CELL MEMBRANE FLUIDITY UNDER FLUID SHEAR STRESS

拟议研究的总体目标是确定哺乳动物细胞对流体流动的主要机械传感器。不同类型的细胞对流体剪切应力的各种反应已经在生化基础上进行了探索，例如一氧化氮的产生或G蛋白的激活。假设是流体剪切应力增加了膜的流动性，从而激活异三量G蛋白。荧光膜探针的初步实验有力地支持了这一假设。拟议中的项目将侧重于基于荧光和实时计算机图像处理的显微技术。三种可选的方法是：光漂白后荧光恢复、荧光偏振各向异性和环境特异性荧光探针。这三种方法都允许在亚细胞尺度上探测流动性特性，尽管物理原理根本不同。因此，个别结果将用于相互支持和完善整体发现。此外，可以研究位置特定效应，例如细胞-细胞连接处的力积累。如果成功，该研究将为内皮细胞如何感知血流动力学力以及细胞内和宏观尺度上的剪切力分布提供基本的理解。

The overall goal of the proposed research is to determine the primary mechanosensor of mammalian cells for fluid flow. Various responses of different cell types to fluid shear stress have been explored on a biochemical basis, e.g. nitric oxide production or activation of G proteins. The hypothesis is that fluid shear stress increases membrane fluidity, thus activating heterotrimetric G proteins. Preliminary experiments using fluorescent membrane probes strongly support the hypothesis. The proposed project will focus on microscopic techniques that build on fluorescence and real-time computer image processing. Three alternative methods well be applied: Fluorescence recovery after photobleaching, fluorescence polarization anisotropy, and environment-specific fluorescent probes. All three methods allow the probing of fluidity properties on a sub- cellular scale, although the physical principles are fundamentally different. Therefore, the individual results will be used to mutually support and complete the overall findings. Furthermore, location specific effects, e.g. force accumulation at the cell-cell junction, can be investigated. If successful, the study will provide a fundamental understanding on how the endothelium senses hemodynamic forces as well as an insight into the shear-induced force distribution within the cell and on a macroscopic scale.