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. The results support the conclusion that AZM directs the docking and priming of synaptic vesicles and essential positioning of Ca 2+ -channels relative to the vesicles' Ca 2+ -sensors. Here we review the findings and comment on their applicability to understanding mechanisms of docking, priming and Ca 2+ -triggering at other synapses, where the arrangement of active zone components differs.

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Hypothesis Relating the Structure, Biochemistry and Function of Active Zone Material Macromolecules at a Neuromuscular Junction

Frontiers in Synaptic Neuroscience ◽

10.3389/fnsyn.2021.798225 ◽

2022 ◽

Vol 13 ◽

Author(s):

Joseph A. Szule

Keyword(s):

Membrane Fusion ◽

Synaptic Vesicle ◽

Active Zone ◽

Structural Integrity ◽

Neuromuscular Junctions ◽

Vesicle Trafficking ◽

Material Structure ◽

Neurotransmitter Secretion ◽

Active Zones ◽

And Function

This report integrates knowledge of in situ macromolecular structures and synaptic protein biochemistry to propose a unified hypothesis for the regulation of certain vesicle trafficking events (i.e., docking, priming, Ca2+-triggering, and membrane fusion) that lead to neurotransmitter secretion from specialized “active zones” of presynaptic axon terminals. Advancements in electron tomography, to image tissue sections in 3D at nanometer scale resolution, have led to structural characterizations of a network of different classes of macromolecules at the active zone, called “Active Zone Material’. At frog neuromuscular junctions, the classes of Active Zone Material macromolecules “top-masts”, “booms”, “spars”, “ribs” and “pins” direct synaptic vesicle docking while “pins”, “ribs” and “pegs” regulate priming to influence Ca2+-triggering and membrane fusion. Other classes, “beams”, “steps”, “masts”, and “synaptic vesicle luminal filaments’ likely help organize and maintain the structural integrity of active zones. Extensive studies on the biochemistry that regulates secretion have led to comprehensive characterizations of the many conserved proteins universally involved in these trafficking events. Here, a hypothesis including a partial proteomic atlas of Active Zone Material is presented which considers the common roles, binding partners, physical features/structure, and relative positioning in the axon terminal of both the proteins and classes of macromolecules involved in the vesicle trafficking events. The hypothesis designates voltage-gated Ca2+ channels and Ca2+-gated K+ channels to ribs and pegs that are connected to macromolecules that span the presynaptic membrane at the active zone. SNARE proteins (Syntaxin, SNAP25, and Synaptobrevin), SNARE-interacting proteins Synaptotagmin, Munc13, Munc18, Complexin, and NSF are designated to ribs and/or pins. Rab3A and Rabphillin-3A are designated to top-masts and/or booms and/or spars. RIM, Bassoon, and Piccolo are designated to beams, steps, masts, ribs, spars, booms, and top-masts. Spectrin is designated to beams. Lastly, the luminal portions of SV2 are thought to form the bulk of the observed synaptic vesicle luminal filaments. The goal here is to help direct future studies that aim to bridge Active Zone Material structure, biochemistry, and function to ultimately determine how it regulates the trafficking events in vivo that lead to neurotransmitter secretion.

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

The Journal of Comparative Neurology ◽

10.1002/cne.21975 ◽

2009 ◽

Vol 513 (5) ◽

pp. 457-468 ◽

Cited By ~ 64

Author(s):

Sharuna Nagwaney ◽

Mark Lee Harlow ◽

Jae Hoon Jung ◽

Joseph A. Szule ◽

David Ress ◽

...

Keyword(s):

Active Zone ◽

Synaptic Vesicles ◽

Neuromuscular Junctions ◽

Presynaptic Membrane

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A presynaptic spectrin network controls active zone assembly and neurotransmitter release

10.1101/812032 ◽

2019 ◽

Author(s):

Qi Wang ◽

Lindsey Friend ◽

Rosario Vicidomini ◽

Tae Hee Han ◽

Peter Nguyen ◽

...

