Structural Diversity within the Endoplasmic Reticulum-From the Microscale to the Nanoscale

Abstract

The endoplasmic reticulum (ER) is a continuous, highly dynamic membrane compartment that is crucial for numerous basic cellular functions. The ER stretches from the nuclear envelope to the outer periphery of all living eukaryotic cells. This ubiquitous organelle shows remarkable structural complexity, adopting a range of shapes, curvatures, and length scales. Canonically, the ER is thought to be composed of two simple membrane elements: sheets and tubules. However, recent advances in superresolution light microscopy and three-dimensional electron microscopy have revealed an astounding diversity of nanoscale ER structures, greatly expanding our view of ER organization. In this review, we describe these diverse ER structures, focusing on what is known of their regulation and associated functions in mammalian cells.

Figure 1.

Endoplasmic reticulum (ER) distribution at the cellular level. (A) Airyscan micrograph of an ER membrane marker (mEmerald-Sec61β) in an interphase COS-7 cell. Dashed lines delineate phenotypically distinct subregions of the ER network. (B) Radial crop, indicating fluorescence intensity of ER membrane across each subregion. (C) Tiled airyscan micrograph of ER distribution across several neighboring U2-OS cells. (D) 3D volume rendering of ER (Halo-Sec61β) in a COS-7 cell. (E) Airyscan micrograph indicating substantial overlap of ER (Halo-Sec61, orange) with microtubules (EMTB-3 × GFP, blue). (F) Airyscan micrograph of ER (Sec61β) in a central slice through a metaphase HeLa cell. Scale bars, 10 µm (A,B); 20 µm, (C); 5 µm (E,F).

Figure 2.

Superresolution microscopy of endoplasmic reticulum (ER) structure. (A) Widefield image of Halo-Sec61β in the periphery of a COS-7 cell (left). Inset region is shown by both widefield (center) and structured illumination microscopy (right). The enhanced resolution of SIM reveals previously unappreciated structural complexity. (B) Laser scanning confocal image of an ER luminal marker (Halo-KDEL) in a metaphase HeLa cell. Inset region is shown by both confocal and stimulated emission depletion (STED) microscopy. Improved resolution in the STED image reveals previously unresolvable stacked ER sheets. (C) 3D SIM of complex peripheral ER structures pseudo-colored by axial position. (D) Lattice light sheet (LLS) paint microscopy of the lipid-binding dye Bodipy-TR. Inset indicates a complex tubular ER structure. (E) FIB-SEM reconstruction of a 3D-ER matrix (white) with simulated confocal image below (orange). Scale bars, 10 µm, 1 µm, 1 µm (A); 5 µm, 500 nm, 500 nm (B); 10 µm (C); 5 µm, 1 µm (D).

Figure 3.

Nanostructure of the endoplasmic reticulum (ER). (A) Location of ribosomes on planar and tubular ER (planar, green; tubular, blue). (Panel A reproduced from Heinrich et al. 2021 with permission from the authors and Springer Nature 2021.) (B) FIB-SEM reconstruction indicating specialized ER domains, including mitochondria-ER contact sites (red) and ER exit sites (ERES, green). (C–J) 3D EM reconstructions of tube-based (C–F) and sheet based (G–J) ER nanostructures. (Panel H is adapted from Terasaki et al. 2013 with permission from Elsevier 2013.) Structures are not shown to scale.

Figure 4.

Endoplasmic reticulum (ER) tubules. (A) Laser scanning confocal image of peripheral ER tubules (Halo-Sec61β) in a live COS-7 cell. (B) FIB-SEM reconstruction of an isolated ER tubule. (C) Cartoon schematic indicating the variability of ER tubule width across different cell types. (D) Structured illumination micrographs of ER tubules in cells fixed with either 0.25% glutaraldehyde at 37°C (left) or methanol at −20°C. Methanol fixed tubules appear vesiculated. (E) Cartoon of schematized reticulon (blue) and REEP (green) family hairpin proteins inserted into a curved ER membrane. (F) Cartoon representing ER tubule pearling observed in 3D reconstructions from high pressure frozen or high-speed imaged cells. (G) Time-lapse montage of ER tubule extension in a live cell. (H) Cartoon indicating distinct mechanisms of ER tubulation/extension. (I) Grazing incidence-structured illumination microscopy (GI-SIM) of peripheral ER tubules, with kymographs at demarcated positions indicating tubule movement over time. Scale bars, 2 µm, 500 nm (A); 2 µm, 2 µm (D); 2 µm (G); 2.5 µm, 500 nm, time bar, 750 msec (I).

Figure 5.

Tubule-based endoplasmic reticulum (ER) structures. (A) Three-dimensional FIB-SEM reconstruction of a three-way junction from a COS7 cell. (B) Cartoon schematic indicating atlastin-mediated tubule fusion to generate lunapark-stabilized three-way junctions. Atlastin is pseudocolored in pink and purple, lunapark in cyan. (C) Three-dimensional schematic of a three-way junction showing flattening of membrane on top and bottom. (D) Structured illumination micrograph showing peripheral ER network (white) and distribution of three-way junctions (red). (E) ER overlaid with tubule skeleton (white) and three-way junction position over time (green). (Panel E reprinted from Nixon-Abell et al. 2016 with permission from the authors who hold the copyright.) (F) Airyscan image of a 3D matrix (left) with inset (center) paired with equivalently sized FIB-SEM reconstruction of a 3D matrix. (G) SIM image of a 2D matrix (left) with inset (center) and equivalently sized FIB-SEM reconstruction of a 2D matrix. (H) GI-SIM of ER matrices, with kymographs at indicated positions indicating tubule rearrangements within the matrix over time. (Panel H reprinted from Nixon-Abell et al. 2016 with permission from the authors who hold the copyright.) Scale bars, 2.5 µm (D); 1 µm (E); 5 µm, 1 µm (F); 2 µm, 500 nm (G); colored bars are 1 µm. The height of each kymograph is 2.5 sec. (H).

Figure 6.

Sheets. (A–D) Distinct sheet-based structures observable in 3D EM data, including (A) stacked helicoidal sheets, (B) twisted sheets (arrow indicates pitch), (C) cisterna with irregular luminal spacing, and (D) flat sheets with fenestration. (E) XZ FIB-SEM slice of a U2-OS cell with zoomed inset of the basal nuclear envelope. Yellow = nucleus, blue = cytoplasm. (F) XY slices of the nuclear face of the inner membrane (INM) showing chromatin (left), the perinuclear space showing nuclear pores (center), and the cytoplasmic face of the outer nuclear membrane (ONM) showing polyribosomes. Scale bars, 2.5 µm, 500 nm (E); 1 µm, 1 µm, 1 µm (F).