Correction: ELKS1 localizes the synaptic vesicle priming protein bMunc13-2 to a specific subset of active zones

Presynaptic active zones (AZs) are unique subcellular structures at neuronal synapses, which contain a network of specific proteins that control synaptic vesicle (SV) tethering, priming, and fusion. Munc13s are core AZ proteins with an essential function in SV priming. In hippocampal neurons, two different Munc13s—Munc13-1 and bMunc13-2—mediate opposite forms of presynaptic short-term plasticity and thus differentially affect neuronal network characteristics. We found that most presynapses of cortical and hippocampal neurons contain only Munc13-1, whereas ∼10% contain both Munc13-1 and bMunc13-2. Whereas the presynaptic recruitment and activation of Munc13-1 depends on Rab3-interacting proteins (RIMs), we demonstrate here that bMunc13-2 is recruited to synapses by the AZ protein ELKS1, but not ELKS2, and that this recruitment determines basal SV priming and short-term plasticity. Thus, synapse-specific interactions of different Munc13 isoforms with ELKS1 or RIMs are key determinants of the molecular and functional heterogeneity of presynaptic AZs.

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Partial purification of presynaptic plasma membrane by immunoadsorption.

The Journal of Cell Biology ◽

10.1083/jcb.94.1.88 ◽

1982 ◽

Vol 94 (1) ◽

pp. 88-96 ◽

Cited By ~ 23

Author(s):

G P Miljanich ◽

A R Brasier ◽

R B Kelly

Keyword(s):

Plasma Membrane ◽

Synaptic Vesicle ◽

Nerve Terminal ◽

Specific Activity ◽

Electric Organ ◽

Partial Purification ◽

Vesicle Membrane ◽

Specific Activities ◽

Active Zones ◽

Presynaptic Plasma Membrane

During transmitter release, synaptic vesicle membrane is specifically inserted into the nerve terminal plasma membrane only at specialized sites or "active zones." In an attempt to obtain a membrane fraction enriched in active zones, we have utilized the electric organ of the marine ray. From this organ, a fraction enriched in nerve terminals (synaptosomes) was prepared by conventional means. These synaptosomes were bound to microscopic beads by an antiserum to purified electric organ synaptic vesicles (anti-SV). The success of this immunoadsorption procedure was demonstrated by increased specific activities of bead-bound nerve terminal cytoplasmic markers and decreased specific activities of markers for contaminating membranes. To obtain a presynaptic plasma membrane (PSPM) fraction, we lysed the bead-bound synaptosomes by hypoosmotic shock and sonication, resulting in complete release of cytoplasmic markers. When the synaptosomal fraction was surface-labeled with iodine before immunoadsorption, 10% of this label remained bead-bound after lysis, compared with 2% of the total protein, indicating an approximately fivefold enrichment of bead-bound plasma membrane. Concomitantly, the specific activity of bead-bound anti-SV increased approximately 30-fold, indicating an enrichment of plasma membrane which contained inserted synaptic vesicle components. This PSPM preparation is not simply synaptic vesicle membrane since two-dimensional electrophoresis revealed that the polypeptides of the surface-iodinated PSPM preparation include both vesicle and numerous nonvesicle components. Secondly, antiserum to the PSPM fraction is markedly different from anti-SV and binds to external, nonvesicle, nerve terminal components.

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Diurnal Changes in Exocytosis and the Number of Synaptic Ribbons at Active Zones of an on-Type Bipolar Cell Terminal

Journal of Neurophysiology ◽

10.1152/jn.00364.2006 ◽

2006 ◽

Vol 96 (4) ◽

pp. 2025-2033 ◽

Cited By ~ 50

Author(s):

Court Hull ◽

Keith Studholme ◽

Stephen Yazulla ◽

Henrique von Gersdorff

Keyword(s):

Synaptic Vesicle ◽

Bipolar Cell ◽

Bipolar Cells ◽

Synaptic Ribbons ◽

Diurnal Changes ◽

Synaptic Vesicle Exocytosis ◽

Pulse Ratio ◽

Vesicle Exocytosis ◽

Active Zones ◽

Vesicle Pool

The number and morphology of synaptic ribbons at photoreceptor and bipolar cell terminals has been reported to change on a circadian cycle. Here we sought to determine whether this phenomenon exists at goldfish Mb-type bipolar cell terminals with the aim of exploring the role of ribbons in transmitter release. We examined the physiology and ultrastructure of this terminal around two time points: midday and midnight. Nystatin perforated-patch recordings of membrane capacitance ( Cm) revealed that synaptic vesicle exocytosis evoked by short depolarizations was reduced at night, even though Ca2+ currents were larger. The efficiency of exocytosis (measured as the Δ Cm jump per total Ca2+ charge influx) was thus significantly lower at night. The paired-pulse ratio remained unchanged, however, suggesting that release probability was not altered. Hence the decreased exocytosis likely reflects a smaller readily releasable vesicle pool at night. Electron microscopy of single sections from intact retinas averaged 65% fewer ribbons at night. Interestingly, the number of active zones did not change from day to night, only the probability of finding a ribbon at an active zone. Additionally, synaptic vesicle halos surrounding the ribbons were more completely filled at night when these on-type bipolar cells are more hyperpolarized. There was no change, however, in the physical dimensions of synaptic ribbons from day to night. These results suggest that the size of the readily releasable vesicle pool and the efficiency of exocytosis are reduced at night when fewer ribbons are present at bipolar cell terminal active zones.

