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

Abstract

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 distinct classes of AZM macromolecules positioned at different distances from the membrane. Establishing the number, arrangement and sequence of association of AZM macromolecules involved in vesicle docking provides an anatomical basis for testing and extending concepts of docking mechanisms provided by biochemistry.

Numbers of presynaptic Ca 2+ channel clusters match those of functionally defined vesicular docking sites in single central synapses

“…Another remaining source of uncertainty concerns SV supply. Docking-site replenishment is a Ca 2+ -driven process (3) that depends on filament-like links between SVs and plasma membrane (20,24,56,57), possibly involving actin and myosin II (58)(59)(60)(61). In PF-MLI synapses, recent results indicate that an empty docking site is resupplied by a replacement SV that moves toward the docking site by a cytoskeleton-driven transition (twostep model) (29).…”

“…junctions, SVs dock externally to the presumed VGCCs, forming two parallel and external rows of docking sites (19,20) whereas in the mouse they dock between the two rows of VGCCs, forming a single central line of docking sites (21); in each case, specific cytoskeletal elements link SVs, VGCCs, and plasma membrane together. Interestingly, in the mouse neuromuscular junction, estimates for the number of docking sites located inside the two rows of VGCCs and for the size of the readily releasable pool of SVs coincide at two per active zone (22).…”

Variable priming of a docked synaptic vesicle

“…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.…”

Release of chemical transmitters from cell bodies and dendrites of nerve cells

“…For example, the time course and magnitude of transmitter release have been measured with extraordinarily high resolution in motoneuron terminals, thanks to the occurrence of miniature endplate potentials and by the measurement of ion currents that occur in the receptors of the postsynaptic muscle membrane [15]. Thus, reliable estimates have been made of the number of acetylcholine molecules in a small (40-50 nm diameter) clear vesicle [16], the timing of calcium in the presynaptic membrane [17], the molecular mechanisms for docking and fusion [18,19] and the number of vesicles that release transmitter per impulse [20]. A major advance is that one can observe presynaptic steps in the release mechanism by electron microscopic tomography [21].…”