Keyword(s):

Active Zone ◽

Neurotransmitter Release ◽

Synaptic Cleft ◽

Structure And Function ◽

Dynamic Changes ◽

Synaptic Structure ◽

Synaptic Boutons ◽

Active Zones ◽

And Function ◽

Spectrin Network

ABSTRACTWe have previously reported that Drosophila Tenectin (Tnc) recruits αPS2/βPS integrin to ensure structural and functional integrity at larval NMJs (Wang et al., 2018). In muscles, Tnc/integrin engages the spectrin network to regulate the size and architecture of synaptic boutons. In neurons, Tnc/integrin controls neurotransmitter release. Here we show that presynaptic Tnc/integrin modulates the synaptic accumulation of key active zone components, including the Ca2+ channel Cac and the active zone scaffold Brp. Presynaptic α-Spectrin appears to be both required and sufficient for the recruitment of Cac and Brp. We visualized the endogenous α-Spectrin and found that Tnc controls spectrin recruitment at synaptic terminals. Thus, Tnc/integrin anchors the presynaptic spectrin network and ensures the proper assembly and function of the active zones. Since pre- and postsynaptic Tnc/integrin limit each other, we hypothesize that this pathway links dynamic changes within the synaptic cleft to changes in synaptic structure and function.

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Synaptic Vesicles Having Large Contact Areas with the Presynaptic Membrane are Preferentially Hemifused at Active Zones of Frog Neuromuscular Junctions Fixed during Synaptic Activity

International Journal of Molecular Sciences ◽

10.3390/ijms20112692 ◽

2019 ◽

Vol 20 (11) ◽

pp. 2692

Author(s):

Jae Hoon Jung

Keyword(s):

Synaptic Vesicles ◽

Neuromuscular Junctions ◽

Synaptic Activity ◽

Membrane Thickness ◽

Intermediate Step ◽

Presynaptic Membrane ◽

Vesicle Membrane ◽

Contact Areas ◽

Contact Sites ◽

Active Zones

Synaptic vesicles dock on the presynaptic plasma membrane of axon terminals and become ready to fuse with the presynaptic membrane or primed. Fusion of the vesicle membrane and presynaptic membrane results in the formation of a pore between the membranes, through which the vesicle’s neurotransmitter is released into the synaptic cleft. A recent electron tomography study on frog neuromuscular junctions fixed at rest showed that there is no discernible gap between or merging of the membrane of docked synaptic vesicles with the presynaptic membrane, however, the extent of the contact area between the membrane of docked synaptic vesicles and the presynaptic membrane varies 10-fold with a normal distribution. The study also showed that when the neuromuscular junctions are fixed during repetitive electrical nerve stimulation, the portion of large contact areas in the distribution is reduced compared to the portion of small contact areas, suggesting that docked synaptic vesicles with the largest contact areas are greatly primed to fuse with the membrane. Furthermore, the finding of several hemifused synaptic vesicles among the docked vesicles was briefly reported. Here, the spatial relationship of 81 synaptic vesicles with the presynaptic membrane at active zones of the neuromuscular junctions fixed during stimulation is described in detail. For the most of the vesicles, the combined thickness of each of their contact sites was not different from the sum of the membrane thicknesses of the vesicle membrane and presynaptic membrane, similar to the docked vesicles at active zones of the resting neuromuscular junctions. However, the combined membrane thickness of a small portion of the vesicles was considerably less than the sum of the membrane thicknesses, indicating that the membranes at their contact sites were fixed in a state of hemifusion. Moreover, the hemifused vesicles were found to have large contact areas with the presynaptic membrane. These findings support the recently proposed hypothesis that, at frog neuromuscular junctions, docked synaptic vesicles with the largest contact areas are most primed for fusion with the presynaptic membrane, and that hemifusion is a fusion intermediate step of the vesicle membrane with the presynaptic membrane for synaptic transmission.

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Synaptic vesicles and microtubules in frog motor endplates

Proceedings of the Royal Society of London Series B Biological Sciences ◽

10.1098/rspb.1978.0102 ◽

1978 ◽

Vol 203 (1152) ◽

pp. 219-227 ◽

Cited By ~ 21

Keyword(s):

Electron Microscopy ◽

Skeletal Muscle ◽

Small Proportion ◽

Active Zone ◽

Synaptic Vesicles ◽

Presynaptic Membrane ◽

Albumin Solution ◽

Motor Endplates ◽

Active Zones ◽

A New Technique

Motor endplates of the cutaneous pectoris skeletal muscle of the frog have been examined by electron microscopy using a new technique. This involves pretreatment with an albumin solution, followed by fixation with 4% unbuffered tetroxide. A small proportion of the endplate axonal ramifications show microtubules clothed in synaptic vesicles and focused on the presynaptic membrane, in particular on the active zones. The microtubules run in the presynaptic cytoplasm either parallel to or across the active zones. These two sets of microtubules cross each other at the active zones, which lie opposite the dips in the post-junctional folds. The possibility that the microtubules are involved in the translocation of synaptic vesicles to the active zone is discussed.

