Dynamics of transitional endoplasmic reticulum sites in vertebrate cells

Abstract

A typical vertebrate cell contains several hundred sites of transitional ER (tER). Presumably, tER sites generate elements of the ER-Golgi intermediate compartment (ERGIC), and ERGIC elements then generate Golgi cisternae. Therefore, characterizing the mechanisms that influence tER distribution may shed light on the dynamic behavior of the Golgi. We explored the properties of tER sites using Sec13 as a marker protein. Fluorescence microscopy confirmed that tER sites are long-lived ER subdomains. tER sites proliferate during interphase but lose Sec13 during mitosis. Unlike ERGIC elements, tER sites move very little. Nevertheless, when microtubules are depolymerized with nocodazole, tER sites redistribute rapidly to form clusters next to Golgi structures. Hence, tER sites have the unusual property of being immobile, yet dynamic. These findings can be explained by a model in which new tER sites are created by retrograde membrane traffic from the Golgi. We propose that the tER-Golgi system is organized by mutual feedback between these two compartments.

Figure 1

Sec13 marks discrete ER subdomains that are distinct from the E1 compartment. (A) HeLa cells were fixed and stained with anti-Sec13 polyclonal antibody followed by Rhodamine Red-X-conjugated anti-rabbit antibody. The image shows a representative cell, which contains several hundred tER sites. (B) HeLa cells transiently expressing myc-tagged Myt1 were examined by double-label immunofluorescence. Sec13 (red) was visualized as in (A), and Myt1-containing ER tubules (green) were visualized using anti-myc monoclonal antibody followed by Cy2-conjugated anti-mouse antibody. This merged image shows a portion of the ER network from the periphery of a cell. All of the tER sites are associated with ER tubules. (C) HeLa-E1 cells, which produce the Rubella virus E1 fusion protein, were examined by double-label immunofluorescence. Sec13 (red) was visualized as in (A), and the E1 fusion protein (green) was visualized using the P5D4 monoclonal antibody followed by Cy2-conjugated anti-mouse antibody. Two cells are present in this merged image, which shows that Sec13 is largely excluded from regions that contain the E1 fusion protein. Scale bars represent 10 μm.

Figure 2

The tER and the ERGIC can be resolved in cells grown at 37°C, but are indistinguishable in cells incubated at 15°C. tER sites (red) were visualized by staining HeLa cells for Sec13 as in Figure 1A. ERGIC elements (green) were visualized in the same cells using anti-ERGIC-53 monoclonal antibody followed by Cy2-conjugated anti-mouse antibody. (A) Cells growing normally at 37°C were fixed and processed for immunofluorescence. The dominant ERGIC-53 signal derives from ER-localized molecules, but as shown in the inset, punctate ERGIC elements can be seen next to some of the tER sites. (B) Cells were incubated at 15°C for 3 h before fixation. As shown in the merged image and the inset, the Sec13 and ERGIC-53 staining patterns are almost identical. (C) Cells were incubated on ice for 15 min, then quickly warmed to 37°C in the presence of 5 μg/ml nocodazole for an additional 2 h. As shown in the merged image, tER sites and ERGIC elements are closely associated, but distinct. The inset contains examples of tER and ERGIC structures that are optically well resolved. Scale bars represent 10 μm.

Figure 3

tER sites primarily exhibit undirected short-range movements. Experiments were performed with CHO-S13G cells, which produce Sec13-GFP. (A) Immunofluorescence of a fixed CHO-S13G cell stained with anti-GFP monoclonal antibody followed by Cy2-conjugated anti-mouse antibody. (B) A living cell of clone CHO-S13G was examined by video confocal microscopy at 37°C. Images were captured every 3.3 s for 15 min and then assembled into a movie. Some photobleaching occurred as a result of the imaging. The panels display a region in the cell periphery; for the figure, image frames were selected at ∼1-min intervals from the first 9 min of the video. Eight tER sites are visible. For the most part, these tER sites exhibited only slow movements. An exception is the uppermost tER site (arrowhead), which underwent a single rapid movement of ∼1.2 μm ∼30 s after the beginning of the video. Scale bars represent 10 μm (A) or 2 μm (B).

