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Nanoscale architecture of endoplasmic reticulum export sites and of Golgi membranes as determined by electron tomography - PubMed

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Nanoscale architecture of endoplasmic reticulum export sites and of Golgi membranes as determined by electron tomography

L Andrew Staehelin et al. Plant Physiol. 2008 Aug.

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Figures

Figure 1.
Figure 1.

Comparison of the spatial resolution of a confocal and an EM image of plant Golgi stacks (A and B). A, Tobacco BY-2 cell expressing a α-1,2 mannosidase I-GFP fusion protein that localizes to Golgi stacks. In this confocal micrograph, the individual Golgi stacks are seen as approximately 1 μm in diameter fluorescent spots. B, Thin section electron micrograph of a Golgi stack in a high-pressure-frozen and freeze-substituted BY-2 cell. Note the differences in architecture and staining of the cis-, medial-, and trans-Golgi cisternae. Ribosomes (approximately 15-nm black dots) and an ER cisterna adjacent to the Golgi stack are also seen (courtesy of A. Nebenführ). C and D, Enhancement of electron microscopy by tomography techniques. Two-nanometer-thick electron tomographic slice image (C) and a 3D reconstruction (D) of an Arabidopsis Golgi stack are shown. The 3D model is generated from tracings of 363 slice images, including the image in C.

Figure 2.
Figure 2.

Thin section electron micrographs of ER export sites with budding COPII vesicle profiles (arrowheads) in a tobacco BY-2 cell (A; from Ritzenthaler et al., 2002) and in an Arabidopsis root columella cell (B).

Figure 3.
Figure 3.

3D tomographic model images of ER export sites in a root meristem cell (A) and in a root columella cell (B). A, Four budding COPII vesicles (arrowheads) are located adjacent to the cis-side of a Golgi stack (stippled structure). Note that these COPII buds are forming on three different tubular ER domains and face in different directions. B, Face-on view of an ER export site associated with the cortical ER of a columella cell in which the cisternae are more sheet-like. Here too the COPII buds (arrowheads) are seen budding from different ER domains in close proximity to the cis-most cisterna (C1, stippled structure) of a docked Golgi stack.

Figure 4.
Figure 4.

Transfer of COPII vesicles and their scaffolds to the cis-Golgi matrix. A and B, Tomographic slice image (A) and a corresponding model (B) of a COPII vesicle budding from an ER membrane. The COPII bud is surrounded by an approximately 40-nm-wide, ribosome-excluding scaffold. C and D, Tomographic slice image (C) and a corresponding model (D) of a COPII bud (arrowhead), which is still attached to the ER but whose scaffold has become connected to the cis-side of the Golgi matrix. E and F, Tomographic slice image (E) and a corresponding model (F) of a released COPII vesicle (arrow) that is still close to the ER membrane. The COPII scaffold is linked to the Golgi matrix. G and H, Tomographic slice image (G) and a corresponding model (H) illustrating a COPII vesicle (arrow) that has been transferred to the cis-face of the Golgi stack together with its COPII scaffold. The distal side of the COPII vesicle is completely surrounded by the Golgi matrix. Scale bars = 300 nm.

Figure 5.
Figure 5.

Electron tomographic model of a Golgi stack and its encompassing, ribosome-excluding scaffold (Golgi matrix). A TGN cisterna is also contained within the trans-side extension of the Golgi matrix.

Figure 6.
Figure 6.

Dock, pluck, and go model of ER-to-Golgi vesicle trafficking. A, Go-phase: Golgi stacks travel along actin filaments propelled by myosin motors. B, Dock- and Pluck-phase: The COPII scaffold of a budding vesicle attaches to the cis-side of the Golgi matrix and pulls the passing Golgi off the actin track. Once the COPII scaffold binds to the Golgi matrix, the wiggling motion of the Golgi stack facilitates release of the COPII vesicles by plucking. After COPII vesicle harvesting, the Golgi is free to resume its movement along the actin track.

Figure 7.
Figure 7.

