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.

Ionic stress enhances ER–PM connectivity via phosphoinositide-associated SYT1 contact site expansion in Arabidopsis

“…Using pharmacological approaches, we established that the stress-induced changes in ER-PM connectivity are cytoskeleton independent, and by using fluorescent lipid sensors we uncovered that the dynamics of S-EPCS expansion closely match the dynamics of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P 2 ] accumulation at the PM. These results support the tethering arrangement model of S-EPCS organization, and highlight temporal and mechanistic differences between the fast (within minutes) stress-induced changes in ER-PM connectivity described in mammalian cells (40)(41)(42) and the slow (within hours) stress-induced increase in ER-PM connectivity observed in Arabidopsis seedlings. We propose that the changes in ER-PM connectivity in response to long-term exposure to ionic stress modulate the activity of EPCS-localized components (e.g., SYT1) and facilitate long-term plant adaptive responses to stress (e.g., exchanges of membrane lipids between the cER and the PM).…”

“…To partially overcome these limitations, we adapted the MAPPER marker, originally developed in mammalian cells (39), for use in Arabidopsis. The original MAPPER is a constitutive EPCS marker that nonselectively labels ER-PM junctions without significantly affecting their size, density, and functions (39,40). The Arabidopsis version of MAPPER includes an internal GFP localized within the ER lumen, and maintains the original flexible linker of ∼25-nm length and the C terminus lysine-rich polybasic domain that enables nonselective electrostatic interactions with negatively charged PM phosphoinositides (39) (Fig.…”

Direct characterization of the evanescent field in objective-type total internal reflection fluorescence microscopy

“…Our calibration slide may serve as a tool to routinely check the quality and reproducibility of the evanescent field generated by the multitude of commercial and custom-built TIRF setups [48], thus simplifying interpretation and comparability of acquired data from TIRF microscopes. Furthermore, approaches where the shape of the axial excitation profile is used to infer the axial position of fluorescently labeled molecules [1][2][3][4][5][6][7][8][9][10][11][12][13][19][20][21][22], in particular when combined with incident angle scanning [14][15][16][17][18], will benefit from a fast and precise single-slide calibration tool.…”

“…The brightness of a fluorophore reflects the local excitation intensity, therefore the axial dependence of the excitation field in TIRF microscopy offers the possibility to infer the z-position of a fluorophore. Observing the position, movement, or distribution of molecules in, or at the cell membrane provides insights into many biological processes, such as endo-and exocytosis [1][2][3][4][5][6] or cellular signaling [7][8][9][10][11], as well as (super-resolved) structural information [12][13][14][15][16][17][18]. However, a precise knowledge of the axial excitation profile is often required to accurately interpret TIRF data .…”

Current and Emerging Approaches for Studying Inter-Organelle Membrane Contact Sites

“…Tandem GFP pairs allowed for detecting the alteration of intracellular Ca 2+ level at mitochondria-ER MCS in HeLa and HEK293 cells, in which tandem expression of BFP-CBD (26-residue containing calmodulin-binding domain)-EGFP was sensitive to change of Ca 2+ flux that causes a structural alteration of CBD and a destroyed FRET pair (Miyawaki et al, 1997;Romoser et al, 1997). Cooperation between FRET and total internal reflection microscopy (TIRFM) was designed to study ER-PM MCS in RBL-2H3 (mice) and HeLa cells (Poteser et al, 2016;Chen et al, 2017;Chang et al, 2018). FRET was applied to study lipid transfer regulated by oxysterol-binding protein (OSBP) at the MCSs between ER and other organelles in human RPE1 cells (Jamecna et al, 2019).…”