Variable priming of a docked synaptic vesicle

Abstract

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. There was no merging of the membranes. For terminals fixed during repetitive evoked synaptic transmission, the normal distribution of contact areas was shifted to the left, due in part to a decreased number of large contact areas, and there was a subpopulation of large contact areas where the membranes were hemifused, an intermediate preceding complete fusion. Thus, fusion probability of a docked vesicle is related to the extent of its VM-PM contact area. For terminals fixed 1 h after activity, the distribution of contact areas recovered to that at rest, indicating the extent of a VM-PM contact area is dynamic and in equilibrium. The extent of VM-PM contact areas in resting terminals correlated with eccentricity in vesicle shape caused by force toward the PM and with shortness of active zone material macromolecules linking vesicles to PM components, some thought to include Ca 2+ channels. We propose that priming is a variable continuum of events imposing variable fusion probability on each vesicle and is regulated by force-generating shortening of active zone material macromolecules in dynamic equilibrium.synapse | hemifusion | priming | active zone material | electron tomography S ynaptic vesicles (SVs) move toward and dock on (are held in contact with) the presynaptic plasma membrane (PM) of a neuron's axon terminal before fusing with the PM and releasing their neurotransmitter into the synaptic cleft to mediate synaptic transmission (1, 2). Docking is achieved by force-generating interactions of the vesicle membrane (VM) protein synaptobrevin with the PM proteins syntaxin and SNAP25 (3, 4). These interactions, which produce force by forming a coiled coil called the SNARE core complex, are regulated by auxiliary proteins (1, 5-7). Such force on the VM-PM contact site may also play a role in their fusion (8). At typical synapses, docking and fusion take place at structurally specialized regions along the PM called active zones (9, 10). Several lines of evidence suggest that the formation of the SNARE core complex occurs in the macromolecules composing the common active zone organelle, active zone material (AZM) (2,(11)(12)(13)(14), which is positioned near Ca 2+ channels concentrated in the PM at active zones (15-17). Influx of Ca 2+ through the channels after the arrival of a nerve impulse triggers fusion of the VM of docked SVs with the PM.Priming is a step in synaptic transmission between the docking of an SV on, and fusion with, the PM and accounts for the observation that relatively few docked SVs fuse with the PM...

The structure and function of ‘active zone material’ at synapses

“…Taken together, our findings lead to a hypothesis for the priming of docked synaptic vesicles at frog neuromuscular junctions based on continually variable AZM-mediated forces (figure 4; fully discussed in [26]). Accordingly, after a synaptic vesicle docks, as a result of its vesicle membrane forming connections to multiple pins and ribs [20], each of the pins and proximal rib segments independently shortens and lengthens in dynamic equilibrium, while vesicle membrane -presynaptic membrane contact is maintained.…”

“…We searched for the structural correlates of priming among docked synaptic vesicles at frog neuromuscular junctions. We found in resting terminals that the extent of the vesicle membrane-presynaptic membrane contact area from docked synaptic vesicle to docked synaptic vesicle varies by more than an order of magnitude (from 50 to 650 nm 2 ) with a normal frequency distribution [26]. In muscles fixed during repetitive nerve stimulation, the frequency distribution of the vesicle membrane-presynaptic membrane contact areas was shifted to the left while from muscles fixed 1 h after such 2þ -triggered vesicle membrane-presynaptic membrane fusion.…”

“…Detailed methods for tissue preparation, sectioning and collection of electron microscopy data have been described elsewhere; we used the software package EM3D (www.em3d. org) for reconstruction of volumetric datasets and generation of virtual slices, segmentation and rendering 3D surface models [17,20,21,26]. Of particular importance for our studies was the magnification at which the data were collected; higher magnification results in better 3D spatial resolution.…”

“…B 370: 20140189 localize proteins known from biochemistry to be involved in the docking of synaptic vesicles to specific classes of AZM macromolecules. We have discussed elsewhere [20,21,26] how certain proteins shown to be involved with docking might be distributed among the AZM macromolecules. Of particular interest here is the location of the vesicle membrane proteins synaptobrevin and synaptotagmin and the presynaptic membrane proteins syntaxin and SNAP-25.…”

Synaptic Homeostasis and Its Immunological Disturbance in Neuromuscular Junction Disorders

“…This signaling leads to presynaptic differentiation to localize active zone proteins, synaptic vesicle proteins and Ca 2+ channels at the nerve terminal, thereby conditioning the release-ready ACh-containing vesicles [55,56,57,58,59]. Besides the Wnts canonical signaling, muscle-derived Lrp4 interacts with an Lrp4-binding protein in motor neurons and acts as a retrograde signal to promote presynaptic differentiation [60,61] (left upper part of Figure 1).…”