title: Live-cell imaging of ER-PM contact architecture by a novel TIRFM approach reveals extension of junctions in response to store-operated Ca2+-entry.
abstract: Nanometer-spaced appositions between endoplasmic reticulum and plasma membrane (ER-PM junctions) stabilized by membrane-joining protein complexes are critically involved in cellular Ca2+-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 Ca2+-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.
authors.affiliation: Institute of Biophysics, Medical University of Graz, Harrachgasse 21/4, 8010 Graz, Austria.
authors.forename: Michael
authors.initials: M
authors.lastname: Poteser
authors.affiliation: Institute of Cell Biology, Histology and Embryology Research Unit "Electron Microscopic Techniques", Medical University of Graz, Harrachgasse 21/7, 8010 Graz, Austria.
authors.forename: Gerd
authors.initials: G
authors.lastname: Leitinger
authors.affiliation: Institute of Cell Biology, Histology and Embryology Research Unit "Electron Microscopic Techniques", Medical University of Graz, Harrachgasse 21/7, 8010 Graz, Austria.
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Pritz
authors.affiliation: Institute of Biophysics, Medical University of Graz, Harrachgasse 21/4, 8010 Graz, Austria.
authors.forename: Dieter
authors.initials: D
authors.lastname: Platzer
authors.affiliation: Institute of Biophysics, Johannes Kepler University of Linz, Austria, Gruberstrasse 40, 4020 Linz, Austria.
authors.forename: Irene
authors.initials: I
authors.lastname: Frischauf
authors.affiliation: Institute of Biophysics, Johannes Kepler University of Linz, Austria, Gruberstrasse 40, 4020 Linz, Austria.
authors.forename: Christoph
authors.initials: C
authors.lastname: Romanin
authors.affiliation: Institute of Biophysics, Medical University of Graz, Harrachgasse 21/4, 8010 Graz, Austria.
authors.forename: Klaus
authors.initials: K
authors.lastname: Groschner
mesh_terms: D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism* / Q000648:ultrastructure*; D004721:Endoplasmic Reticulum / Q000378:metabolism* / Q000648:ultrastructure*; D006801:Humans; D000069416:Intravital Microscopy / Q000379:methods*; D008407:Mast Cells / Q000166:cytology* / Q000378:metabolism / Q000648:ultrastructure; D062211:Spatio-Temporal Analysis
chemical_list: D002118:Calcium