Dynamic nanoscale morphology of the ER surveyed by STED microscopy

• Published in: The Journal of cell biology Vol. 218; no. 1; pp. 83 - 96
• Main Authors: Schroeder, Lena K., Barentine, Andrew E.S., Merta, Holly, Schweighofer, Sarah, Zhang, Yongdeng, Baddeley, David, Bewersdorf, Joerg, Bahmanyar, Shirin
• Format: Journal Article
• Language: English
• Published: United States Rockefeller University Press 07.01.2019
• Subjects: Animals; Cells (biology); Cellular biology; Chlorocebus aethiops; COS Cells; Cytoskeleton; Cytoskeleton - drug effects; Cytoskeleton - metabolism; Cytoskeleton - ultrastructure; Depletion; Emission analysis; Endoplasmic reticulum; Endoplasmic Reticulum - drug effects; Endoplasmic Reticulum - metabolism; Endoplasmic Reticulum - ultrastructure; Gene Expression Regulation; Intracellular Membranes - drug effects; Intracellular Membranes - metabolism; Intracellular Membranes - ultrastructure; Light microscopy; Membrane structure; Membrane structures; Microscopy; Microscopy - methods; Microtubules - drug effects; Microtubules - metabolism; Microtubules - ultrastructure; Molecular Imaging - methods; Morphology; Nocodazole - pharmacology; Nogo Proteins - genetics; Nogo Proteins - metabolism; Nuclear Pore Complex Proteins - genetics; Nuclear Pore Complex Proteins - metabolism; Quantitative analysis; Receptors, Cell Surface - genetics; Receptors, Cell Surface - metabolism; Sheets; Stimulated emission; Time-Lapse Imaging - statistics & numerical data; Tubules; Tubulin Modulators - pharmacology
• ISSN: 0021-9525; 1540-8140; 1540-8140
• DOI: 10.1083/jcb.201809107

The endoplasmic reticulum (ER) is composed of interconnected membrane sheets and tubules. Superresolution microscopy recently revealed densely packed, rapidly moving ER tubules mistaken for sheets by conventional light microscopy, highlighting the importance of revisiting classical views of ER structure with high spatiotemporal resolution in living cells. In this study, we use live-cell stimulated emission depletion (STED) microscopy to survey the architecture of the ER at 50-nm resolution. We determine the nanoscale dimensions of ER tubules and sheets for the first time in living cells. We demonstrate that ER sheets contain highly dynamic, subdiffraction-sized holes, which we call nanoholes, that coexist with uniform sheet regions. Reticulon family members localize to curved edges of holes within sheets and are required for their formation. The luminal tether Climp63 and microtubule cytoskeleton modulate their nanoscale dynamics and organization. Thus, by providing the first quantitative analysis of ER membrane structure and dynamics at the nanoscale, our work reveals that the ER in living cells is not limited to uniform sheets and tubules; instead, we suggest the ER contains a continuum of membrane structures that includes dynamic nanoholes in sheets as well as clustered tubules.