Collaborative Research: Mechanisms of Gradient Sensing by 'Feel' in Cell Migration Directed by Extracellular Matrix

细胞迁移是体内许多过程中的重要步骤，如伤口愈合和对感染的反应。这些细胞与允许它们爬行和探索的基质接触。要做到这一点，细胞必须通过触摸和感觉来主动感知基质属性。这个合作项目的首要目标是统一或区分细胞对与基质接触的数量(触摸粘性)和质量(感觉硬度)的感知。多学科研究将与研究生培训、工程课程教学以及促进科学交流的推广相结合。细胞迁移是组织发育和动态平衡的基本过程。化学趋化性的分子决定因素，即细胞迁移受到可溶引诱剂的梯度的影响，已经被很好地理解，并出现了一种范式，将某些细胞内信号通路描述为分子“指南针”。相比之下，趋触性和趋杜性分别由固定化配体密度梯度和机械刚性引导的迁移，人们对此知之甚少，需要一个新的范式，考虑到细胞必须主动地遇到和响应物理提示(即，通过“感觉”)，而不是通过被动地感知可扩散的配体。在目标1下，目标是定义控制迁移细胞中耦合的细胞骨架动力学的反馈机制。据推测，黏附介导的信号通路控制板脂突起的持续时间，并由F-肌动蛋白束在空间上协调。高分辨率的活细胞显微镜和假定的信号和细胞骨架过程的分子扰动将被用来系统地将调节肌动蛋白细胞骨架的假定机制的扰动与板脂突出动力学的变化联系起来。这些研究有望揭示细胞运动是如何受到物理信号的影响的。在目标2下，建议在黏附、信号和细胞骨架动力学的水平上阐明突触偏向的性质。假设F-肌动蛋白束/丝状伪足直接和片状伪足传播，突触探索。一个相关的假说是，突触偏向(比较上下梯度)表现为信号和/或细胞骨架动力学的差异，由粘连整合。建议分析在使用微流控装置产生的趋化梯度上迁移过程中黏附、信号和细胞骨架结构的动力学。这些研究将描述趋触性的前沿运动动力学，这是一种不同于趋化性的、鲜为人知的定向迁移模式。在目标3下，拟议的工作将阐明趋多性的分子和生物物理决定因素，并确定趋上性和趋多性之间的共同或不同特征。将产生具有交联度梯度的水凝胶，并将其与牵引力显微镜相结合，牵引力显微镜是一种测量弹性凝胶中局部应力的方法。这些研究将测试富含发束素的丝状足作为感觉器引导趋向性迁移的作用。这一结果将进一步阐明细胞突起、局灶性黏附动力学和耐药细胞施加的牵引力之间的关系。该项目的跨学科性质为培训两名研究生提供了独特的环境，他们将从事研究和促进科学交流的外联活动。

Cell migration is an important step in many processes in the body, such as the healing of wounds and the response to infection. The cells are in contact with a matrix that allows them to crawl and explore. To do so, cells must actively sense matrix properties by touch and feel. An overarching goal of this collaborative project is to unify or distinguish cell sensing of the quantity (stickiness by touch) versus quality (stiffness by feel) of contacts with the matrix. The multidisciplinary research will be integrated with graduate student training, teaching of engineering courses, and outreach that will foster science communication.Cell migration is a fundamental process in tissue development and homeostasis. The molecular determinants of chemotaxis, cell migration biased by a gradient of a soluble attractant, are reasonably well understood, and a paradigm has emerged characterizing certain intracellular signaling pathways as a molecular "compass". In contrast, haptotaxis and durotaxis, migration directed by gradients of immobilized ligand density and of mechanical stiffness, respectively, are poorly understood and require a new paradigm, considering that cells must actively encounter and respond to physical cues (i.e., by "feel") rather than by passive sensing of diffusible ligands. Under Objective 1, the aim is to define feedback mechanisms that control coupled cytoskeletal dynamics in migrating cells. It is hypothesized that adhesion-mediated signaling pathways control the duration of lamellipodial protrusion and are spatially coordinated by F-actin bundles. High-resolution, live-cell microscopy and molecular perturbations of the putative signaling and cytoskeletal processes will be applied to systematically relate perturbations of putative mechanisms regulating the actin cytoskeleton to changes in lamellipodial protrusion dynamics. These studies are expected to show how cell motility is biased by physical cues. Under Objective 2, it is proposed to elucidate the nature of haptotactic bias at the level of adhesion, signaling, and cytoskeletal dynamics. It is hypothesized that F-actin bundles/filopodia direct, and lamellipodia propagate, haptotactic exploration. A related hypothesis is that the haptotactic bias (comparing up- vs. down-gradient) manifests as differences in signaling and/or cytoskeletal dynamics, integrated by adhesions. It is proposed to analyze the dynamics of adhesion, signaling, and cytoskeletal structures during migration on haptotactic gradients generated using microfluidic devices. These studies will characterize leading-edge motility dynamics during haptotaxis, a poorly understood mode of directed migration that is distinct from chemotaxis. Under Objective 3, the proposed work will elucidate the molecular and biophysical determinants of durotaxis and define features that are common or distinct between haptotaxis and durotaxis. Hydrogels with gradients of crosslinking will be generated and integrated with traction force microscopy, a method for measuring local stress in an elastic gel. These studies will test the role of fascin-rich filopodia as sensory organelles guiding durotactic migration. The results will further elucidate the relationship between cell protrusion, focal adhesion dynamics, and traction force exerted by durotaxing cells. The interdisciplinary nature of the project offers a unique environment for the training of two graduate students, who will be engaged in research and an outreach activity fostering science communication.