Munc13

A key feature of excitatory synapses is the existence of subsynaptic protein nanoclusters (NCs) whose precise alignment across the cleft in a transsynaptic nanocolumn influences the strength of synaptic transmission. However, whether nanocolumn properties vary between excitatory synapses functioning in different cellular contexts is unknown. We used a combination of confocal and DNA-PAINT super-resolution microscopy to directly compare the organization of shared scaffold proteins at two important excitatory synapses-those forming onto excitatory principal neurons (Ex→Ex synapses) and those forming onto parvalbumin-expressing interneurons (Ex→PV synapses)...

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Regulation of neurotransmitter release during presynaptic plasticity underlies varied forms of information processing in the brain. Munc13s play essential roles in release via their conserved C-terminal region, which contains a MUN domain involved SNARE complex assembly, and control multiple presynaptic plasticity processes. Munc13s also have a variable N-terminal region, which in Munc13-1 includes a calmodulin binding (CaMb) domain involved in short-term plasticity and a C2 A domain that forms an inhibitory homodimer...

Regulation of neurotransmitter release during presynaptic plasticity underlies varied forms of information processing in the brain. Munc13s play essential roles in release via their conserved C-terminal region, which contains a MUN domain involved SNARE complex assembly, and control multiple presynaptic plasticity processes. Munc13s also have a variable N-terminal region, which in Munc13-1 includes a calmodulin binding (CaMb) domain involved in short-term plasticity and a C 2 A domain that forms an inhibitory homodimer...

Familial hemophagocytic lymphohistiocytosis (FHL) is an inherited, often fatal immune deficiency characterized by severe systemic hyperinflammation. Although allogeneic bone marrow transplantation can be curative, more effective therapies are urgently needed. FHL is caused by inactivating mutations in proteins that regulate cellular immunity. Here, we used an adeno-associated virus-based CRISPR-Cas9 system with an inhibitor of nonhomologous end joining to repair such mutations in potentially long-lived T cells ex vivo...

UNLABELLED: Nanoscale protein organization within the active zone (AZ) and post-synaptic density (PSD) influences synaptic transmission. Nanoclusters of presynaptic Munc13-1 are associated with readily releasable pool size and neurotransmitter vesicle priming, while postsynaptic PSD-95 nanoclusters coordinate glutamate receptors across from release sites to control their opening probability. Nanocluster number, size, and protein density vary between synapse types and with development and plasticity, supporting a wide range of functional states at the synapse...

Tight coordination of the spatial relationships between protein complexes is required for cellular function. In neuronal synapses, many proteins responsible for neurotransmission organize into subsynaptic nanoclusters whose trans-cellular alignment modulates synaptic signal propagation. However, the spatial relationships between these proteins and NMDA receptors (NMDARs), which are required for learning and memory, remain undefined. Here, we mapped the relationship of key NMDAR subunits to reference proteins in the active zone and postsynaptic density using multiplexed super-resolution DNA-PAINT microscopy...

Neurotransmitters are released from synaptic vesicles, the membrane of which fuses with the plasma membrane upon calcium influx. This membrane fusion reaction is driven by the formation of a tight complex comprising the plasma membrane SNARE proteins syntaxin-1a and SNAP-25 with the vesicle SNARE protein synaptobrevin. The neuronal protein Munc18-1 forms a stable complex with syntaxin-1a. Biochemically, syntaxin-1a cannot escape the tight grip of Munc18-1, so formation of the SNARE complex is inhibited. However, Munc18-1 is essential for the release of neurotransmitters in vivo...

Action potentials trigger neurotransmitter release with minimal delay. Active zones mediate this temporal precision by co-organizing primed vesicles with Ca V 2 Ca 2+ channels. The presumed model is that scaffolding proteins directly tether primed vesicles to Ca V 2s. We find that Ca V 2 clustering and vesicle priming are executed by separate machineries. At hippocampal synapses, Ca V 2 nanoclusters are positioned at variable distances from those of the priming protein Munc13. The active zone organizer RIM anchors both proteins, but distinct interaction motifs independently execute these functions...

Munc13-1 is essential for vesicle docking and fusion at the active zone of synapses. Here, we report that Munc13-1 self-assembles into molecular clusters within diacylglycerol-rich microdomains present in phospholipid bilayers. Although the copy number of Munc13-1 molecules in these clusters has a broad distribution, a systematic Poisson analysis shows that this is most likely the result of two molecular species: monomers and mainly hexameric oligomers. Each oligomer is able to capture one vesicle independently...

A key feature of excitatory synapses is the existence of subsynaptic protein nanoclusters whose precise alignment across the cleft in a trans-synaptic nanocolumn influences the strength of synaptic transmission. However, whether nanocolumn properties vary between excitatory synapses functioning in different cellular contexts is unknown. We used a combination of confocal and DNA-PAINT super-resolution microscopy to directly compare the organization of shared scaffold proteins at two important excitatory synapses - those forming onto excitatory principal neurons (Ex→Ex synapses) and those forming onto parvalbumin-expressing interneurons (Ex→PV synapses)...

