I began by examining by electron tomography the relationship of the membrane of docked vesicles to the presynaptic membrane in tissue sections from active zones of resting frog and mouse neuromuscular junctions fixed in aldehyde and stained with heavy metals with the aim of determining its constancy. Because the relationship could not be readily determined by eye owing to staining irregularities and other factors, I devised an averaging method for measuring the distance from the external surface of the presynaptic membrane to the luminal surface of the vesicle membrane for 64 docked frog vesicles and 23 docked mouse vesicles. The results were the same for all vesicles. The distance across the membrane at the site of contact of docked vesicle was similar to the sum of the distances across the presynaptic membrane and the vesicle membrane away from the region of apposition. Given the spatial resolution for membranes in my data sets I conclude that for most, if not all, of the docked vesicles in frog and mouse neuromuscular junctions the external surface of the vesicle membrane is in close apposition with, i.e. within 2-3 nm from, the cytoplasmic surface of the presynaptic membrane. Next, I measured the size of the area of close apposition between the vesicle membrane and the presynaptic membrane. For frog it ranged from ∼150nm2 to ∼650nm2; for mouse it ranged from ∼150 nm2 to ∼650 nm 2, while the proximity between docked vesicles and presynaptic membrane is constant, the size of the area of such close apposition is highly variable. I found in axon terminals of frog neuromuscular junctions that had been fixed during tetanic electrical stimulation that docked vesicles having large areas of close apposition were absent and that for those vesicles that were hemifused with the presysynaptic membrane the size of the area of membrane interaction was similar to the largest areas of close apposition of docked vesicles at rest. These findings indicate that at resting neuromuscular junctions there are vesicles in different stages of docking: docked vesicles having a small area of close apposition to the presynaptic membrane are in early stages of docking while docked vesicles having a large area of contact are in later stages that will lead to the fusion their membrane with the presynaptic membrane during synaptic activity.
Next I participated in electron tomography experiments aimed at extending studies on the structure of active zone material and its relationship to docked vesicles. Previous studies had shown that within 15 nm of the presynaptic membrane the active zone material is composed of three sets of elongate macromolecules called ribs, beams and pegs. Beams are connected to beams and ribs, ribs are connected to docked vesicles and pegs and pegs are connected to macromolecules in the presynaptic membrane including calcium channels. The study done by my colleagues and me showed that deep to the ribs there were two additional sets of macromolecules connected to docked vesicles: spars and booms. Spars were more superficial than booms and both arose from macromolecules vertically linked to beams. We also observed structures linking the docked vesicles to the presynaptic membrane called pins.
When I measured the length of ribs from docked vesicles to their attachment to those pegs proximal to the vesicles, I found that the length was less for docked vesicles having large areas of close apposition with the presynaptic membrane than for docked vesicles having small areas of close apposition. This indicates that ribs shorten during increasing stages of docking. I also obtained evidence for a similar shortening of pins during increasing stages of docking. I found that the distance between those pegs proximal to the docked vesicles and those pegs distal to the vesicles was greater for docked vesicles having large areas of close apposition than for docked vesicles having small areas of close apposition. Thus the shortening of ribs and pins may lead to the large areas of close apposition between the vesicle membrane and presynaptic membrane during docking, while the shortening of ribs may also lead to the movement of the proximal pegs with their attached presynaptic membrane macromolecules, which very likely include calcium channels that are involved in triggering fusion during synaptic transmission, toward the vesicles.
In sum, my results show that different stages in the docking and fusion of synaptic vesicle membrane with presynaptic membrane are regulated by orderly physical changes in the synaptic vesicle (shape) and in specific macromolecules of active zone material (length and position). The identification of these changes provides a physical basis for advancing our understanding of the biochemical mechanisms that underlie synaptic transmission.
|School Location:||United States -- California|
|Source:||DAI-B 70/10, Dissertation Abstracts International|
|Keywords:||Docked vesicles, Macromolecules, Neuromuscular junction, Presynaptic, Synaptic transmission, Synaptic vesicle|
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