Fig 1.
The Golgi complex of epididymal principal cells.
A-C. Transmission electron microscopy of chemical fixed samples of the initial segment from rat epididymis. A. Imaging of this 80 nm ultrathin section shows that the large GC of this cell type is composed of several stacks (S) connected by heteromorphic structures forming a ribbon. Most of these stacks run parallel to the plasma membrane (PM) while others are transversally arranged. B. Detail of the stacks (S) adjacent to the nucleus (N) in a 50 nm ultrathin section. C. This picture shows the complex nature of elements connecting the stacks (S) formed by heterogeneous membranes representing tubular networks and/or fenestrated cisternae (arrows). D. Immunolocation of GM130. This matrix protein is restricted to cis-Golgi elements. E. Immunolocation of Rab6. This small GTP is mostly located in tubule-vesicular elements at the trans-Golgi side. Scale bars: 200 nm.
Fig 2.
3D overview of the Golgi ribbon.
Sequential tomogram slices of a dual-axis tomogram showing an overview of the Golgi area. In Fig A, the GC displays an S-like shape apparently formed by several independent stacks (S), although the analysis of sequential tomogram slices show they were all connected (B-F). The non-compact regions (delimited by parentheses) contain clusters of vesicles (stars) and microtubules (arrows). The inset in Fig F shows a COP-coated bud (arrowhead) close to a clathrin-coated vesicle bearing the characteristic spikes. S, stack; m, mitochondria; asterisks, pores. Volume of the calculated tomogram (x,y,z): 4095 x 4095 x 360 pixels, pixel size 0.59 nm. See also S1 Movie for this tomographic reconstruction.
Fig 3.
Models in 3D of the GC of principal cells.
3D models generated from dual-axis (A) or single-axis (B,C) tomographic data of the Golgi region. A,B Image of the complete model including CGN (light purple), TGN (salmon), cis- and trans-ER (blue), microtubules (fuchsia), small vesicles (green), clathrin-coated vesicles (dark green) and the Golgi stack, whose separated cisternae are in different colors. C1 (light pink), C2 (orange), C3 (dark red), C4 (yellow), C5 (pink), C6 (light orange), C7 (red) C8 (light yellow). C. In this model the same colors were used except for cisternae that are displayed in yellow. Volumes of the calculated tomograms (x,y,z): A: 4095 x 4095 x 340 pixels, B: 4095 x 4095 x 336 pixels; C: 4095 x 4095 x 385 pixels; pixel size: 0.59 nm. See also S2, S3 and S4 Movies.
Fig 4.
A. 3D model generated from Fig 3A by removing all features except the cisternae. B. Modeled cisternae are display from cis (C1) to trans side (C7). Arrowheads point to tubular structures at the non-compact region of the ribbon.
Fig 5.
A,B. Tomographic slices of a portion of the Golgi ribbon from chemically fixed (A) and cryofixed (HPF-FS) (B) samples showing branches (arrows). C. Slice of an electron tomographic reconstruction of the stack that shows an Y-shape cisterna (arrow). D. Three-dimensional model of the Golgi region shown in C (the complete model is shown in Fig 3C). Apart from the Y-shape cisterna (arrow) there is a connection between two adjacent cisternae (arrowhead). E. Slice of a reconstruction of a stack made up of dilated cisternae, where a cisterna projects a branch passing through neighbouring cisternae (arrow). F. 3D model of the tomogram reconstruction shown in E (the complete model is shown in Fig 3B). Volumes of the calculated tomograms (x,y,z): A: 4095 x 4095 x 186 pixels, B: 4095 x 4095 x 400 pixels, pixel size 0.59 nm.
Fig 6.
Pocket-like cisterna invaginations.
A-C. Three sequential slices through the tomogram showing that Golgi membranes are folded forming pocket-like invaginations. The complete 3D model for this GC is shown in Fig 3A. D. Detail corresponding to the box in Fig A. E. The 3D model in E shows a left side view of the pocket hole shown in D. F. Detail of the box in Fig C. G. The 3D model shows a right side view of the pocket hole shown in F. H. An x/y-plane of the 3D model of the reconstruction showing the inner holes of the pockets (yellow and orange) and the hole in the middle cisterna (pink) are connected forming a corridor (asterisk). The model has the same orientation as the tomographic slices shown in A-C. I. Tomographic slice showing a pocket. J. 3D model of the reconstruction shown in I. The inset shows an ER tubule (blue) crossing the trans cisternae and approaching to the pocket.
Fig 7.
Association endoplasmic reticulum-Golgi complex.
A-C. Five consecutive tomogram slices (A) and the 3D model corresponding to this tomogram (B, C). ER membranes run parallel to the cis and trans cisternae. Flattened trans-ER lies closely over the trans face of the two trans-most cisternae (arrow). Tubules emerge from the ER inwards, through the trans cisternae (large and small arrowhead). An ER cisterna crosses regions that contain numerous vesicles and tubules and also microtubules (asterisks). D. 3D model illustrating an ER tubule (asterisk) crossing a gap (non-compact region) between two stacks at the cis side. E, F. Ultrathin cryosections immunolabelled for the ER marker PDI. ER elements surround the stacks (S) at both cis and trans sides but they cross the ribbon through the non-compact regions (arrow). Scale bars: 200 nm.
Fig 8.
A, B. TEM of chemical fixed principal cells from the epididymis. Micrographs of an ultrathin section show microtubules crossing the Golgi ribbon. C, D. 3D models simplified from Fig 3A and 3B, showing the relationship between microtubules and ER (C) and Golgi cisternae (D). E-G. Immunolocation of tubulin in ultrathin cryosections. The Golgi area of this cell type shows a high immunoreactivity for tubulin. Arrowheads in Fig E point to a putative microtubule that runs very close to the edge of stack. Tubulin immunoreactivity observed between tubule-vesicular elements may represent cross-sectioned microtubules or soluble tubulin. The inset in Fig E shows a pair of centrioles in the Golgi region of epididymal cells. Immunoreactive microtubules cross the non-compact regions of the ribbon (F) but are also directly associated with cisternae (G). S = stack. Scale bars: 200 nm.
Fig 9.
Tubular transport intermediates.
A. Conventional electron microscopy micrograph showing a tubule (arrow) emerging from the lateral rims of a cisterna. B. Face view of a cisterna showing pores and one tubule (arrow) extending outwards, roughly parallel to the plane of the cisterna. C 3D-model (obtained from Fig 3C) showing a tubule (arrow) that extends from the edge of the stack into a region surrounded by ER and vesicles. D. Sequential z axis slices illustrating a heterotypic connection (arrow) between two cisternae of the same stack. Volume of the calculated tomogram (x,y,z): 4095 x 4095 x 262 pixels, pixel size 0.59 nm. Scale bars: 200 nm.