogels to compare with 3D cell-derived matrix. 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 the precise manner in which cells move and interact with their matrix environment will also facilitate tissue engineering studies.