Golgi structure in three dimensions: functional insights from the normal rat kidney cell

Abstract

Three-dimensional reconstructions of portions of the Golgi complex from cryofixed, freeze-substituted normal rat kidney cells have been made by dual-axis, high-voltage EM tomography at approximately 7-nm resolution. The reconstruction shown here ( approximately 1 x 1 x 4 microm3) contains two stacks of seven cisternae separated by a noncompact region across which bridges connect some cisternae at equivalent levels, but none at nonequivalent levels. The rest of the noncompact region is filled with both vesicles and polymorphic membranous elements. All cisternae are fenestrated and display coated buds. They all have about the same surface area, but they differ in volume by as much as 50%. The trans-most cisterna produces exclusively clathrin-coated buds, whereas the others display only nonclathrin coated buds. This finding challenges traditional views of where sorting occurs within the Golgi complex. Tubules with budding profiles extend from the margins of both cis and trans cisternae. They pass beyond neighboring cisternae, suggesting that these tubules contribute to traffic to and/or from the Golgi. Vesicle-filled "wells" open to both the cis and lateral sides of the stacks. The stacks of cisternae are positioned between two types of ER, cis and trans. The cis ER lies adjacent to the ER-Golgi intermediate compartment, which consists of discrete polymorphic membranous elements layered in front of the cis-most Golgi cisterna. The extensive trans ER forms close contacts with the two trans-most cisternae; this apposition may permit direct transfer of lipids between ER and Golgi membranes. Within 0.2 microm of the cisternae studied, there are 394 vesicles (8 clathrin coated, 190 nonclathrin coated, and 196 noncoated), indicating considerable vesicular traffic in this Golgi region. Our data place structural constraints on models of trafficking to, through, and from the Golgi complex.

Figure 1

HVEM tomographic reconstruction of a portion of the Golgi ribbon from a fast frozen, freeze-substitution fixed NRK cell. Two serial 4-nm slices extracted from the tomogram are shown in a and b. Comparison of the images shows how little is changed from one such slice to its neighbor; e.g., the position of the microtubule (red arrow). (a) Membranes of individual Golgi and ER cisternae are clearly seen. (b) In analyzing the data, different cisternae were modeled by placing points along the membranes that delimit them, connecting the points with colored line segments, and building closed contours that model the different membrane compartments of a given slice. c and d are renditions of the surfaces fit to all of the contours for each object modeled in this Golgi region. The model viewed in c is in the same orientation as the tomographic slices. d shows a cis-side view with the cis ER removed to provide a better view of the ERGIC elements and the underlying Golgi cisternae. The 44 elements of the ERGIC are discontinuous, display no coated budding profiles, and do not appear to be flattened against the cis-most cisterna. Free vesicles in wells and the NCR (white) have unrestricted access to the cis side of the stack. The colors used to represent different components of the model are the same in all figures: ER, blue-gray; ribosomes, small purple spheres; ERGIC, yellow; Golgi cisterna: C1, green; C2, purple; C3, rose; C4, olive; C5, pink; C6, bronze; C7, red. Polymorphic structures in the NCR are light pink and gold. Non–clathrin-coated budding profiles on cisternae C1–C6, blue stippling. Clathrin-coated buds on C7, yellow stippling. Bars, 250 nm.

Figure 2

Identification of membranes and coat structures in tomographic images. Due to the thinness of tomographic slices (4 nm) and the limitations of the tomographic technique, membranes appear different from conventional thin sections (40–80 nm). Here we illustrate membranes and coats in composite images, each the sum of three adjacent tomographic slices. Panels on the right include interpretive diagrams. a and a′ show detail of a Golgi cisternal membrane that shows the two leaflets of the lipid bilayer with some clarity. b, c, and c′ show noncoated vesicles; the membrane thickness appears similar to that in a, and the two leaflets of the bilayer are occasionally discernible. d, e, and e′ show a non–clathrin-coated vesicle and bud, respectively; the two leaflets are easily seen, but the darkly stained coat structure is indistinguishable from the outer leaflet. f and f′ show a clathrin-coated bud in cross-section with characteristic spike structures. g and g′ show the cagelike structure on the surface of a clathrin-coated vesicle. The arrow in g indicates a single clathrin triskelion. Bar, 50 nm.

