Live-cell imaging of ER-PM contact architecture by a novel TIRFM approach reveals extension of junctions in response to store-operated Ca(2+)-entry

Nanometer-spaced appositions between endoplasmic reticulum and plasma membrane (ER-PM junctions) stabilized by membrane-joining protein complexes are critically involved in cellular Ca(2+)-handling and lipid trafficking. ER-PM junctional architecture and plasticity associated with inter-membrane communication are as yet barely understood. Here, we introduce a method to precisely characterize ER-PM junction morphology and dynamics with high temporal resolution and minimal disturbance of junctional intermembrane communication. We show that expression of soluble cytosolic fluorophores in combination with TIRFM enables to delineate ER and PM distance in the range of 10–150 nm. Live-cell imaging of sub-plasmalemmal structures in RBL-2H3 mast cells by this method, designated as fluorescence density mapping (FDM), revealed profound dynamics of ER-PM contact sites in response to store-depletion. We report the existence of a Ca(2+)-dependent process that expands the junctional ER to enlarge its contact surface with the PM, thereby promoting and stabilizing STIM1-Orai1 competent ER-PM junctions.