Analysis of Phosphatidylinositol Transfer at ER-PM Junctions in Receptor-Stimulated Live Cells

Phosphatidylinositol (PI) is an inositol-containing phospholipid synthesized in the endoplasmic reticulum (ER). PI is a precursor lipid for PI 4,5-bisphosphate (PI(4,5)P₂) in the plasma membrane (PM) important for Ca²⁺ signaling in response to extracellular stimuli. Thus, ER-to-PM PI transfer becomes essential for cells to maintain PI(4,5)P₂ homeostasis during receptor stimulation. In this chapter, we discuss two live-cell imaging protocols to analyze ER-to-PM PI transfer at ER-PM junctions, where the two membrane compartments make close appositions accommodating PI transfer. First, we describe how to monitor PI(4,5)P₂ replenishment following receptor stimulation, as a readout of PI transfer, using a PI(4,5)P₂ biosensor and total internal reflection fluorescence microscopy. The second protocol directly visualizes PI transfer proteins that accumulate at ER-PM junctions and mediate PI(4,5)P₂ replenishment with PI in the ER in stimulated cells. These methods provide spatial and temporal analysis of ER-to-PM PI transfer during receptor stimulation and can be adapted to other research questions related to this topic.