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doi: 10.1242/10.1242/jcs.00115

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Ultrastructural characterization of endoplasmic reticulum — Golgi transport containers (EGTC)

¹ Membrane Biology Laboratory, Institute of Molecular and Cell Biology, 30 Medical Drive, Singapore 117609, Republic of Singapore
² NCA Laboratory, Institute of Molecular and Cell Biology, 30 Medical Drive, Singapore 117609, Republic of Singapore

View larger version (98K): [in a new window]   Fig. 1. Concentration of VSVG in large, pleiomorphic structures upon release from the ER. Transmission electron micrographs of VSV-infected NRK cells subjected to the rapid ethane-freezing procedure 10 minutes after release from 40°C. (A) Low magnification micrograph (top panel) illustrating the presence of large, pleiomorphic structures (*) with extensive bud profiles enriched with VSVG immunogold labeling (9 nm gold particles). Bar, 0.5 µm. The boxed region is magnified (bottom panel) to illustrate a single structure and its various features, including bud-profiles (BP) and the matrix (M). (B,C) These structures (*) are segregated from COPII-labeled (Sec13 antibody, 9 nm gold particles) ER exit sites (Hong, 1998) (arrowheads). Bar, 0.2 µm. (D) These structures (*) are devoid of markers of the Golgi stack (G), such as GS28 (Subramaniam et al., 1996), 9 nm gold particles (arrowheads). (E) Some of these structures (*) can be found in the vicinity of the Golgi stack seemingly in the process of aligning and tethering with the Golgi stack for presumably a fusion event (G). Bar, 0.2 µm. (F) The structure (*) shown here is in the process of fusing with a Golgi stack (G) and appears to be emptying its VSVG (9 nm gold particles) content into it. Bar, 0.2 µm.

View larger version (78K): [in a new window]   Fig. 2. The ER-Golgi transport container (EGTC) is an isolated, membrane-bound entity. (A) Transmission electron micrographs of six serial 70 nm sections of VSV-infected NRK cells subjected to the rapid ethane-freezing procedure 10 minutes after release from 40°C. An EGTC is shown with the surrounding membranes in view, demonstrating the lack of membranous connection between the EGTC and the surrounding membranes. Bar, 0.1 µm. (B) Three-dimensional reconstruction of the serial sections in (A) using a contour alignment reconstruction (CAR) program (Saetzler et al., 2002) viewed from two different angles. The spheres are projections of the vesicular profiles at the edges of the EGTC. Scale: 1 mm represents 10 nm.

View larger version (80K): [in a new window]   Fig. 3. The EGTC is enriched in the markers expected for an ER-Golgi transport intermediate. Transmission electron micrographs of VSV-infected NRK cells subjected to the rapid ethane freezing procedure 10 minutes after release from 40°C. (A) Double immunogold labeling of VSVG (9 nm gold particles, small arrowheads) and COPI (ß-COP antibody, 14 nm gold particles, large arrowheads). Although found within the same structures (*), VSVG labeling is more concentrated at the central matrix whereas ß-COP labeling (14 nm gold particles, arrowheads) is mostly associated with the bud profiles at the periphery. This is also illustrated in the single ß-COP labeling (10 nm gold particles, arrowheads) in B. This is also the case for p58 (p58 antibody, 10 nm gold particles, arrowheads) labeling as shown in C. Bars, 0.2 µm. (D) Double immunogold labeling for ß-COP (14 nm gold particles, large arrowheads) and the tether protein p115 (p115 antibody, 9 nm gold particles, small arrowheads), demonstrating that p115 is also found at a significant level in the EGTC. (E) Immunogold labeling for the soluble cargo albumin (albumin antibody, 10 nm gold particles) in HepG2 cells. Bars, 0.2 µm.

View larger version (124K): [in a new window]   Fig. 4. VSVG is first found in COPII-positive vesicle chains/clusters upon release from the ER at 15°C. Transmission electron micrographs of three 70 nm serial sections (A, B and C) of VSV-infected NRK cells subjected to the rapid ethane freezing procedure for 3 hours upon transfer from 40°C to 15°C. The sections were double-labeled for VSVG (9 nm gold particles, small arrowheads) and Sec13 (14 nm gold particles, large arrowheads). Bar, 0.1 µm.

View larger version (198K): [in a new window]   Fig. 5. EGTCs have distinct morphological profiles that may correspond to different chronological stages of its biogenesis. Transmission electron micrographs of uninfected NRK cells subjected to the rapid ethane freezing procedure followed by osmium tetroxide treatment to enhance the morphology of membrane structures. Two morphologically distinct EGTCs are on view here. The upper left structure (I) appears to have extensive electron-dense membrane structures within it (white arrowheads) that are not found in the lower right structure. These membrane structures may reflect COPII coats that are frequently seen to line budding profiles and vesicles (black arrowheads) associated with the ERES. Bar, 0.2 µm.

View larger version (149K): [in a new window]   Fig. 6. EGTCs may be derived from COPII vesicle cluster/chains that coalesce by homotypic fusion processes. Transmission electron micrographs of uninfected NRK cells subjected to the rapid ethane-freezing procedure followed by osmium enhancement as in Fig. 5. (A) Electron-dense material, probably corresponding to COPII, can be found on vesicles and bud profiles associated with the ER transitional elements (arrowheads). (B,C) Vesicles with this electron-dense coat are frequently found in chains or clusters (arrowheads), and these chains and clusters may well be fusing with a nearby, existing EGTC (*).

View larger version (37K): [in a new window]   Fig. 7. (A) Schematic model of ER-Golgi transport mediated by EGTC. COPII vesicles that bud from the ERES quickly coalesce to form clusters. These could either fuse with an existing EGTC or aggregate with each other to form a nascent EGTC (I). This process is apparently inhibited or slowed at 15°C. Within the EGTC, anterograde cargo is segregated from retrograde cargo via the action of COPI, and the EGTC `matures' by COPI-mediated retrograde transport of its components back to the ER (II). `Mature' EGTCs travel to the perinuclear region via microtubule tracks where it eventually fuses with the Golgi (III).