PMC

3D model of the ER, showing a tubular network in a metaphase CHO-K1 cell prepared using HPF/FS. The ER model was derived from five 250-nm sections. Tubules are clearly the predominant morphology, and the nominal persisting sheets (*) are very small. Bars are 0.5 μm in the tomogram and 2 μm in the inset, which shows an overall view of the cell.

Relative distribution of thin-section ER profiles by length category demonstrates that mitotic Huh-7 and NRK-52E cells have more short profiles and fewer long profiles than their respective interphase cells. Similar, albeit smaller, differences are observed between Huh-7 and NRK-52E interphase cells. Error bars indicate ± SEM. Numbers (n) in the image refer to the number of cells analyzed.

The reorganization of ER into concentric layers seems to be more predominant toward the end of metaphase. Huh-7 cells were cotransfected with pHsp47-GFP and pH2B-monomeric red fluorescent protein and imaged by confocal microscopy at 10-min intervals starting from early metaphase (time 00:00) and progressing through telophase. A deconvoluted optical section of consecutive time points demonstrates the progressive reorganization of ER from the reticular appearance at early metaphase (arrowheads) toward concentric layers at the end of metaphase (00:20, arrows). Bar, 10 μm.

Modeling of ER and ribosomes from tomograms. Samples of Huh-7 (A, C, D) and NRK-52E (B, E, F) cells were prepared by HPF/FS, and individual sheets with ribosomes were modeled from the resulting tomograms. An example of a fenestrated sheet of the Huh-7 cells and a more intact sheet in NRK-52E cells are marked with a right bracket (}) in the tomogram slice. The corresponding models show that highly curved membranes (sheet edges in D and E, fenestrations in C, and tubules in E) tend to have fewer ribosomes. Bars, 0.5 μm (A, B).

Model of mitotic sheet-to-tubule transformation. The transformation of rough ER starts from intact or fenestrated sheets and proceeds toward a more fenestrated state that can eventually produce structures resembling tubular networks. The extent of the shape change correlates with the density of membrane-bound ribosomes. The starting and ending points vary among cell types, but the direction of transformation remains the same in all cultured mammalian cells analyzed here. The arrow with the color gradient symbolizes the progression from interphase to metaphase. ER structures on the color scale and cell types are marked with dark colors in interphase and light colors during mitosis.

NRK-52E cells show fenestrated ER sheets and tubules during mitosis. The flat shape of a metaphase NRK-52E cell helps to expose many ER sheets in a confocal optical section (arrows in A). (B) A thin-section image of cytochemically stained ER in a metaphase NRK-52E cell shows longitudinal profiles of sheets with perforations (arrows) and long lines of transverse sheet profiles with multiple gaps (arrowheads) parallel to the PM. Cells transiently expressed Hsp47-GFP in A and ssHRP-KDEL in B as ER markers. (C) Two successive 250-nm sections of a wt metaphase NRK-52E cell prepared using HPF/FS were subjected to ET (also see Supplemental Video S7). Models of two neighboring ER structures (marked in the tomographic slice as C1 and C2) are composed of extensively fenestrated sheets, and tubules and are shown separately to allow easier visualization. Bars, 5 μm (A and insets of B and C) and 0.5 μm (the rest).

Correlative-light EM of a metaphase Huh-7 cell. A Huh-7 cell transiently coexpressing ssGFP-KDEL and ssHRP-KDEL was cultured on a glass-bottom dish (A, phase contrast image) and imaged by confocal microscopy (C, D) before chemical fixation and cytochemical staining for EM (E, F). (C) An optical section from the adhering side of the cell (see illustration in B) shows sheet profiles and some ER network. Highly fenestrated ER sheet profiles (arrows) are found in a thin-section EM image (E) from the same height (derived from the boxed region in inset). (D) An optical section from the middle of the cell shows multiple layers of long ER profiles coaligned with the PM. (F) The corresponding thin-section image (from the boxed region in inset) shows similar but gapped ER profiles (arrowheads). Thick sections for ET were cut in between the thin sections (E, F), and the tomogram is provided in Supplemental Video S4. Bars, 5 μm (A, C, D, and insets of E and F), 0.5 μm (E), and 2 μm (F).

Interphase ER in Huh-7 and NRK-52E cells consists mainly of sheets; however, sheet profiles in thin sections of interphase Huh-7 cells are segmented and fenestrated as compared with NRK-52E. Huh-7 and NRK-52E cells expressing Hsp47-GFP were subjected to wide-field microscopy. Huh-7 cells have abundant peripheral sheets (arrows), as shown in A. NRK-52E cells (B) also have many sheets (arrows) and small areas of tubular networks (arrowheads) in the cell periphery. In EM micrographs, transverse sections of sheets in wt (C) and cytochemically stained (E) Huh-7 cells show multiple gaps (arrowheads), and many fenestrations are exposed in longitudinal sections of sheets (arrows). In contrast, transverse sections reveal long sheet profiles (open arrowheads) in NRK-52E cells, and longitudinal sections (open arrows) show sheets that are often completely intact in wt (D) and cytochemically stained (F) cells. ET analysis of the ER in Huh-7 and NRK-52E interphase cells clearly show the fenestrated nature of the ER sheets in Huh-7 cells (G, arrowheads) as compared with the intact sheets in NRK-52E cells (H, open arrowheads). The samples for ET were prepared by HPF/FS. The modeled volumes are derived from two 250-nm-thick sections. NE, nuclear envelope. Bars, 5 μm (A, B), and 1 μm (C–H).

Model of a metaphase Huh-7 cell imaged with the SBF-SEM technique. A whole Huh-7 cell transiently expressing ssHRP-KDEL was chemically fixed, cytochemically stained, and imaged with SBF-SEM (Supplemental Video S6A), providing a voxel size of 27 × 27 × 50 nm, and the ER was modeled. (A) An image of one block-face overlaid with the modeled ER. For a clear view, a small part of the cell and ER model (A–D, Supplemental Video S6B) is shown starting from the metaphase plate of the chromosomes (A, B), including the spindle region (A, D) and the cortical parts (B, C). Cutting surface is marked with red color. Organization of the ER in cortical layers is visible in the block-face image (A) and views of the 3D model (C, D). The cortical layers are composed of fenestrated sheets and short tubules (C; tubules are marked with arrows). Long tubules originating from the cortical ER extend toward the middle of the cell (D, E). A view covering the complete middle part of the cell shows that this tubular part is quite extensive (E). Bars, 5 μm (A, B, and inset of E) and 1 μm (C–E).