Real-time microviscosity measurement tools for the cell

描述(申请人提供)：细胞膜和细胞质中的微粘度是细胞信号和疾病的重要参数。目前，微观尺度上的粘度由荧光各向异性、光漂白后荧光恢复(FRAP)或磁性纳米颗粒决定。一组粘度敏感的荧光分子，通常被称为分子转子，允许以最小的仪器要求和非常高的时间和空间分辨率来实现一种新的、快速和方便的方法。然而，分子旋转器有一个缺点--它们是基于强度的，局部浓度梯度可能会降低测量精度。拟议的研究计划建立在最近开发的比率计量型染料(J.Am.Chem.Soc.2006；128：398-399)，其中分子转子和荧光参考单元形成共价连接的染料对，用于共振能量转移(RET)。这种比率计量型染料有可能克服浓度梯度和光学性质带来的限制。拟议研究的总体目标是开发用于磷脂双层、细胞膜和细胞质的特定荧光比率粘度传感器。我们提出了膜靶向和细胞质靶向比率计量型分子旋转器的合成、表征和优化。实验方法包括新型粘度敏感染料在模型磷脂双层膜、红细胞膜和活细胞中的测试和应用。这种测试方法使我们能够在日益复杂的环境中表征新的探测器。 拟议研究的结果将是一系列适用于蜂窝环境的实时微尺度粘度探头的可用性，应用范围广泛。新的粘度探头将有用的一些例子包括： -分析在流体切应力下细胞膜参与细胞信号传递的情况(血管内皮细胞) 动脉粥样硬化中细胞膜生物力学变化的分析 -涉及膜脂筏的研究 -分析细胞质粘度在细胞冷冻保存中的作用 虽然本申请的目的不是在上面的例子中实际应用分子旋转器，但我们将为涉及上述任何领域或相关领域的研究人员提供必要的探针及其使用方法。 公共卫生相关声明：细胞膜和细胞质粘度与细胞信号(例如，血压调节)和各种疾病状态(例如，与动脉粥样硬化、细胞恶性肿瘤、高胆固醇血症和糖尿病相关的细胞膜粘度改变)高度相关。我们建议开发新的、超快和超高分辨率的方法，利用荧光分子旋转器来确定膜和细胞质粘度的变化。有了这些新工具，涉及细胞粘度的研究将被加速或首先成为可能，从而能够更快地研究和更好地了解细胞信号传递过程和各种疾病状态的细胞基础。细胞膜和细胞质粘度与细胞信号(例如血压调节)和各种疾病状态(例如与动脉粥样硬化、细胞恶性肿瘤、高胆固醇血症和糖尿病相关的膜粘度改变)高度相关。我们建议开发新的、超快和超高分辨率的方法，利用荧光分子旋转器来确定膜和细胞质粘度的变化。有了这些新工具，涉及细胞粘度的研究将被加速或首先成为可能，从而能够更快地研究和更好地了解细胞信号传递过程和各种疾病状态的细胞基础。

DESCRIPTION (provided by applicant): Microviscosity in the cell membrane and the cell cytoplasm are important parameters in cell signaling and disease. Presently, viscosity on the microscopic scale is determined by fluorescence anisotropy, fluorescence recovery after photobleaching (FRAP), or magnetic nanoparticles. A group of viscosity-sensitive fluorescent molecules, generally termed molecular rotors, allows a new, fast, and convenient approach with minimum requirements of instrumentation and very high temporal and spatial resolution. However, molecular rotors pose one disadvantage - they are intensity based, and local concentration gradients may reduce measurement accuracy. The proposed research program builds on a recently developed ratiometric dye (J.Am.Chem.Soc. 2006; 128: 398-399) in which a molecular rotor and a fluorescent reference unit form a covalently linked dye pair for resonance energy transfer (RET). This ratiometric dye has the potential to overcome limitations posed by concentration gradients and optical properties. The overall goal of the proposed research is to develop specific fluorescent ratiometric viscosity sensors to be used in phospholipid bilayers, cell membranes, and the cell cytoplasm. We propose the synthesis, characterization, and optimization of membrane-targeted as well as cytoplasm-targeted ratiometric molecular rotors. The experimental approach includes the testing and application of the new viscosity sensitive dyes in model phospholipid bilayers, red cell ghosts, and living cells. This testing approach allows us to characterize the new probes in environments of increasing complexity. The outcome of the proposed research will be the availability of a series of real-time, microscale viscosity probes for cellular environments with a wide range of applications. Some examples where the new viscosity probes will be useful are: - Analysis of the involvement of the cell membrane in cell signaling under fluid shear stress (vascular endothelial cells) - Analysis of changes in cell membrane biomechanics in atherosclerosis - Studies involving membrane lipid rafts - Analysis of the role of cytoplasmic viscosity in the cryopreservation of cells While it is not the goal of this application to actually apply molecular rotors in the above examples, we will provide the necessary probes as well as their methods of use for investigators involved in any fields listed above or related. Public Health Relevance Statement: Cell membrane and cytoplasmic viscosity are of high relevance to cell signaling (e.g. blood pressure regulation) and to various disease states (e.g. altered membrane viscosity related to atherosclerosis, cell malignancy, hypercholesterolemia, and diabetes). We propose to develop new, ultrafast and ultra-high resolution methods to determine changes in membrane and cytoplasm viscosity using fluorescent molecular rotors. With these new tools, studies involving viscosity in the cell will be accelerated or made possible in the first place, thus enabling faster study and better understanding of cell signaling processes and the cellular foundations of various disease states. Cell membrane and cytoplasmic viscosity are of high relevance to cell signaling (e.g. blood pressure regulation) and to various disease states (e.g. altered membrane viscosity related to atherosclerosis, cell malignancy, hypercholesterolemia, and diabetes). We propose to develop new, ultrafast and ultra-high resolution methods to determine changes in membrane and cytoplasm viscosity using fluorescent molecular rotors. With these new tools, studies involving viscosity in the cell will be accelerated or made possible in the first place, thus enabling faster study and better understanding of cell signaling processes and the cellular foundations of various disease states.