Alkandari Mariam, McMaster Christopher R, Tavasoli Mahtab
Department of Pharmacology, Dalhousie University, Halifax, NS, Canada.
Bio Protoc. 2025 Jun 5;15(11):e5336. doi: 10.21769/BioProtoc.5336.
Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P] is a phospholipid enriched on the cytoplasmic leaflet of the plasma membrane, where it plays important roles in membrane trafficking and cytoskeletal dynamics through proteins that directly bind to it. PI(4,5)P can be metabolized to other phosphorylated forms of phosphatidylinositol to regulate numerous processes such as cell growth and development. PI(4,5)P can also be hydrolyzed to generate the second messengers diacylglycerol (DAG) and inositol triphosphate (IP). Altered metabolism or mislocalization of PI(4,5)P can perturb one or more of its functions and contribute to disease states. Here, we present a protocol to visualize and quantify the localization of PI(4,5)P in live cells. The protocol uses a highly specific PI(4,5)P protein binding domain coupled to enhanced green fluorescence protein (PH-PLCD1-GFP), enabling localization and quantification of cytosol-facing PI(4,5)P to be determined. Localization and quantification of the PH-PLCD1-GFP, PI(4,5)P specific probe, is enabled by fluorescence imaging and confocal microscopy. This approach can be used to study the dynamics of PI(4,5)P localization temporally in live cells under both physiological and pathological conditions. Key features • Protocol for the quantification of PI(4,5)P membrane localization in live cells. • Uses the expression of the highly specific PH-PLCD1-GFP, PI(4,5)P probe, in cells, followed by fluorescence image acquisition using confocal microscopy and subsequent image processing. • Adaptable to various cell types and experimental conditions. • Presents detailed instructions for reagent preparation, fluorescence measurement, and quantification.
磷脂酰肌醇-4,5-二磷酸[PI(4,5)P]是一种富集于质膜胞质侧小叶的磷脂，它通过直接与之结合的蛋白质在膜运输和细胞骨架动力学中发挥重要作用。PI(4,5)P可代谢为磷脂酰肌醇的其他磷酸化形式，以调节细胞生长和发育等众多过程。PI(4,5)P也可被水解生成第二信使二酰基甘油（DAG）和肌醇三磷酸（IP）。PI(4,5)P代谢改变或定位错误会扰乱其一项或多项功能，并导致疾病状态。在此，我们提供了一种在活细胞中可视化和定量PI(4,5)P定位的方案。该方案使用与增强型绿色荧光蛋白偶联的高度特异性PI(4,5)P蛋白结合结构域（PH-PLCD1-GFP），能够确定面向胞质溶胶的PI(4,5)P的定位和定量。通过荧光成像和共聚焦显微镜可实现PH-PLCD1-GFP（PI(4,5)P特异性探针）的定位和定量。这种方法可用于研究在生理和病理条件下活细胞中PI(4,5)P定位的时间动态。关键特性 • 活细胞中PI(4,5)P膜定位定量方案。 • 使用细胞中高度特异性的PH-PLCD1-GFP（PI(4,5)P探针）表达，随后使用共聚焦显微镜采集荧光图像并进行后续图像处理。 • 适用于各种细胞类型和实验条件。 • 提供试剂制备、荧光测量和定量分析详细说明。
