The structure and function of 'active zone material' at synapses

Abstract

The docking of synaptic vesicles on the presynaptic membrane and their priming for fusion with it to mediate synaptic transmission of nerve impulses typically occur at structurally specialized regions on the membrane called active zones. Stable components of active zones include aggregates of macromolecules, 'active zone material' (AZM), attached to the presynaptic membrane, and aggregates of Ca(2+)-channels in the membrane, through which Ca(2+) enters the cytosol to trigger impulse-evoked vesicle fusion with the presynaptic membrane by interacting with Ca(2+)-sensors on the vesicles. This laboratory has used electron tomography to study, at macromolecular spatial resolution, the structure and function of AZM at the simply arranged active zones of axon terminals at frog neuromuscular junctions. The results support the conclusion that AZM directs the docking and priming of synaptic vesicles and essential positioning of Ca(2+)-channels relative to the vesicles' Ca(2+)-sensors. Here we review the findings and comment on their applicability to understanding mechanisms of docking, priming and Ca(2+)-triggering at other synapses, where the arrangement of active zone components differs.

Keywords: active zone material; docking; electron tomography; priming; synaptic transmission; synaptic vesicle.

Figure 1.

Arrangement of AZM at active zones on the presynaptic membrane of frog neuromuscular junctions. (a) A 2D electron micrograph from an approximately 80 nm thick tissue section cut in the transverse plane of an active zone. A dense aggregate of macromolecules, constituting the main body of AZM, is seated in the presynaptic membrane's active zone ridge and flanked by synaptic vesicles (asterisks) docked on the presynaptic membrane. The area in the box approximates the area shown in b. (b) Composite diagram of the relationships of AZM macromolecules exposed by electron tomography and viewed in the active zone's transverse plane. Ribs, spars and booms arise from beams, steps and masts in the main body of the AZM to connect to specific domains of the membrane of docked synaptic vesicles (SV) according to their depth/distance from the presynaptic membrane (PM). Pegs link ribs to macromolecules, thought to include Ca²⁺-channels and Ca²⁺-activated K⁺-channels in the presynaptic membrane, while beyond the main body of AZM, the pins link the vesicle membrane directly to the presynaptic membrane. The topmast links the deep end of the mast to an undocked synaptic vesicle. Non-AZM macromolecules connect the membrane of docked synaptic vesicles to the membrane of nearby undocked synaptic vesicles and are similar to filamentous macromolecules that connect undocked synaptic vesicles to each other. (c) A 2D electron micrograph from an approximately 50 nm thick tissue section cut in the horizontal plane of an active zone. The main body of the AZM in this plane is a band. The docked synaptic vesicles are arranged in a single row on each side of it. Scale bar, 100 nm for a and c. (d) A 3D schematic of the active zone showing the same structures, with the same colour code, that are labelled in b, with indicators of the active zone's horizontal, transverse and median planes. (a) and (c) adapted from [20]; (b) and (d) from [21].

Figure 2.

Linkage of macromolecules in docked synaptic vesicles to AZM macromolecules. (a) A schematic profile of a docked synaptic vesicle viewed in the transverse plane of the active zone. The interconnected assembly of macromolecules in the synaptic vesicle lumen is linked by its nubs to macromolecules that span the vesicle membrane and connect to AZM and non-AZM macromolecules. (b) The same docked synaptic vesicle rotated 90^o and viewed from the main body of AZM in the median plane. The coloured spots on the radial arms of the luminal assembly mark regions connected by nubs and their membrane-spanning macromolecules to specific classes of AZM and non-AZM macromolecules.

Figure 3.

Rotation of undocked synaptic vesicles and alignment of synaptic vesicle macromolecules with AZM macromolecules during docking. The chiral shape of the luminal assembly of macromolecules is stereotypic for both docked and undocked synaptic vesicles. Represented by 3D arrows, the orientation of the shape of the luminal assemblies in docked synaptic vesicles (red arrows) is the same with respect to the presynaptic membrane and the midline of the main body of AZM. The orientation of the shape of the luminal assemblies in undocked synaptic vesicles (blue arrows) varies from vesicle to vesicle. Thus, undocked synaptic vesicles must rotate in order for the appropriate vesicle membrane-spanning macromolecules, anchored by the luminal assembly (figure 2), to align with and connect to AZM macromolecules. (From [21].)

Figure 4.

Model for AZM-mediated docking, priming and Ca²⁺-triggered vesicle membrane–presynaptic membrane fusion. After a docked synaptic vesicle fuses with and flattens into the presynaptic membrane (blue and white stippled membrane), an undocked synaptic vesicle is directed towards and held in contact with the vacated docking site on the presynaptic membrane by a stepwise progression of stable interactions between it and multiple booms, spars and ribs of the AZM (colour-coded as in figure 1b). Once a synaptic vesicle is docked it undergoes variable priming: the pins and proximal rib segments that link it to the presynaptic membrane shorten and lengthen in dynamic equilibrium (copper and gold double-headed arrows), generating variable force that brings about coordinated variation in (1) the extent of the vesicle membrane–presynaptic membrane contact area and the stability of the lipid bilayers at the contact site (double-headed black arrow), in (2) the proximity of proximal pegs and their associated calcium channels (double-headed green arrow) to the vesicle membrane's Ca²⁺-sensors and in (3) the synaptic vesicle's eccentricity (blue double-headed arrow) in the plane of the presynaptic membrane. Accordingly, docked synaptic vesicles are most primed when their pins and proximal rib segments are shortest, their vesicle membrane–presynaptic membrane contact areas are largest, their lipid bilayers are most destabilized towards fusion threshold, their associated Ca²⁺-channels are, on average, in closest proximity to it and they are most eccentric in shape. The membrane of docked synaptic vesicles that are most primed at the moment a nerve impulse arrives has the greatest probability of merging with the presynaptic membrane to form a fusion pore. Fusion occurs while the synaptic vesicle is still attached to the AZM macromolecules. Figure adapted from [20] and [26].