Development of a Fluorescence-Based Force Sensor Molecule

描述（由申请人提供）：细胞运动是癌症发展、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物化学表征。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。我们建议通过开发荧光力传感器分子来测量作为力的函数的细胞迁移力学。基于DNA发夹结构，该分子具有模块化设计，其中发夹的良好表征的双链体用作力传感组件，而发夹基部的FRET对用作解压缩发夹所需的力的报告物。我们已经校准了分子，并测量了同时发生的构象和荧光转换在18 pN。为了在分子水平上研究细胞粘附力，我们将含有RGD的粘附肽，常见的细胞外基质蛋白，纤连蛋白，整合素介导的粘附传感器。在初步结果中，我们已经验证了在盖玻片上合成力传感器分子的化学。我们将在传统的荧光显微镜下通过分子内FRET的损失对基质施加粘附力的位置进行成像。因为力的信息是可视的，我们将非计算地生成一个运动细胞的动态“分子力图”。由于其简单性和缺乏计算，力可视化技术有可能成为缺乏复杂力学，计算或光学专业知识的细胞生物学家进行基于力的测定的基础。公共卫生相关性：细胞迁移是胚胎组织和器官发育、成体组织维持和癌症转移的基本过程。在我们对迁移的理解中，一个很大的差距是作为运动机制特征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，从而绘制出细胞移动通过组织纤维的3D矩阵时产生的力场。

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.