Abstract
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.
Highlights
The endoplasmic reticulum (ER) is the largest membrane-bound organelle in eukaryotic cells
We characterize an understudied yet prominent feature of ER membranes—dynamic, nanoscale-sized holes in ER sheets that we call “nanoholes.” We demonstrate the effect of reticulons, Climp63, and the microtubule cytoskeleton on ER membrane structures that we conclude exist within a continuum between flat sheets and curved tubules
We imaged the periphery of live COS-7 cells expressing the genetically encoded fusion protein Halo-KDEL or SNAP-KDEL (Keppler et al, 2003; Los et al, 2008), which exclusively localizes to the ER lumen and can be labeled with organic dyes compatible with stimulated emission depletion (STED) imaging (Fig. 1 A; Bottanelli et al, 2016)
Summary
The ER is the largest membrane-bound organelle in eukaryotic cells. ER membranes are spread throughout the cytoplasm to perform essential functions in protein and lipid synthesis as well as calcium signaling. Acquiring STED and confocal images of the same region within the cell periphery revealed ER structures that were not detected by conventional microscopy approaches (Fig. 1 B).
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
