Figure 1.
A) Viewed in the active zone’s transverse plane by conventional 2-D electron microscopy of a tissue section, the AZM is a small electron dense patch between a pair of synaptic vesicles (asterisks) docked on the presynaptic membrane (arrow). The AZM’s superficial surface is attached to the presynaptic membrane where the membrane curves into the synaptic cleft forming the active zone ridge just opposite the mouth of a junctional fold (JF) in the muscle fiber’s surface. The AZM’s irregular deep surface extends about 75 nm into the cytoplasm. A cloud of undocked vesicles lies lateral and deep to the docked vesicles. B) Viewed in the active zone’s horizontal plane by conventional 2-D electron microscopy of a tissue section, the AZM is a narrow, irregularly dense band. This band of AZM has a slight angular change midway along its length (dashed box). The section includes a row of docked synaptic vesicles on each side of the AZM band in the lower half of the image. It passed superficial to the rows of docked vesicles along much of the band in the upper half of the image, where it includes only that portion of the band in the active zone ridge. At the tip of the AZM band in the upper half of the image, the section includes only the superficial layer of the AZM, exposing a series of ribs extending from each side of a beam, which are outlined in gold in the inset. Scale bar in A and B = 50 nm. C) 3-D schematic of the active zone, showing the active zone ridge in the presynaptic membrane (pale blue), rows of docked synaptic vesicles (dark blue), the AZM’s ribs (yellow gold), beams (brown gold) (from electron tomography on tissue sections; [11], [13]), and indicators of the active zone’s horizontal, transverse and median planes.
Figure 2.
Composite diagram of layers of AZM macromolecules at resting active zones exposed by electron tomography.
Shown in the transverse plane of the active zone, the superficial layer of macromolecules in the main body of the AZM includes beams, ribs and pegs; the intermediate layer includes steps and spars; and the deep layer includes masts, booms and topmasts. Ribs, spars and booms connect to docked synaptic vesicles (SV), topmasts connect to nearby undocked vesicles, and pegs connect to macromolecules in the presynaptic membrane (PM). Pins are positioned away from the main body of the AZM and link docked vesicles to the presynaptic membrane. All AZM components are shown in virtual slices and surface models from reconstructed tissue sections in subsequent Figures except for pegs, which were not included in this study (but see [11], [13]). The color code is the same for all Figures.
Figure 3.
Transverse distribution of macromolecules in the main body of the AZM at resting active zones.
A,D) Two regions 10 nm apart along the depth axis of the reconstructed volume of an active zone sectioned near its transverse plane. Each of the images was formed by the summation of three 1.2 nm thick serial virtual slices made through the volume in the same plane as the section. B,E) The same regions shown in A and D with the summed outlines of the AZM macromolecules in each of the virtual slices overlaid. C,F) Surface models, 10 nm thick, of the AZM macromolecules shown in A and D derived from segmentation of eight adjacent virtual slices and rotated to the transverse plane. G,H,I) Surface models generated as for those in C and F. Tissue used for each of the surface models was from a different frog. The tissue was chemically fixed except for that used for the surface model in I, which was fixed by rapid freezing. Ribs (yellow gold) extend from beams (brown gold) in the superficial layer of AZM, spars (red) extend from steps (gray) in the intermediate layer, and booms (purple) extend from masts (dark green) in the deep layer, while the ribs, spars and booms connect to synaptic vesicles (dark blue) docked on the presynaptic membrane (pale blue). Topmasts (light green) in C,F,G and I link masts to undocked vesicles near the active zone’s midline. The linkage of some topmasts to masts is incomplete in C,F, and H because discontinuous staining made segmentation uncertain or the topmast extended beyond the edge of the tissue section (arrowheads). Scale bars = 50 nm.
Figure 4.
Longitudinal distribution of the AZM’s main body macromolecules at resting active zones.