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Cryo-electron tomography of cells: connecting structure and function

Histochemistry and Cell Biology ◽

10.1007/s00418-008-0459-y ◽

2008 ◽

Vol 130 (2) ◽

pp. 185-196 ◽

Cited By ~ 72

Author(s):

Vladan Lučić ◽

Andrew Leis ◽

Wolfgang Baumeister

Keyword(s):

Electron Tomography ◽

Structure And Function ◽

Cryo Electron Tomography ◽

And Function

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Release and Recycling of the Readily Releasable Vesicle Population in a Synapse Possessing No Reserve Population

Journal of Neurophysiology ◽

10.1152/jn.01258.2006 ◽

2007 ◽

Vol 97 (6) ◽

pp. 4048-4057 ◽

Cited By ~ 3

Author(s):

J. H. Koenig ◽

Kazuo Ikeda

Keyword(s):

High Frequency ◽

Active Zone ◽

Neuromuscular Junctions ◽

Recycling Rate ◽

Spontaneous Release ◽

Electron Microscopic ◽

Single Action ◽

Active Zones ◽

Evoked Activity ◽

Microscopic Techniques

We previously demonstrated that the tergotrochanteral muscle (TTM) of Drosophila is innervated by unique synapses that possess a small readily releasable/recycling vesicle population (active zone population), but not the larger reserve vesicle population observed at other neuromuscular junctions in this animal. Using light and electron microscopic techniques and intracellular recording from the G1 muscle fiber of the TTM, the release and recycling characteristics of the readily releasable/recycling population were observed without any possible contribution from a reserve population. Our results indicate 1) the total number of vesicles in synapses presynaptic to the G1 fiber correlates with the total number of quanta that can be released onto this fiber; 2) the number of quanta released by a single action potential onto the G1 fiber is about one half the number of morphologically “docked” vesicles in active zones onto the G1, and this ratio decreases in a partially depleted state; 3) the recycling rate at 1-Hz stimulation, a frequency that does not cause any depression, is 0.24 recycled vesicle/active zone/s; and 4) normal-appearing spontaneous release occurs from the active zone vesicle population and, unlike synapses that possess a reserve population, the frequency of this release is reduced after high-frequency evoked activity.

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Molecular dissection of the photoreceptor ribbon synapse

The Journal of Cell Biology ◽

10.1083/jcb.200408157 ◽

2005 ◽

Vol 168 (5) ◽

pp. 825-836 ◽

Cited By ~ 263

Author(s):

Susanne tom Dieck ◽

Wilko D. Altrock ◽

Michael M. Kessels ◽

Britta Qualmann ◽

Hanna Regus ◽

...

Keyword(s):

Active Zone ◽

Direct Interaction ◽

Specific Protein ◽

Molecular Assembly ◽

Ribbon Synapse ◽

Molecular Dissection ◽

Presynaptic Membrane ◽

Retinal Photoreceptor ◽

And Function ◽

Α1 Subunit

The ribbon complex of retinal photoreceptor synapses represents a specialization of the cytomatrix at the active zone (CAZ) present at conventional synapses. In mice deficient for the CAZ protein Bassoon, ribbons are not anchored to the presynaptic membrane but float freely in the cytoplasm. Exploiting this phenotype, we dissected the molecular structure of the photoreceptor ribbon complex. Identifiable CAZ proteins segregate into two compartments at the ribbon: a ribbon-associated compartment including Piccolo, RIBEYE, CtBP1/BARS, RIM1, and the motor protein KIF3A, and an active zone compartment including RIM2, Munc13-1, a Ca2+ channel α1 subunit, and ERC2/CAST1. A direct interaction between the ribbon-specific protein RIBEYE and Bassoon seems to link the two compartments and is responsible for the physical integrity of the photoreceptor ribbon complex. Finally, we found the RIBEYE homologue CtBP1 at ribbon and conventional synapses, suggesting a novel role for the CtBP/BARS family in the molecular assembly and function of central nervous system synapses.

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