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Endocytosis of synaptic vesicle membrane at the frog neuromuscular junction.

The Journal of Cell Biology ◽

10.1083/jcb.98.2.685 ◽

1984 ◽

Vol 98 (2) ◽

pp. 685-698 ◽

Cited By ~ 190

Author(s):

T M Miller ◽

J E Heuser

Keyword(s):

Plasma Membrane ◽

Synaptic Vesicle ◽

Thin Section ◽

Nerve Terminal ◽

Motor Nerve ◽

Freeze Fracture ◽

Frog Neuromuscular Junction ◽

Vesicle Membrane ◽

Coated Pits ◽

Active Zones

Frog nerve-muscle preparations were quick-frozen at various times after a single electrical stimulus in the presence of 4-aminopyridine (4-AP), after which motor nerve terminals were visualized by freeze-fracture. Previous studies have shown that such stimulation causes prompt discharge of 3,000-6,000 synaptic vesicles from each nerve terminal and, as a result, adds a large amount of synaptic vesicle membrane to its plasmalemma. In the current experiments, we sought to visualize the endocytic retrieval of this vesicle membrane back into the terminal, during the interval between 1 s and 2 min after stimulation. Two distinct types of endocytosis were observed. The first appeared to be rapid and nonselective. Within the first few seconds after stimulation, relatively large vacuoles (approximately 0.1 micron) pinched off from the plasma membrane, both near to and far away from the active zones. Previous thin-section studies have shown that such vacuoles are not coated with clathrin at any stage during their formation. The second endocytic process was slower and appeared to be selective, because it internalized large intramembrane particles. This process was manifest first by the formation of relatively small (approximately 0.05 micron) indentations in the plasma membrane, which occurred everywhere except at the active zones. These indentations first appeared at 1 s, reached a peak abundance of 5.5/micron2 by 30 s after the stimulus, and disappeared almost completely by 90 s. Previous thin-section studies indicate that these indentations correspond to clathrin-coated pits. Their total abundance is comparable with the number of vesicles that were discharged initially. These endocytic structures could be classified into four intermediate forms, whose relative abundance over time suggests that, at this type of nerve terminal, endocytosis of coated vesicles has the following characteristics: (a) the single endocytotic event is short lived relative to the time scale of two minutes; (b) earlier forms last longer than later forms; and (c) a single event spends a smaller portion of its lifetime in the flat configuration soon after the stimulus than it does later on.

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A stochastic model of active zone material mediated synaptic vesicle docking and priming at resting active zones

Scientific Reports ◽

10.1038/s41598-017-00360-z ◽

2017 ◽

Vol 7 (1) ◽

Cited By ~ 2

Author(s):

Jae Hoon Jung ◽

Sebatian Doniach

Keyword(s):

Stochastic Model ◽

Synaptic Vesicle ◽

Active Zone ◽

Vesicle Docking ◽

Active Zones

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Structural changes after transmitter release at the frog neuromuscular junction.

The Journal of Cell Biology ◽

10.1083/jcb.88.3.564 ◽

1981 ◽

Vol 88 (3) ◽

pp. 564-580 ◽

Cited By ~ 309

Author(s):

J E Heuser ◽

T S Reese

Keyword(s):

Plasma Membrane ◽

Synaptic Vesicle ◽

Structural Changes ◽

Frog Neuromuscular Junction ◽

Vesicle Membrane ◽

Intramembrane Particles ◽

Synaptic Vesicle Exocytosis ◽

Quick Freezing ◽

Vesicle Exocytosis ◽

Active Zones

The sequence of structural changes that occur during synaptic vesicle exocytosis was studied by quick-freezing muscles at different intervals after stimulating their nerves, in the presence of 4-aminopyridine to increase the number of transmitter quanta released by each stimulus. Vesicle openings began to appear at the active zones of the intramuscular nerves within 3-4 ms after a single stimulus. The concentration of these openings peaked at 5-6 ms, and then declined to zero 50-100 ms late. At the later times, vesicle openings tended to be larger. Left behind at the active zones, after the vesicle openings disappeared, were clusters of large intramembrane particles. The larger particles in these clusters were the same size as intramembrane particles in undischarged vesicles, and were slightly larger than the particles which form the rows delineating active zones. Because previous tracer work had shown that new vesicles do not pinch off from the plasma membrane at these early times, we concluded that the particle clusters originate from membranes of discharged vesicles which collapse into the plasmalemma after exocytosis. The rate of vesicle collapse appeared to be variable because different stages occurred simultaneously at most times after stimulation; this asynchrony was taken to indicate that the collapse of each exocytotic vesicle is slowed by previous nearby collapses. The ultimate fate of synaptic vesicle membrane after collapse appeared to be coalescence with the plasma membrane, as the clusters of particles gradually dispersed into surrounding areas during the first second after a stimulus. The membrane retrieval and recycling that reverse this exocytotic sequence have a slower onset, as has been described in previous reports.