Surface models were generated from 1.0 nm thick serial virtual slices through a reconstructed active zone sectioned near its horizontal plane. The slice series was in the same plane. A) The ribs (yellow gold) and beams (brown gold) of the superficial layer of the AZM extend throughout the length of the active zone except at the gap, where the superficial layer was not included in the tissue section. Multiple ribs connect to each docked vesicle except at the upper right, where the vesicle(s) was too close to the edge of the section to be clearly discerned in the reconstruction. Ribs in some regions are not distinguishable from their neighbors because of the model’s angle of rotation. B) The intermediate layer shown together with the superficial layer. Steps (gray) are centered between opposing pairs of docked vesicles (as in solid box) along straight stretches of the active zone. The positioning of the steps is less regular where there is an angular change in the active zone’s long axis (dashed boxes). Typically, each docked vesicle is connected to spars (red) arising from two steps. C) The deep layer is shown together with the superficial and intermediate layers. Masts (dark green) overlay steps (compare with B). Multiple booms (purple) extend from each mast to connect to docked vesicles. D,E,F) Near transverse views of the surface models shown in A,B, and C from the regions in those panels marked d, e and f respectively. Arrows in A indicate the vesicles and associated AZM macromolecules used in Figure 5 to demonstrate our method for measuring the angle of approach of different classes of AZM macromolecules to docked vesicles. Topmasts were not included in the tissue section.
Table 1.
Averaged Dimensions of AZM Macromolecules in Resting Active Zones.
Figure 5.
Angle of approach of main body AZM macromolecules to docked vesicles.
The docked vesicles shown at the arrows in Figure 4 together with selected members of their associated AZM macromolecules were projected and traced onto a two dimensional plane. Lines drawn parallel to the ribs (yellow gold; A), spars (red; B) and booms (purple; C) approach a vesicle at different angles relative to perpendicular lines drawn to the plane of the beam (brown gold). Measurements of such angles from many docked vesicles reveal that the average angle of approach is significantly different for each class of AZM macromolecule, as detailed in the text and as shown here for single macromolecules from each class: A, 5°; B, 17°; and C, 27°.
Figure 6.
Connection sites of main body AZM macromolecules on docked vesicles at resting active zones.
A) Median view of 20 docked vesicles with ∼3 nm long stretches of ribs (yellow gold), spars (red) and booms (purple) attached to mark their connection sites on the vesicles. B) The centroids of AZM connection sites on all vesicles shown in A were normalized for small variations in vesicle diameter. They were, then, aligned to the relative position of the rib connections using a cross-correlation algorithm, and plotted on an idealized sphere. C) The rib, spar and boom connection sites on the idealized sphere in B plotted according to their distance from the rib connections. There is little overlap between vesicle domains connected to ribs and spars and moderate overlap between domains connected to spars and booms, but the domains are distinct from each other with a significance level of p<0.0001.
Figure 7.
Arrangement of pins at resting active zones.
A) A surface model, 9 nm thick, from an active zone sectioned in the transverse plane. A pin (copper) links the hemisphere of a docked vesicle that faces away from the active zone ridge (asterisk) to the presynaptic membrane. B) A virtual slice, 0.5 nm thick, from the series of virtual slices segmented to generate the surface model in A. The pin, docked vesicle and presynaptic membrane are outlined. An asterisk marks the main body of the AZM in the active zone ridge. C) A surface model, 20 nm thick, from an active zone sectioned in the median plane. The docked vesicles are from a row of such vesicles flanking the main body of AZM. A pin between the vesicles bifurcates to link the vesicles to the presynaptic membrane. D) The connection sites of pins and ribs (marked by ∼3 nm long segments of each: ribs, yellow gold; pins, copper) on docked synaptic vesicles viewed in the horizontal plane. The vesicles are from four active zones that were sectioned in the median or transverse planes after fixation by glutaraldehyde or rapid freezing. The connection sites surround the region of contact between the vesicle membrane and presynaptic membrane (dashed outline).
Figure 8.
Connection of main body AZM macromolecules to synaptic vesicles at activated active zones.