Face-on views of three Golgi stack models in which the cis-most (C1) cisternae are colored orange (arrows) and the underlying C2 cis-cisternae are colored green. The cis-most cisternae vary in size from a small blob (A), to two intermediate-sized, branched tubules (B), and to a slightly larger disc with tubular extensions around its margins (C). These highly variable shapes are consistent with the de novo assembly of cis-Golgi cisternae from COPII vesicle (yellow spheres) as postulated by the cisternal progression model of Golgi trafficking.

Figure 8.
Figure 8.

Immunoelectron tomography localization of native α-1,2 mannosidase I (ManI) in an Arabidopsis meristem Golgi stack. A to C, Electron micrographs of three serial thin sections labeled with anti-ManI 15-nm immunogold particles. D, 3D tomographic model of the Golgi stack and immunogold particles seen in A to C. Note that nearly all of the immunogold particles are associated with the C3 and C4 medial-cisternae (arrowheads). The Golgi cisternae models were rendered semitransparent to make all of the gold particles visible. Scale bars = 100 nm.

Figure 9.
Figure 9.

COPII-, COPIa-, and COPIb-type vesicles of S. dubia. A to C, A gallery of tomographic slice images showing COPII-type vesicles. D to F, A gallery of tomographic slice images showing COPIa-type vesicles. G to I, A gallery of tomographic slice images depicting COPIb-type vesicles. In C, F, and I, models of COPII, COPIa, and COPIb vesicles are overlaid on tomographic slices, respectively. J, 3D model of Golgi associated vesicles superimposed on a single slice of an S. dubia Golgi stack illustrating the nonoverlapping distribution of COPIa-type (light green spheres) and COPIb-type vesicles (light purple spheres). COPII-type vesicles (gold spheres) colocalize with COPIa-type vesicles, though. Light blue and pink spheres in the trans-side of the Golgi represent secretory and clathrin-coated vesicles (Donohoe et al., 2007). Scale bar = 100 nm.

Figure 10.
Figure 10.

Schematic diagram illustrating the sites of origin and the trafficking routes of five types of ER/Golgi/TGN-associated vesicles. COPIa-type vesicles bud from assembling cis-cisternae and recycle molecules back to the ER, whereas the COPIb-type vesicles are produced by medial, trans, and early TGN cisternae and recycle molecules between these cisternae.

Figure 11.
Figure 11.

3D tomographic model of a dividing Golgi stack docked to an ER export site in an Arabidopsis root meristem cell. A, The two sets of cis- and medial-cisternae are held together by a larger trans-Golgi cisterna (pink). The Golgi stack is connected to budding COPII vesicles via interactions between the COPII scaffolds and the cis-Golgi matrix (arrowheads). B, Face-on view of the dividing Golgi stack shown in A. The C1 (orange) and C2 (green) cisternae of the two cis-side stacks (C1, C2, C1', and C2' marked with arrows) display varying shapes resembling the assembling cis-most cisternae in Figure 7. C, On the trans-side, three TGN cisternae (TGN1–TGN3) at different stages of maturation are seen.

Figure 12.
Figure 12.

3D models (face-on views) of a trans-most Golgi, an early TGN, and a late TGN cisterna of meristem cell Golgi stacks illustrating the TGN maturation process. CCV, Clathrin-coated vesicle; SV, secretory vesicle.

Figure 13.
Figure 13.

A, Tomographic slice image of an Arabidopsis meristem cell with two Golgi stacks (Golgi 1, Golgi 2). Golgi stack 1 (B and C) is associated with a late TGN cisterna that is budding mostly secretory vesicles (SV). In contrast, the late TGN cisterna associated with Golgi 2 (D and E) is producing mostly clathrin-coated buds (CCV). This latter type of TGN is also known as partially coated reticulum (Pesacreta and Lucas, 1985). Scale bar in A = 250 nm, scale bars in B to E = 200 nm.

Figure 14.
Figure 14.

3D model images of a Golgi stack and its TGN cisternae. A, The late TGN cisterna has fragmented into a cluster of secretory vesicles (SV) that surround small, residual membrane fragments. CCV, Clathrin-coated vesicle. B, Face-on view of the late TGN model shown in A in which the secretory and clathrin-coated vesicles have been omitted to illustrate the morphologies of the residual fragments of the cisternal membrane (arrowheads).

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