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传导和疾病的重要参数。目前，微观尺度上的粘度是由荧光各向异性、光漂白后荧光恢复（FRAP）或磁性纳米颗粒决定的。一组粘度敏感的荧光分子，通常被称为分子转子，允许一种新的，快速，方便的方法，仪器要求最低，非常高的时间和空间分辨率。然而，分子转子有一个缺点——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例染料（j.a.m chem . soc）上。2006年;128: 398-399)，其中分子转子和荧光参考单元形成共价连接染料对共振能量转移（RET）。这种比例染料有潜力克服浓度梯度和光学性质的限制。提出的研究的总体目标是开发特定的荧光比例粘度传感器，用于磷脂双层，细胞膜和细胞质。我们提出了合成、表征和优化膜靶向以及细胞质靶向的比例分子转子。实验方法包括新型黏度敏感染料在模型磷脂双层、红细胞幽灵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对新探针进行表征。

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.