A) 10 nm thick surface model from an active zone sectioned in the median plane. It includes a row of vesicles from one side of an active zone ridge and the presynaptic membrane. Two of the vesicles are docked on the presynaptic membrane, one is fused with the membrane and one is an undocked vesicle 16 nm from a vacated docking site on the membrane. The color boundary at the interface of the fused vesicle membrane with the presynaptic membrane is arbitrary. B) An 80 nm thick surface model of the same vesicles in A that includes the vesicle surface facing the AZM. Connection sites of ribs (yellow gold), spars (red) and booms (purple) are indicated by ∼3 nm long stretches of each. C) A 25 nm thick surface model from an active zone sectioned in the transverse plane. A former docked vesicle that has fused with the presynaptic membrane is connected to ribs, a spar and booms. D) A 2.4 nm thick virtual slice, which was summed from 2 successive 1.2 nm thick virtual slices, of an activated active zone sectioned in the transverse plane. A former docked vesicle that is fused with the presynaptic membrane is connected not only to main body AZM macromolecules (asterisk), but also to non-AZM macromolecules (arrows) that link it to undocked vesicles (V1 and V2). One of the vesicles (V2) was linked to a topmast in other virtual slices. E) The undocked vesicle on the right of the AZM is 10 nm from a vacated docking site on the presynaptic membrane and has rib, spar, and boom connections as does the docked vesicle on the left. F) Undocked vesicles 10 nm and 15 nm from vacated docking sites on the presynaptic membrane with connection sites of ribs, spars and booms marked by ∼3 nm long stretches of each.
Figure 9.
Classes of AZM macromolecules connected to undocked vesicles at various distances from vacated docking sites in activated terminals.
A) Based on 3-D measurements in surface models from activated active zones, undocked synaptic vesicles (SV) >∼30 nm from vacated docking sites on the presynaptic membrane (PM) have relatively few connections to main body AZM macromolecules. The number of connection sites gradually increases with decreasing distance of the undocked vesicles from the presynaptic membrane to <∼15 nm, where it is similar to that for docked vesicles at resting active zones (indicated by the open symbol on the Y-axis ± SD). B) Viewed from the median plane of the active zone, undocked synaptic vesicles 42–29 nm from the presynaptic membrane have connection sites formed mostly by booms (purple), vesicles 24–17 nm from the presynaptic membrane have connection sites formed mostly by booms and spars (red), while vesicles 16–4 nm from the presynaptic membrane have connection sites formed by similar numbers of booms, spars and ribs (yellow gold) as on docked vesicles (compare with Figure 6A). C) Viewed in the horizontal plane, undocked vesicles 15–4 nm from the presynaptic membrane have connection sites formed by pins (copper) as well as ribs.
Figure 10.
Non-AZM macromolecules connected to a docked and nearby undocked synaptic vesicles at a resting NMJ.
A surface model 20 nm thick with a docked vesicle linked to AZM macromolecules colored as in previous Figures. The docked vesicle is near four undocked vesicles. Non-AZM macromolecules (pewter) link the undocked vesicles to each other and variously to the presynaptic membrane and the docked vesicle.
Figure 11.
Schematized 3-D arrangement of classes of AZM macromolecules throughout the depth of the resting active zone.
A) View includes the transverse, horizontal and median planes of the active zone (see Figure 1C). Core macromolecules include beams (brown gold), steps (grey) and masts (dark green). Macromolecules connecting core macromolecules to synaptic vesicles (dark blue) and the presynaptic membrane (pale blue) along with channels (frosted green) in the membrane, include ribs (yellow gold), pegs (orange gold), pins (copper), spars (red), booms (purple), and topmasts (light green).The presynaptic membrane and the docked vesicles in the row on the right are transparent to expose the extent of the AZM connections. B) View from the median plane of the active zone toward the left row of docked vesicles in A. C) View from beyond the active zone toward the left row of docked vesicles in A.
Figure 12.
Model of the sequential association of undocked synaptic vesicles with different AZM macromolecules leading to docking.
When a docked synaptic vesicle (A) fuses with the presynaptic membrane (B), booms (purple), spars (red), and ribs (yellow gold) remain attached to the vesicle membrane until it undergoes flattening into the presynaptic membrane (C). An undocked vesicle within 50 nm of the docking site on the presynaptic membrane first forms connections with booms that have dissociated from the fused vesicle (C) and then sequentially with dissociated spars (D) and ribs-pins (E) which direct it to the docking site on the presynaptic membrane (A). Pins are not included in B, C and D because we were unable to distinguish them from other presynaptic membrane linked macromolecules as the fused vesicles flattened.