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.

Variable priming of a docked synaptic vesicle.

The priming of a docked synaptic vesicle determines the probability of its membrane (VM) fusing with the presynaptic membrane (PM) when a nerve impulse arrives. To gain insight into the nature of priming, we searched by electron tomography for structural relationships correlated with fusion probability at active zones of axon terminals at frog neuromuscular junctions. For terminals fixed at rest, the contact area between the VM of docked vesicles and PM varied >10-fold with a normal distribution.

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

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.

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.

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.

Macromolecular connections of active zone material to docked synaptic vesicles and presynaptic membrane at neuromuscular junctions of mouse.

Electron tomography was used to view macromolecules composing active zone material (AZM) in axon terminals at mouse neuromuscular junctions. Connections of the macromolecules to each other, to calcium channels in the presynaptic membrane, and to synaptic vesicles docked on the membrane prior to fusing with it during synaptic transmission were similar to those of AZM macromolecules at frog neuromuscular junctions previously examined by electron tomography and support the hypothesis that AZM regulates vesicle docking and fusion. A species difference in the arrangement of AZM relative to docked vesicles may help account for a greater vesicle-presynaptic membrane contact area during docking and a greater probability of fusion during synaptic transmission in mouse.

Methods for generating high-resolution structural models from electron microscope tomography data.

Reconstructed volumes generated by tilt-image electron-microscope tomography offer the best spatial resolution currently available for studying cell structures in situ. Analysis is often accomplished by creating surface models that delineate grayscale contrast boundaries. Here, we introduce a specialized and convenient sequence of segmentation operations for making such models that greatly improves their reliability and spatial resolution as compared to current approaches, providing a basis for making accurate measurements.