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Separation of presynaptic Cav2 and Cav1 channel function in synaptic vesicle exo- and endocytosis by the membrane anchored Ca2+ pump PMCA

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2106621118 ◽

2021 ◽

Vol 118 (28) ◽

pp. e2106621118

Author(s):

Niklas Krick ◽

Stefanie Ryglewski ◽

Aylin Pichler ◽

Arthur Bikbaev ◽

Torsten Götz ◽

...

Keyword(s):

Synaptic Vesicle ◽

Neurotransmitter Release ◽

Calcium Currents ◽

Short Term ◽

Triggered Release ◽

Presynaptic Terminals ◽

Dynamic Regulation ◽

Short Term Plasticity ◽

Separate Control ◽

Active Zones

Synaptic vesicle (SV) release, recycling, and plastic changes of release probability co-occur side by side within nerve terminals and rely on local Ca2+ signals with different temporal and spatial profiles. The mechanisms that guarantee separate regulation of these vital presynaptic functions during action potential (AP)–triggered presynaptic Ca2+ entry remain unclear. Combining Drosophila genetics with electrophysiology and imaging reveals the localization of two different voltage-gated calcium channels at the presynaptic terminals of glutamatergic neuromuscular synapses (the Drosophila Cav2 homolog, Dmca1A or cacophony, and the Cav1 homolog, Dmca1D) but with spatial and functional separation. Cav2 within active zones is required for AP-triggered neurotransmitter release. By contrast, Cav1 localizes predominantly around active zones and contributes substantially to AP-evoked Ca2+ influx but has a small impact on release. Instead, L-type calcium currents through Cav1 fine-tune short-term plasticity and facilitate SV recycling. Separate control of SV exo- and endocytosis by AP-triggered presynaptic Ca2+ influx through different channels demands efficient measures to protect the neurotransmitter release machinery against Cav1-mediated Ca2+ influx. We show that the plasma membrane Ca2+ ATPase (PMCA) resides in between active zones and isolates Cav2-triggered release from Cav1-mediated dynamic regulation of recycling and short-term plasticity, two processes which Cav2 may also contribute to. As L-type Cav1 channels also localize next to PQ-type Cav2 channels within axon terminals of some central mammalian synapses, we propose that Cav2, Cav1, and PMCA act as a conserved functional triad that enables separate control of SV release and recycling rates in presynaptic terminals.

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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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The Morphological and Molecular Nature of Synaptic Vesicle Priming at Presynaptic Active Zones

Neuron ◽

10.1016/j.neuron.2014.11.003 ◽

2014 ◽

Vol 84 (4) ◽

pp. 882 ◽

Cited By ~ 1

Author(s):

Cordelia Imig ◽

Sang-Won Min ◽

Stefanie Krinner ◽

Marife Arancillo ◽

Christian Rosenmund ◽

...

Keyword(s):

Synaptic Vesicle ◽

Active Zones ◽

Molecular Nature

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(M)Unc13s in Active Zone Diversity: A Drosophila Perspective

Frontiers in Synaptic Neuroscience ◽

10.3389/fnsyn.2021.798204 ◽

2022 ◽

Vol 13 ◽

Author(s):

Chengji Piao ◽

Stephan J. Sigrist

Keyword(s):

Synaptic Vesicle ◽

Active Zone ◽

Stoichiometric Composition ◽

Release Site ◽

Homeostatic Plasticity ◽

Release Factor ◽

Loosely Coupled ◽

Action Potential Frequency ◽

Active Zones ◽

Potential Frequency

The so-called active zones at pre-synaptic terminals are the ultimate filtering devices, which couple between action potential frequency and shape, and the information transferred to the post-synaptic neurons, finally tuning behaviors. Within active zones, the release of the synaptic vesicle operates from specialized “release sites.” The (M)Unc13 class of proteins is meant to define release sites topologically and biochemically, and diversity between Unc13-type release factor isoforms is suspected to steer diversity at active zones. The two major Unc13-type isoforms, namely, Unc13A and Unc13B, have recently been described from the molecular to the behavioral level, exploiting Drosophila being uniquely suited to causally link between these levels. The exact nanoscale distribution of voltage-gated Ca2+ channels relative to release sites (“coupling”) at pre-synaptic active zones fundamentally steers the release of the synaptic vesicle. Unc13A and B were found to be either tightly or loosely coupled across Drosophila synapses. In this review, we reported recent findings on diverse aspects of Drosophila Unc13A and B, importantly, their nano-topological distribution at active zones and their roles in release site generation, active zone assembly, and pre-synaptic homeostatic plasticity. We compared their stoichiometric composition at different synapse types, reviewing the correlation between nanoscale distribution of these two isoforms and release physiology and, finally, discuss how isoform-specific release components might drive the functional heterogeneity of synapses and encode discrete behavior.

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