Imaging the Nanoscale Distribution of Phosphoinositides in the Cell Plasma Membrane with Single-Molecule Localization...

Phosphoinositides make up only a small fraction of cellular phospholipids yet control cell function in a fundamental manner. Through protein interactions, phosphoinositides define cellular organelle identity and regulate protein function and organization and recruitment at the cytosol-membrane interface. As a result, perturbations on phosphoinositide metabolism alter cell physiology and lead to a wide range of human diseases, including cancer and diabetes. Among seven phosphoinositide members, phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P₂, also known as PI(4,5)P₂ or PIP₂) is abundant in the plasma membrane. Besides its role in the second messenger pathway of phospholipase C that cleaves PtdIns(4,5)P₂ to form diacylglycerol and inositol-1,4,5-trisphosphate (IP₃), PtdIns(4,5)P₂ regulates membrane trafficking and the function of the cytoskeleton, ion channels, and transporters. The nanoscale organization of PtdIns(4,5)P₂ in the plasma membrane becomes essential to understand cellular signaling specificity in time and space. Here, we describe a single-molecule method to visualize the nanoscale distribution of PtdIns(4,5)P₂ in the plasma membrane by using super-resolution microscopy and the dual-color fluorescent probes based on the PLC_δ1 pleckstrin homology (PH) domain. This approach can be extended to image other phosphoinositides by changing the specific probes.