Development of a Fluorescence-Based Force Sensor Molecule

描述（申请人提供）：细胞运动是癌症发生、免疫和转移的关键基础过程。直到最近，大多数研究都集中在这些现象的生化表征上。然而，来自细胞外基质的机械信号在细胞运动的调节中起着重要作用。我们建议通过开发一种荧光力传感器分子来测量细胞迁移力学作为力的函数。基于DNA发夹结构，该分子具有模块化设计，发夹具有良好的双极性，可作为力传感组件，而发夹底部的FRET对可作为打开发夹所需的力的报告器。我们已经校准了分子，并测量了同时构象和荧光跃迁发生在18pn。为了在分子水平上研究细胞粘附力，我们加入了一种含有rgd的粘附肽，与细胞外基质蛋白纤维连接蛋白共同，用于整合素介导的对传感器的粘附。在初步结果中，我们已经验证了在盖盖上合成力传感器分子的化学反应。我们将成像的附着力是通过损失分子内FRET与传统荧光显微镜施加到矩阵。因为力信息是可视化的，我们将非计算生成一个动态的“分子力图”的运动细胞。由于其简单性和缺乏计算性，力可视化技术有潜力成为缺乏复杂力学、计算或光学专业知识的细胞生物学家进行基于力的分析的基础。公共卫生相关性：细胞迁移是胚胎组织和器官发育、成人组织维持和癌症转移的基本过程。我们对迁移的理解有一个很大的差距，那就是描述迁移机制的细胞力的起源和大小。分子荧光力传感器将提供原位检测和报告分子力的能力，从而绘制出细胞在组织纤维的3D矩阵中移动时产生的力场。

DESCRIPTION (provided by applicant): Cell motility is a key fundamental process in development, immunity, and metastasis of cancers. Until recently, most studies have focused on the biochemical characterization of these phenomena. However, mechanical cues from the extracellular matrix play an important role in the regulation of cell locomotion. We propose to measure cell migration mechanics as a function of force by developing a fluorescent force sensor molecule. Based on a DNA hairpin structure, the molecule has a modular design with the well characterized duplex of the hairpin serves as the force sensing component, while a FRET pair at the base of the hairpin serves as a reporter of the force necessary to unzip the hairpin. We have calibrated the molecule and measured the simultaneous conformational and fluorescence transitions occur at 18 pN. To study cellular adhesion forces at the molecular level, we have incorporated an RGD-containing adhesion peptide, common to the extracellular matrix protein, fibronectin, for integrin-mediated adhesion to the sensor. In preliminary results, we have validated the chemistry for synthesis of the force sensor molecule on a coverslip. We will image where adhesive force is applied to the matrix by a loss of intramolecular FRET with a conventional fluorescence microscope. Because the force information is visual, we will non-computationally generate a dynamic 'molecular force map' of a motile cell. Because of its simplicity and lack for computation, the force visualization technology has the potential to be a basis for force-based assays by cell biologists who lack expertise in sophisticated mechanics, computational, or optics. Public Health Relevance: Cell migration is a fundamental process in the development of embryonic tissues and organs, the maintenance of adult tissues, and the transition to metastasis in cancer. A large gap in our understanding of migration is the origin and magnitude of cellular forces that characterize the mechanics of movement. A molecular fluorescent force sensor would provide the capability of detecting and reporting molecular forces in situ and thus map out the force fields generated when a cell moves through a 3D matrix of tissue fibers.