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.
Highlights
Communication of the endoplasmic reticulum (ER) with other organelles is typically mediated by specialized contact sites displaying a membrane nano-architecture that enables efficient exchange of substrates and information
As the method is based on the reduction of overall cytosolic fluorophore density by local non-fluorescent sub-plasmalemmal structures, we designated this method as fluorescence density mapping (FDM)
We introduce fluorescence density mapping (FDM) as a simple and versatile extension of total internal reflection microscopy (TIRFM)
Summary
Communication of the endoplasmic reticulum (ER) with other organelles is typically mediated by specialized contact sites (junctions) displaying a membrane nano-architecture that enables efficient exchange of substrates and information. Despite an increasing awareness of the (patho)physiological significance of ER-PM junctions and their architectural plasticity, suitable methods for live-cell fluorescence imaging of dynamic morphological changes in these nano-structures have not been developed. This important experimental advance is so far hindered by the lack of fluorescent probes, which reliably report junctional morphology without interfering with architecture and function. We utilized FDM in combination with common TIRFM and TIRFM-FRET to investigate the store depletion-induced dynamics of ER-PM junctions in RBL-2H3 mast cells These experiments provided the first direct observation of SOCE-associated remodeling of ER-PM junctions in living rat mast cells and indicate a tight link between junctional Ca2+ handling and architectural dynamics
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