Active Zone Material-Directed Orientation, Docking, and Fusion of Dense Core Vesicles Alongside Synaptic Vesicles at Neuromuscular Junctions

Active zone material is an organelle that is common to active zones along the presynaptic membrane of chemical synapses. Electron tomography on active zones at frog neuromuscular junctions has provided evidence that active zone material directs the docking of synaptic vesicles (SVs) on the presynaptic membrane at this synapse. Certain active zone material macromolecules connect to stereotypically arranged macromolecules in the membrane of undocked SVs, stably orienting a predetermined fusion domain of the vesicle membrane toward the presynaptic membrane while bringing and holding the two membranes together. Docking of the vesicles is required for the impulse-triggered vesicle membrane-presynaptic membrane fusion that releases the vesicles’ neurotransmitter into the synaptic cleft. As at other synapses, axon terminals at frog neuromuscular junctions contain, in addition to SVs, vesicles that are larger, are much less frequent and, when viewed by electron microscopy, have a distinctive electron dense core. Dense core vesicles at neuromuscular junctions are likely to contain peptides that are released into the synaptic cleft to regulate formation, maintenance and behavior of cellular apparatus essential for synaptic impulse transmission. We show by electron tomography on axon terminals of frog neuromuscular junctions fixed at rest and during repetitive impulse transmission that dense core vesicles selectively dock on and fuse with the presynaptic membrane alongside SVs at active zones, and that active zone material connects to the dense core vesicles undergoing these processes in the same way it connects to SVs. We conclude that undocked dense core vesicles have a predetermined fusion domain, as do undocked SVs, and that active zone material directs oriented docking and fusion of these different vesicle types at active zones of the presynaptic membrane by similar macromolecular interactions

Alignment of Synaptic Vesicle Macromolecules with the Macromolecules in Active Zone Material that Direct Vesicle Docking

Synaptic vesicles dock at active zones on the presynaptic plasma membrane of a neuron’s axon terminals as a precondition for fusing with the membrane and releasing their neurotransmitter to mediate synaptic impulse transmission. Typically, docked vesicles are next to aggregates of plasma membrane-bound macromolecules called active zone material (AZM). Electron tomography on tissue sections from fixed and stained axon terminals of active and resting frog neuromuscular junctions has led to the conclusion that undocked vesicles are directed to and held at the docking sites by the successive formation of stable connections between vesicle membrane proteins and proteins in different classes of AZM macromolecules. Using the same nanometer scale 3D imaging technology on appropriately stained frog neuromuscular junctions, we found that ∼10% of a vesicle’s luminal volume is occupied by a radial assembly of elongate macromolecules attached by narrow projections, nubs, to the vesicle membrane at ∼25 sites. The assembly’s chiral, bilateral shape is nearly the same vesicle to vesicle, and nubs, at their sites of connection to the vesicle membrane, are linked to macromolecules that span the membrane. For docked vesicles, the orientation of the assembly’s shape relative to the AZM and the presynaptic membrane is the same vesicle to vesicle, whereas for undocked vesicles it is not. The connection sites of most nubs on the membrane of docked vesicles are paired with the connection sites of the different classes of AZM macromolecules that regulate docking, and the membrane spanning macromolecules linked to these nubs are also attached to the AZM macromolecules. We conclude that the luminal assembly of macromolecules anchors in a particular arrangement vesicle membrane macromolecules, which contain the proteins that connect the vesicles to AZM macromolecules during docking. Undocked vesicles must move in a way that aligns this arrangement with the AZM macromolecules for docking to proceed

Regulation of Synaptic Vesicle Docking by Different Classes of Macromolecules in Active Zone Material

The docking of synaptic vesicles at active zones on the presynaptic plasma membrane of axon terminals is essential for their fusion with the membrane and exocytosis of their neurotransmitter to mediate synaptic impulse transmission. Dense networks of macromolecules, called active zone material, (AZM) are attached to the presynaptic membrane next to docked vesicles. Electron tomography has shown that some AZM macromolecules are connected to docked vesicles, leading to the suggestion that AZM is somehow involved in the docking process. We used electron tomography on the simply arranged active zones at frog neuromuscular junctions to characterize the connections of AZM to docked synaptic vesicles and to search for the establishment of such connections during vesicle docking. We show that each docked vesicle is connected to 10–15 AZM macromolecules, which fall into four classes based on several criteria including their position relative to the presynaptic membrane. In activated axon terminals fixed during replacement of docked vesicles by previously undocked vesicles, undocked vesicles near vacated docking sites on the presynaptic membrane have connections to the same classes of AZM macromolecules that are connected to docked vesicles in resting terminals. The number of classes and the total number of macromolecules to which the undocked vesicles are connected are inversely proportional to the vesicles’ distance from the presynaptic membrane. We conclude that vesicle movement toward and maintenance at docking sites on the presynaptic membrane are directed by an orderly succession of stable interactions between the vesicles and distinc

Pairing of connection sites of nubs with connection sites of AZM and non-AZM macromolecules.

<p>(A,B) Surface model of the membrane of a docked vesicle in one of the primary reconstructions; (A) shows the hemisphere facing the main body of the AZM and (B) shows the hemisphere facing away from the main body. (A-left, B-left) Three-voxel-thick terminal portions of the AZM’s ribs (gold), spars (red), booms (purple) and pins (copper) and of non-AZM macromolecules (pewter), mark the connection site of each on the outer cytosolic surface of the vesicle membrane (pale blue). The vesicle’s fusion domain is indicated by the dashed line (B). (A-right, B-right) The AZM and non-AZM macromolecules are disabled on the surface model and the membrane has been made almost transparent. Three-voxel-thick terminal portions of the luminal assembly’s nubs (blue) mark the connection site of each on the luminal surface of the vesicle membrane. The distribution of the connection sites of the nubs is non-uniform and appears similar to the asymmetric distribution of the connection sites of the AZM and non-AZM macromolecules, although some of the similarity is obscured in these 2D images by the vesicle’s 3D curvature and the difference in the vesicle’s luminal and outer diameters. (C) Using <i>x,y,z</i> coordinates of the connection sites in (A,B), after correcting for the difference between luminal and outer diameters of the vesicle membrane, the relative positions of the centroids of the connection sites of the AZM and non-AZM macromolecules and of the nubs were plotted on a 2D Robinson map. The centroids were overlaid by filled circles slightly smaller in diameter than the connection sites and color-coded as in (A,B). The hemisphere of the vesicle facing the main body of the AZM lies between the bold longitudinal lines. The main AZM binding domain, which covers about 20% of the outer surface area of this vesicle and includes about half of the total nub and AZM/non-AZM macromolecule connection sites on the membrane, is encircled by the solid white line. Each nub is paired with an AZM or non-AZM macromolecule lying opposite to, or slightly offset from, it.</p