Conical Electron Tomography of a Chemical Synapse: Polyhedral Cages Dock Vesicles to the Active Zone

The rendered volume of the reconstructed synapse. Visualization of the 3D structure of the synapse by volume rendering involves assigning light intensities to each voxel of the reconstructed volume. Rendering requires inverting the contrast of the planes so that matter is white and the lack thereof is black. Volume rendering shows vesicles as spheres instead of circles bounded by the unit membrane pattern (yellow). The plasma membranes appear as wavy surfaces (white bands) and the syndesome as a cage instead of a dense particle (green). Vesicles at the active zone are arranged as a corona (yellow) around the syndesome. The cytomatrix appears as ubiquitous densities, which are associated to the plasma membranes and which occupy the cytoplasm of the terminal. The thick layer of density located underneath the plasma membrane corresponds to the PSD. Scale bar, 75 nm.

Semiautomatic segmentation of the reconstructed synapse. To understand the relationships that vesicles and plasma membranes establish with the cytomatrix, the two elements were split and presented in “complementary” maps. A shows the membrane map. Vesicles appear as spherical bodies, and plasma membranes appear as wavy planes highlighting the boundaries of the terminals. The unit membrane pattern is observed in the small region (left side) that was oriented perpendicular to the plane of the membrane. At the active zone, vesicles are arranged in arches, circumscribing empty spaces, indicated by green stars. At the cytoplasm, the vesicles exhibited a more random orientation, although, at places, they appear to be arranged in single-file rows. B shows the cytomatrix map. At the active zone, large particles representing the syndesomes comprised the cytomatrix (green). These structures occupy the empty center of the coronas of vesicles seen in A (green stars). The surface of the syndesomes contains interruptions indicative of their cage-like structure. Note that, in the membrane maps, the middle syndesome was obscured by three vesicles in “front” of it. Note also the presence of the syndesome on the right, unresolved in the plane section of this synapse in Figure 1. The white arrows in both panels indicate a vesicle and the void created in the cytomatrix by the segmentation. Scale bar, 95 nm.

Segmentation of a syndesome and its corona of vesicles. This figure illustrates the relationships between the syndesome, its corona of vesicles, and the plasma membrane. A shows the syndesome comprising the central particle connected to an outer ring by radial spokes. The corona, comprising five synaptic vesicles, is intimately associated with the external ring. B shows a view oriented perpendicular to the plasma membrane, as determined by the unit membrane structure of the plasma membrane and three of the vesicles. The cage exhibits three faces and a series of spikes linking the vesicles of the corona to its surface (small arrows). The vesicle indicated by the white arrow is hemifused to the plasma membrane. C shows a fully fused vesicle (red) associated to the syndesome (green). The corona of vesicles appears incomplete and some are difficult to appreciate because the viewing angle emphasizes the opening with the plasma membrane. Scale bar, 60 nm.

Hemifused vesicles associated with two cages. A is a plane of a small region of the active zone in which a synaptic vesicle (red) is attached to two cages (green). B is a portion of the rendered volume of the same region. Four classes of vesicles were labeled with arrows: (1) those attached to two cages and the active zone (arrow 1); (2) those attached to one syndesome and the active zone (first tier; arrow 2); (3) those attached to a syndesome but separated from the plasma membrane (second tier; arrow 3); and (4) those attached to filaments of the cytomatrix (arrow 4). Scale bar, 60 nm.

Center-to-center spacing of neighboring polyhedral cages. The values of the center-to-center were first binned and then plotted with the program fityk (http://www.unipress.waw.pl/fityk/). The x-axis represents the spacing between cages in nanometers, and the y-axis represents the number of measurements after binning. Visual inspection indicated the presence of three peaks that were refined until reaching convergence. The parameters of the Gaussian curves (center, height, and width) provided the area under the curve from which the size of the different subpopulations was obtained and expressed as percentage of the total area under the peaks. The two peaks from the left are centered at 106 and 162 nm and represent, respectively, 52 and 32% of the total area. Thus, 52% of the area of the active zone contains syndesomes separated by a single vesicle, whereas 32% contains syndesomes separated by two vesicles (indicated by the schematic drawings under the respective curves). The predominance of measurements in these bins suggests significant hexagonal close packing in the arrangement of units on the active zone. The remaining small peak is centered at 232 nm and represents 13% of the total area, suggesting that regions that deviate from perfect hexagonal close packing also occur in the active zone (see Fig. 9).

Syndesomes are polyhedral cages. A small region of the active zone (white band), a syndesome (green) and its corona of synaptic vesicles (yellow), was segmented and rotated around an axis represented by the plane of the plasma membrane. A shows a view oriented perpendicular to the active zone, as determined by the trilayer structure of the plasma membrane (white band). The syndesome appears as a cage with polygons, discernable by the four dark regions of their centers, arranged in a row. B shows a view rotated ∼30° from the perpendicular plane, as determined from the disappearance of the trilayer structure. A larger region of the surface of the cage is revealed demonstrating the series of polygons tessellating its surface. C shows the en face view of the plasma membrane (∼0°). This view shows the entire thickness of the section. Only a part of the cage comprising three polygons meeting at a vertex is clearly visible (white arrow). To view the surface of the polyhedral cage, the vesicles of the corona were excluded. The straight line and the arrow show the direction of the rotation used to generate the views. Scale bar, 90 nm.

Comparison with a clathrin model. We compared the structure of the polyhedral cages ( A ) with the faces of the clathrin cage model available in the Protein Structure Data Bank for the hexagonal barrel structure, reconstructed from cryomicroscopy data by averaging thousands of cages and by applying D6 symmetry (1XI4.pdb and 1XI4.pdb1) (Fotin et al., 2004). In the model of the hexagonal barrel, there are two types of hexagons: those lining the top (or the bottom) of the barrel, which is surrounded only by pentagons, and the hexagons of the central belt, which are in contact with both pentagons and hexagons. B , The hexagon coming from the belt produced a slightly better fit. Although it was not possible to observe a precise correspondence between the two lattices, both were comparable in size and shape. Scale bar, 60 nm.

Relationships between cages, vesicles, and the cytomatrix. This figure illustrates the interactions between polyhedral cages (green), the vesicles (yellow), and the filaments (red) in the cytoplasm of the terminal. This region seen here was cropped from the map in Figure 3. A shows two cages (green) associated to the active zone and surrounded by vesicles (yellow). The densities occupying the spaces between these organelles contain components of the cytoskeleton (the cytomatrix). These on top of the cages were colored red for emphasis. PSD indicates the layer of density associated to the postsynaptic membrane. B shows the same region after removing the cytoskeleton, including PSD. The filaments colored red attached to the syndesomes were maintained to demonstrate the location of vesicles within the terminal. C shows the region after removing the synaptic vesicles. This view shows the syndesomes associated to the plasma membrane and the interconnecting filaments. Vesicles were attached along the entire length of filament. At places, the cytoskeleton appeared as islands in which filaments formed a web decorated with small particles. Arrows indicate the relationship of vesicles to cytomatrix. Scale bar, 60 nm.