A Fluorescence-Based Biosensor for Measurement of Cell Derived Forces

描述（由申请人提供）：过去几十年的研究已经证明了机械线索在细胞功能中的重要性。然而，很少有工具可以测量细胞产生的力，而且许多工具都有很大的局限性：细胞必须被镀在二维表面上；细胞必须被镀在柔软的、可变形的基底上；在多细胞的体内环境中，细胞力是无法测量的。当前提案的目标是开发一种生物传感器蛋白，它将有效地将机械信号转换为荧光信号。荧光信号的变化将在广泛的环境中测量，包括三维分析、不同硬度的底物和体内系统。这种生物传感器蛋白将由两个结构域构成：一个细胞结合结构域和一个力传导结构域。细胞结合区域将由细胞外基质蛋白纤维连接蛋白的片段组成，纤维连接蛋白已被证明可以介导细胞粘附。力传导结构域将由一段非结构化多肽组成，两侧是荧光蛋白。一种现象称为福斯特共振能量转移（FRET）发生在两个荧光蛋白之间，当他们在近距离。这种能量转移随着两者被进一步拉开而减少，并且可以在荧光显微镜上测量。FRET信号可以用来量化连接它们的多肽的端到端距离。施加在非结构化多肽上的力可以用聚合物动力学的数学模型来计算。施加的力和FRET信号之间的关系也将测量实验使用剪切流分析和磁场分析。随后，力生物传感器将用于测量细胞产生的力，其分辨率达到单个整合素键的水平。公共卫生相关性：细胞产生收缩力，拉动周围的组织和细胞。越来越多的证据表明，机械环境在组织的功能和生长中起着重要作用；例如，转移性肿瘤的组织明显比周围的健康组织硬；在肝脏中，驱动肝硬化的星状细胞只有在周围有坚硬的组织时才能茁壮成长；间充质干细胞的命运可以通过改变它们附着的表面的机械特性来引导到骨骼、大脑或肌肉上。然而，测量这些力的工具有限。当前提案的目标是开发一种蛋白质生物传感器，将机械信号转换为荧光信号，允许在广泛的设置中测量这些细胞来源的力。

DESCRIPTION (provided by applicant): Insights over the past decades have demonstrated the importance of mechanical cues in cellular function. However, few tools exist to measure cell-generated forces, and many of these have significant limitations: cells must be plated onto a 2-D surface; cells must be plated onto soft, deformable substrates; and cell forces cannot be measured in multicellular, in vivo environments. The goal of the current proposal is to develop a biosensor protein which would effectively convert a mechanical signal to a fluorescence signal. Changes in the fluorescence signal will be measurable in a wide range of environments, including 3-D assays, substrates of varying stiffness, and in vivo systems. This biosensor protein will be constructed of two domains: a cell-binding domain and a force-transducing domain. The cell- binding domain will consist of a fragment of the extracellular matrix protein, fibronectin, that has been shown to mediate cell adhesion. The force-transducing domain will consist of a length of unstructured polypeptide, flanked on either side by fluorescent proteins. A phenomenon known as Forster Resonance Energy Transfer (FRET) occurs between the two fluorescent proteins when they are in close proximity. This energy transfer diminishes as the two are pulled further apart, and can be measured on a fluorescence microscope. The FRET signal can be used to quantify the end-to-end distance of the polypeptide that links them. The force applied to the unstructured polypeptide can be calculated using mathematical models of polymer dynamics. The relationship between the applied force and the FRET signal will also be measured experimentally using shear flow assays and magnetic field assays. Subsequently, the force biosensor will be used to measure cell-generated forces, with resolution at the level of single integrin bonds. PUBLIC HEALTH RELEVANCE: Cells generate contractile forces that pull on surrounding tissues and cells. There is increasing evidence that the mechanical environment plays an important role in the function and growth of tissues; for example, the tissue of metastatic tumors is significantly stiffer than surrounding healthy tissue; in the liver, stellate cells that drive cirrhosis can only thrive when they have stiff tissue surrounding them; and the fate of mesenchymal stem cells can be directed towards bone, brain, or muscle simply by altering the mechanical properties of the surface to which they are attached. However, limited tools exist to measure these forces. The goal of the current proposal is to develop a protein biosensor that converts mechanical signals into fluorescence signals, allowing for measurement of these cell-derived forces in a wide range of settings.