Figure 3

(Top) Successive layers of the Golgi model, each viewed from its cis side. cis-ER, the ER on the cis side; ERGIC, the ERGIC layer; C1–C7, the cisternae in cis-to-trans order; trans-ER, the ER on the trans side. C6′ is a partial cisterna, positioned between C6 and C7, which we interpret as ER. The starred object in C5 represents a membrane tubule of unknown origin. The noncompact region (NCR) displays all the elements lying between the two compact regions but not connected to either. All coated vesicles budding from cisternal margins are highlighted with stippling. The trans-most cisterna, C7, is the only one that is budding clathrin-coated vesicles (yellow stippling). All fenestrae and holes within each cisterna are included. (Bottom) Stereo image of the complete model including the free vesicles that surround the stacks (not shown in Fig. 1). Yellow, blue, and green spheres represent clathrin-coated, non–clathrin-coated, and noncoated vesicles, respectively. Bars, 250 nm.

Figure 4

Stereo images of the cis-Golgi well in the right compact region of the model. a shows a view from C1 to C6, revealing the depth of this well and the extent to which it is filled with buds and vesicles. The probable reason why vesicles are not round is discussed in Materials and Methods. b shows the same well as defined by holes in C3–C5, displaying the arrangement of the tapered tubule that enters this model from the side (Fig. 3, C5, star). Bar, 100 nm.

Figure 5

Relationships among different Golgi layers. (a) A higher magnification view of the cis side of the stack, showing parts of the ERGIC, the C1, and C2 cisternae oriented to display two tubules that extend from C2 past C1 and project into the ERGIC region. Both tubes end in non–clathrin-coated buds (short arrows). A tubule also projects from C1 and runs roughly parallel to the longer C2 tubule (long arrow). (b) Higher magnification view of the NCR. Many free non–clathrin-coated and uncoated vesicles (60–80 nm) and polymorphic elements fill this region. Some polymorphic elements resemble those of the ERGIC (gold), whereas the longer, rounded elements are different (pink). (c) A higher magnification view of the region where C5 projects a tubule past C6 and C7 into the space trans to the Golgi stacks (arrow). A tubule from C6 is also evident (arrowhead) and a lobule is protruding from C7 (star). (d and e) Two views of the trans Golgi showing the position of the trans ER relative to C6 and C7. The trans ER has ribosomes on only its free side (see also Fig. 3, bottom), and it is flattened against regions of both C6 (e, arrow) and C7 in a manner similar to the association between adjacent Golgi cisternae. The trans ER also wraps around C7 (d, arrow). The C7 cisterna covers most of the NCR. (e) Two tubules extend from C6: one projects in the trans direction, over C7 and the trans ER, where it branches and produces three non–clathrin-coated buds. The other projects in the cis direction, past all five previous cisternae, into the ERGIC region, where it ends in a non–clathrin-coated bud (see also Fig. 1 c). Bars, 250 nm.

Figure 6

Components of the NCR attached to cisternae, including bridges, buds, and projecting tubules. a–c show views of the relevant parts of C1, C3, and C6, respectively. The complexity of these structures suggests that they are highly dynamic. Bar, 100 nm.

Figure 7

Comparative data on (a) surface area, (b) volume, (c) density of fenestrae, and (d) number of coated buds for the ERGIC and C1–C7 cisternae.

Figure 8

Histograms describing the average diameters of openings in Golgi cisternae: (a) all cisternae in the model, (b) the cis cisternae (C1 and C2), (c) the medial cisternae (C3–C5), and (d) the trans-most cisternae (C6 and C7). The area of each opening was measured and used to determine the diameter of an equivalent circle. Most of the fenestrae in C1 and C2 (b) have diameters between 25 and 50 nm (mode 34 nm), while those in C6 and C7 (d) range from 10 to 30 nm (mode 18 nm). The distributions for C1–C5 (b and c) show a distinct population of very small fenestrae (∼8 nm), which are either absent or merged with the main peak in C6 and C7.

Figure 9

Interpretative model of the structural organization of the mammalian Golgi apparatus based on the data presented in this paper.