PI(4,5)P₂ Imaging Using a GFP Reporter in Living Cells

Background

Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P₂] is a phospholipid that belongs to the phosphoinositide family. Phosphoinositides are phosphorylated derivatives of phosphatidylinositol that can be further phosphorylated at different positions on the inositol ring to generate a variety of distinct lipid signaling molecules [1,2]. PI(4,5)P₂ is a phosphoinositide that is preferentially localized to the inner leaflet of the plasma membrane [3–5]. PI(4,5)P₂ can bind to numerous proteins, often through a distinct PI(4,5)P₂ binding domain, with exemplars including Pleckstrin homology (PH) domain, Phox (PX) homology domain, Postsynaptic Density-95 (PSD-95)/Discs Large (Dlg)/Zonula Occludens-1 (PDZ), Protein Kinase C conserved Region 2 (C2), Src Homology 2 (SH2), Plant Homeodomain (PHD), and Myristoylated Alanine-Rich C-Kinase Substrate (MARCKS) domains [2,6–10]. This binding often aids in the localization of a protein to the plasma membrane, where they regulate numerous processes such as membrane trafficking and cytoskeletal dynamics [1,5,7–11]. PI(4,5)P₂ also serves as a substrate for phospholipase Cs that convert PI(4,5)P₂ to diacylglycerol and inositol triphosphate (IP₃). Diacylglycerol can itself recruit proteins to membranes, including members of the protein kinase C family, while IP₃ releases Ca²⁺ from the endoplasmic reticulum [12,13]. Perturbation of the level or localization of PI(4,5)P₂ can have profound effects on cell dynamics and cell signaling and contribute to disease states [14–16].

Numerous phosphoinositide binding domains have varying affinities and specificities for each phosphoinositide [2,3,17]. The PH domain of phospholipase C delta (PH-PLCD1) has high affinity and specificity for PI(4,5)P₂, and a chimeric PH-PLCD1-GFP fusion is an accurate reporter for PI(4,5)P₂ localization [5,6,11,17,18]. This interaction has been validated in multiple species and cell types, including mammalian cells, Xenopus oocytes, Drosophila, and yeast, supporting its broad applicability [19–22]. Our optimized protocol allows for the visualization of PI(4,5)P₂ and enables the quantification of the ratio of plasma membrane to intracellular PI(4,5)P₂. Using the PH-PLCD1-GFP fusion allows for real-time high-resolution tracking of PI(4,5)P₂ in live cells. This protocol serves as a comprehensive step-by-step guide providing a standardized approach to quantify PI(4,5)P₂ plasma membrane localization.