Conical electron tomography of a chemical synapse: vesicles docked to the active zone are hemi-fused

Abstract

We have used thin sectioning and conical electron tomography to determine the three-dimensional structure of synaptic vesicles that were associated (docked) at release sites of the presynaptic membrane, called active-zones. Vesicles docked at the active zone occupied a strategic location: they formed regions of contact with the plasma membrane on one side and with that of one or more vesicles located deeper within the presynaptic terminal on the other side. The region of contact with the active zone measured approximately 15 nm in diameter ( approximately 2% of the vesicle's surface) and contained a smaller approximately 6 nm region where the proximal leaflets merged (hemi-fused). Hemi-fusion was only observed on the side of vesicles in contact with the active zone; at the side of contact between neighboring vesicles, the membranes were not hemi-fused. Approximately three-fourths of the docked vesicles contained hemi-fused regions. Vesicles fully fused to the active zone (exhibiting pores that appeared as interruptions of a single membrane) were less frequently observed ( approximately 1 of 10 hemi-fused vesicles). In conclusion, our observations in cortical synapses strengthen the hypothesis that hemi-fusion is a stable intermediary that precedes full fusion and release.

FIGURE 1

Three-dimensional map of a synapse visualized by volumetric rendering and density segmentation. (A) The synapse is comprised of a presynaptic (upper half) and a postsynaptic (lower half) terminal. Spherical profiles of different diameters represent the membranous organelles in the presynaptic terminal. The synaptic vesicles appear as small spheres that cluster at the active zone, which itself faces a layer of density associated with the postsynaptic plasma membrane. Other membrane-bound organelles include a group of three coated vesicles, a coated pit (lower right side) and a large endosome with vesicles inside the lumen (upper right corner). Docked synaptic vesicles are closest to the membrane of the active zone. This reconstruction has six docked vesicles. The rectangle at the center of the panel encloses the volume of the docked vesicle that is shown in Fig. 2. The inset shows an individual plane of another docked vesicle. To underscore the closeness of the region of contact, the vesicle was colored red and the plasma membrane white. The volume of the reconstruction was ∼0.1 μm³ and the resolution, estimated from the unit-membrane pattern, ∼4 nm. (B) The densities of two of the different membranous organelles were extracted (segmented) and reconstructed independently (color-coded). The parallel white bands represent the pre- and postsynaptic plasma membranes separated by the extracellular space. The thick brown layer associated to the plasma membrane represents the postsynaptic densities that define the active zone in the presynaptic terminal. The three orange particles associated to the plasma membrane between the vesicles belong to the presynaptic web (–51). Depending on their distance to the active zone, the synaptic vesicles were located at ∼15 nm from the active zone (docked and in red) or deeper within the terminal (in blue). (Note that the viewing angle induces the illusion that some of the red vesicles seem farther from the active zone while some of the blue vesicles seem closer than the 50-nm separation.) The organelles colored yellow outside the active zone include a coated pit, three coated vesicles, and a large endosome with vesicles in its lumen. The distinctive structure in conjunction with its location outside the active zone allowed us to identify these profiles as unambiguously belonging to classical clathrin-related retrieval apparatuses. Bar: 265 nm.

FIGURE 2

The unit membrane pattern is resolved along all directions. (A) The unit membrane pattern is comprised of two dense layers flanking an electron lucent center, the whole of which measures ∼6 nm in overall thickness. The pattern is seen in the membranes of the pre- and postsynaptic terminals and vesicles. The red and blue lines indicate the approximated location of planes oriented perpendicular to the x,y plane. (B, C) The unit membrane pattern is also visualized comprising the membrane of synaptic vesicles viewed in the x,z and y,z planes. Since the overall thickness of the pattern is ∼6 nm, it follows that the resolution of the map (3–4 nm) allowed our resolving the pattern in all directions. The red and blue arrows point to the same vesicle viewed in the two planes. Incomplete vesicles appear as hemi-cups, an effect resulting from sectioning. All panels in the figure are individual planes presented in positive contrast. Bar: 50 nm.

FIGURE 3

Vesicles docked with the active zone. (A–C) Small volume cropped from the reconstruction in Fig. 1 A (rectangle). The densities correspond to a vesicle docked to the plasma membrane on one side and associated to an adjacent vesicle on the other. The rectangles slicing through the densities indicate the approximate location of the three planes that were selected to illustrate the structure of this docked vesicle. Two side views (A, C) and one edge-on view (B) of the densities, colored green, are shown. (D–F) Images of individual planes along the z-direction (depth) indicated by the rectangles in the upper panels. (G–I) The same images cropped to include images of only a small region of the active zone (green lines) and a docked vesicle (red lines). The densities were displayed in reverse contrast. (D, G) (plane #46) Shows that the side contacting the plasma membrane exhibited a pronounced curvature and was comprised of two closely apposed unit membranes (red and green in G). The side contacting the adjacent vesicle immediately above was less deformed but it was also comprised of two closely apposed unit membranes. The docked vesicle is linked to the plasma membrane by a long fibril (arrowhead) and a shorter fibril, oriented perpendicular to each other. (E, F, H, and I) (planes #50 and #52) Shows planes through the hemi-fused region. The side contacting the plasma membrane is comprised of a single unit membrane. This feature is also underscored in panels H–I by the fact that at the region of contact (vertical arrows, E and F), the unit membrane is comprised of a red line (the distal leaflet of the vesicle) and a green line (the distal leaflet of the active zone). The docked vesicle is thus hemi-fused to the active zone but not with the vesicle immediately above. The densities connecting this docked vesicle to the active zone (arrowheads, D and F) might represent a trans-SNARE complex. Bar: 50 nm.

