Wuttke Anne, Idevall-Hagren Olof, Tengholm Anders
Department of Medical Cell Biology, Uppsala University, Uppsala, Sweden.
Methods Mol Biol. 2010;645:219-35. doi: 10.1007/978-1-60327-175-2_14.
To improve our understanding of the important roles played by inositol lipid derivatives in signalling and other cellular processes, it is crucial to measure phosphoinositide concentration changes in individual cells with high spatial and temporal resolution. A number of protein domains that interact with inositol lipids in a specific manner have been identified. Tagged with the green fluorescent protein or its colour variants, these protein modules can be used as probes to visualize various phosphoinositide species in different sub-cellular compartments. Here, we present protocols for fluorescence imaging of phosphoinositide dynamics in single living cells. Total internal reflection fluorescence microscopy is particularly powerful for time-lapse recordings of phosphoinositides in the plasma membrane. We demonstrate how this technique can be used to record phospholipase C- and PI3-kinase-induced changes in inositol lipids in insulin-secreting cells. These procedures should be applicable to studies of the spatio-temporal regulation of phosphoinositide metabolism in many types of cells.
为了增进我们对肌醇脂质衍生物在信号传导及其他细胞过程中所起重要作用的理解，至关重要的是要以高空间和时间分辨率测量单个细胞中磷酸肌醇浓度的变化。已经鉴定出许多以特定方式与肌醇脂质相互作用的蛋白质结构域。这些蛋白质模块用绿色荧光蛋白或其颜色变体标记后，可用作探针来可视化不同亚细胞区室中的各种磷酸肌醇种类。在此，我们介绍在单个活细胞中对磷酸肌醇动力学进行荧光成像的方案。全内反射荧光显微镜对于质膜中磷酸肌醇的延时记录特别有效。我们展示了如何使用该技术记录胰岛素分泌细胞中磷脂酶C和PI3激酶诱导的肌醇脂质变化。这些程序应适用于多种类型细胞中磷酸肌醇代谢的时空调节研究。
