Idevall-Hagren Olof, Decamilli Pietro
Department of Cell Biology, Program in Cellular Neuroscience, Neurodegeneration and Repair, Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, CT, 06510, USA.
Methods Mol Biol. 2014;1148:109-28. doi: 10.1007/978-1-4939-0470-9_8.
Phosphoinositides, the phosphorylated products of inositol phospholipids, play critical regulatory roles in cell physiology. The elucidation of their functions will greatly benefit from the methodology to manipulate their local concentrations within membranes with high spatial and temporal precision. Recently developed genetically encoded and light-regulated dimerization modules, in combination with the use of fluorescence-tagged lipid-binding domains and live-cell imaging, provide an attractive means to achieve this goal. Here we describe a protocol for blue light-dependent conversion of one phosphoinositide species into another based on the light-regulated dimerization between cryptochrome 2 (CRY2) and its ligand, CIB1. We describe the development of these tools using the dephosphorylation of plasma membrane phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) as an example and show how they can be used to rapidly and reversibly deplete the plasma membrane of this lipid. We also provide instructions for image analysis. The CRY2-CIB1 dimerization method has also already been adapted for the acute and spatially restricted generation of PI(3,4,5)P3 in the plasma membrane. More generally, this methodology should be broadly applicable to studies of the spatiotemporal regulation of membrane lipid metabolism in many types of cells.
磷酸肌醇是肌醇磷脂的磷酸化产物，在细胞生理过程中发挥着关键的调节作用。对其功能的阐明将极大地受益于能够在高空间和时间精度下操纵其在膜内局部浓度的方法。最近开发的基因编码且受光调控的二聚化模块，与荧光标记的脂质结合结构域的使用以及活细胞成像相结合，为实现这一目标提供了一种有吸引力的手段。在此，我们描述了一种基于隐花色素2（CRY2）与其配体CIB1之间的光调控二聚化，将一种磷酸肌醇物种蓝光依赖性转化为另一种的方案。我们以质膜磷脂酰肌醇4,5-二磷酸（PI(4,5)P2）的去磷酸化为例描述了这些工具的开发，并展示了它们如何用于快速且可逆地耗尽质膜中的这种脂质。我们还提供了图像分析的说明。CRY2-CIB1二聚化方法也已适用于在质膜中急性且空间受限地生成PI(3,4,5)P3。更普遍地，这种方法应广泛适用于多种类型细胞中膜脂代谢的时空调节研究。