FIGURE 4

Vesicles fully fused with the active zone. (A–C) Small volume containing densities of a vesicle associated to the plasma membrane on one side and to an adjacent vesicle on the other. In contrast to the volume in Fig. 3, this docked vesicle is fully fused to the plasma membrane. The next vesicle in the chain was hemi-fused to the dimple created by the fusion event. The rectangles cutting through the densities indicate the approximate location of the planes that were selected to illustrate the structure of the region. The densities (green) are shown in side views (A, C) and an edge-on view (B). The upper vesicle is located to the side of the one fused to the plasma membrane (C). In the lower panels, to assist in the visualization of the fusion event, the plasma membrane was colored blue, the fully fused vesicle was colored yellow, and the hemi-fused vesicle was colored red. (D) An oblique plane indicating that the docked vesicle is in fact fully fused to the plasma membrane. A key feature of this obliquely sectioned plane is the region of contact between the vesicle in the second tier (red) and the dimple created by the fusion event (yellow), as illustrated by the mixing of the colors. Measurements of this region along the x,y plane at different points in the z direction have been as low as ∼6 nm, suggesting that there may be areas of hemi-fusion at the region of contact between these two vesicles. Panels E and F (planes #13 and 15) show a discontinuity of the membrane (arrow) that represents the pore spanning the entire region of contact between the fully fused vesicle and the active zone. It is important to note that due to the pore, the previously independent vesicle must now be considered as an extension of the plasma membrane inside the terminal. Bar: ∼50 nm.

FIGURE 5

Vesicles deeper in the terminal are arranged in chains. Synaptic vesicles establishing close contacts formed chains that interlinked vesicles docked to the active zone. (A) Segmentation comprised of five synaptic vesicles (red and blue) and the nearest region of the active zone (white band). The vesicles that are docked to the active zone are hemi-fused (red), while the vesicles comprising the crook of the arch, though they are in direct contact, do not hemi-fuse (blue). The center of the arch appears empty but in the complete reconstruction, it is in fact occupied by particles of the synaptic grid structure. This vesicular chain was segmented from the map in Fig. 1 A, where the synaptic grid particles are represented by the orange volumes. (B) Vesicular chain interlinking a fully fused vesicle (white) to a hemi-fused vesicle (red). A key feature of this chain is that the next tier vesicle (red) hemi-fused at the dimple created by the previous fusion event. Panels C and D compare reconstructions of the region of contact of a hemi-fused vesicle (C) and a fully fused vesicle (D). Both views show the region of contact viewed from the extracellular space. The region of contact of the fully fused vesicle exhibits a pore (arrow). The segmentations of panels C and D were visualized using triangulation instead of volume-rendering methods. Bar: 60 nm.

FIGURE 6

Projecting the volume of the section on a single plane prevents visualization of the hemi-fused regions in docked vesicles. (A–C) Individual planes from different three-dimensional reconstructions of synaptic vesicles that are hemi-fused to the active zone (red arrows). (D–F) Projection calculated by averaging the volume of 64 planes or ∼50 nm along the z-direction (thickness). Instead of a hemi-fused region comprised of single unit membranes, the vesicles and regions of contact become diffuse (yellow arrows). In one example (E), the projection of the vesicle hemi-fused to the active zone is barely visible. Another vesicle, not present in the individual plane in panel B, is now the most prominent vesicle in the projected image. Therefore, the projection artifact would make visualization of a small feature, such as the hemi-fused region of docked vesicles, impossible in images gathered using conventional thin-sectioning electron microscopy. All images are in reverse contrast. Bar: 50 nm.

FIGURE 7

Some of the states leading to the formation of the fusion pore (modified from (52)). Three different states of the area of contact between the membranes of synaptic vesicles and the active zone are illustrated in the panels. (A) Region of close membrane apposition where both membranes are separated by a small aqueous space. This state is proposed in both the direct-fusion and the fusion-through-hemifusion models. (B) Region of contact exhibiting hemi-fusion. In this state, the proximal leaflets (red) merge and the distal leaflets (green and white) form the bilayer. This state is predicted by the fusion-through-hemifusion model and was frequently seen in the vesicles docked to the active zone. (C) Fusion-pore at the perimeter of the hemi-fused region. In this state, both the proximal (red) and distal (green) leaflets of both membranes have merged. This fusion pore was infrequently seen in our study. For clarity, the leaflets of the phospholipid bilayer of the upper membrane were colored green (distal) and red (proximal).