Figure 4

tER sites become clustered near Golgi structures after the addition of nocodazole. (A) A control plate of NRK cells was left at 37°C in the absence of nocodazole. (B and C) NRK cells were incubated on ice for 15 min. Nocodazole was then added to 5 μg/ml, and the cells were quickly warmed to 37°C and incubated for an additional 10 min (B) or 2 h (C). tER sites were visualized by staining for Sec13 as in Figure 1A. The Golgi was visualized using anti-giantin monoclonal antibody followed by Cy2-conjugated anti-mouse antibody. As shown in the merged images, tER sites become clustered near Golgi structures within 10 min after nocodazole addition, and this association persists after 2 h of nocodazole treatment. Scale bars represent 10 μm.

Figure 5

tER clusters form even when Golgi structures reemerge from the ER. NRK cells were incubated with 5 μg/ml BFA for 1 h; this treatment caused the complete redistribution of giantin into the ER. Nocodazole was then added to 5 μg/ml, and after 30 min the BFA was washed out in the continued presence of nocodazole. Sec13 and giantin were visualized as in Figure 4. (A) Fifteen minutes after BFA removal, small giantin-containing structures are associated with nearly all of the tER sites. (B) Two hours after BFA removal, large giantin-containing structures are associated with clustered tER sites, but many of the individual tER sites lack adjacent giantin staining. Scale bars represent 10 μm. (C) Quantitation of the localization data, as described in Materials and Methods. The graph shows the percentage of the Sec13 staining that is within 0.3 μm of a giantin-containing structure at various times after BFA washout in the presence of nocodazole. The bar on the right gives the corresponding value for NRK cells that were treated with nocodazole alone for 2 h. SDs are indicated.

Figure 6

tER sites proliferate during interphase. As described in Materials and Methods, fixed and stained cells from an unsynchronized NRK culture were identified as being in G1, G2, early prophase, or cytokinesis. After a cell was assigned to a stage of the cell cycle, its tER sites were counted. Bars indicate the average number of tER sites per cell. SDs are indicated.

Figure 7

The tER and the Golgi undergo parallel transformations during mitosis. An unsynchronized population of CHO cells was fixed and stained for Sec13 and giantin as in Figure 4. In addition, chromosomes were stained with Hoechst dye to determine the stage of the cell cycle. (A–C) Representative cells from late prophase (A), telophase (B), and cytokinesis (C). Sec13 staining of tER sites is greatly reduced in the late prophase and telophase cells but is strong in the cell undergoing cytokinesis. Giantin staining reveals that multiple large Golgi fragments are present in the late prophase cell and in the cell undergoing cytokinesis, but only a few optically resolvable structures are present in the telophase cell. (D) Quantitation of the mitosis data, as described in Materials and Methods. The total cellular intensity of the Sec13 or giantin signal from optically resolvable structures is plotted, with the interphase values being defined as 100%. SDs are indicated. The large deviations reflect a substantial cell-to-cell variability in the tER and Golgi patterns at each stage of the cell cycle.

Figure 8

Model of a proposed mutual feedback relationship between the tER and the Golgi. (Left) In normal cells, tER sites (red) and Golgi structures (green) exchange material by microtubule-directed transport (bidirectional arrows). ERGIC elements arise at tER sites and move inward to the juxtanuclear Golgi, while retrograde traffic from the Golgi moves outward toward the cell periphery. Retrograde transport events terminate either at tER sites (as drawn) or at random locations on the ER. This recycling of proteins to the ER causes tER sites to proliferate. Because most of the retrograde transport events terminate near their point of origin, ER membranes in the Golgi region contain a high concentration of recycling proteins and a correspondingly high density of tER sites. (Right) When microtubules are disrupted with nocodazole, each Golgi structure receives input only from adjacent tER sites, and the number of these sites determines the size and stability of the Golgi structure. Conversely, membrane recycling from a given Golgi structure induces a localized proliferation of the tER. This positive feedback loop generates intermediate-sized Golgi structures that are next to tER clusters.