The distance between CaV 2.1 voltage-gated Ca2+ channels and the Ca2+ sensor responsible for vesicle release at presynaptic terminals is critical for determining synaptic strength. Yet, the molecular mechanisms responsible for a loose coupling configuration of CaV 2.1 in certain synapses or developmental periods and a tight one in others remain unknown. Here, we examine the nanoscale organization of two CaV 2.1 splice isoforms (CaV 2.1[EFa] and CaV 2.1[EFb]) at presynaptic terminals by superresolution structured illumination microscopy...

Munc13-1 is a key protein necessary for vesicle fusion and neurotransmitter release in the brain. Diacylglycerol (DAG)/phorbol ester binds to its C1 domain in the plasma membrane and activates it. The C1 domain of Munc13-1 and protein kinase C (PKC) are homologous in terms of sequence and structure. In order to identify small-molecule modulators of Munc13-1 targeting the C1 domain, we studied the effect of three DAG-lactones, ( R,Z )-(2-(hydroxymethyl)-4-(3-isobutyl-5-methylhexylidene)-5-oxotetrahydrofuran-2-yl)methyl pivalate (JH-131e-153), ( E )-(2-(hydroxymethyl)-4-(3-isobutyl-5-methylhexylidene)-5-oxotetrahydrofuran-2-yl)methyl pivalate (AJH-836), and ( E )-(2-(hydroxymethyl)-4-(4-nitrobenzylidene)-5-oxotetrahydrofuran-2-yl)methyl 4-(dimethylamino)benzoate (130C037), on Munc13-1 activation using the ligand-induced membrane translocation assay...

Evidence from biochemistry, genetics, and electron microscopy strongly supports the idea that a ring of Synaptotagmin contributes importantly to clamping and release of synaptic vesicles for synchronous neurotransmission. Recent direct measurements in cell-free systems suggest there are 12 SNAREpins in each ready-release vesicle, consisting of 6 peripheral and 6 central SNAREpins. The 6 central SNAREpins are directly bound to the Synaptotagmin ring, are directly released by Ca++ , and they initially open the fusion pore...

Neurotransmitters are released from synaptic and secretory vesicles following calcium-triggered fusion with the plasma membrane. These exocytotic events are driven by assembly of a ternary SNARE complex between the vesicle SNARE synaptobrevin and the plasma membrane-associated SNAREs syntaxin and SNAP-25. Proteins that affect SNARE complex assembly are therefore important regulators of synaptic strength. In this chapter, we review our current understanding of the roles played by two SNARE interacting proteins: UNC-13/Munc13 and UNC-18/Munc18...

Neurotransmitters are stored in small membrane-bound vesicles at synapses; a subset of synaptic vesicles is docked at release sites. Fusion of docked vesicles with the plasma membrane releases neurotransmitters. Membrane fusion at synapses, as well as all trafficking steps of the secretory pathway, is mediated by SNARE proteins. The SNAREs are the minimal fusion machinery. They zipper from N-termini to membrane-anchored C-termini to form a 4-helix bundle that forces the apposed membranes to fuse. At synapses, the SNAREs comprise a single helix from syntaxin and synaptobrevin; SNAP-25 contributes the other two helices to complete the bundle...

The Cytomatrix Assembled at the active Zone (CAZ) of a presynaptic terminal displays electron-dense appearance and defines the center of the synaptic vesicle release. The protein constituents of CAZ are multiple-domain scaffolds that interact extensively with each other and also with an ensemble of synaptic vesicle proteins to ensure docking, fusion, and recycling. Reflecting the central roles of the active zone in synaptic transmission, CAZ proteins are highly conserved throughout evolution. As the nervous system increases complexity and diversity in types of neurons and synapses, CAZ proteins expand in the number of gene and protein isoforms and interacting partners...

Here, we introduce the full functional reconstitution of genetically validated core protein machinery (SNAREs, Munc13, Munc18, Synaptotagmin, and Complexin) for synaptic vesicle priming and release in a geometry that enables detailed characterization of the fate of docked vesicles both before and after release is triggered with Ca2+ . Using this setup, we identify new roles for diacylglycerol (DAG) in regulating vesicle priming and Ca2+ -triggered release involving the SNARE assembly chaperone Munc13. We find that low concentrations of DAG profoundly accelerate the rate of Ca2+ -dependent release, and high concentrations reduce clamping and permit extensive spontaneous release...

Dendritic spines are crucial for excitatory synaptic transmission as the size of a spine head correlates with the strength of its synapse. The distribution of spine head sizes follows a lognormal-like distribution with more small spines than large ones. We analysed the impact of synaptic activity and plasticity on the spine size distribution in adult-born hippocampal granule cells from rats with induced homo- and heterosynaptic long-term plasticity in vivo and CA1 pyramidal cells from Munc13-1/Munc13-2 knockout mice with completely blocked synaptic transmission...

The critical presynaptic protein Munc13 serves numerous roles in the process of docking and priming synaptic vesicles. Here we investigate the functional significance of two distinct oligomers of the Munc13 core domain (Munc13C) comprising C1-C2B-MUN-C2C. Oligomer interface point mutations that specifically destabilized either the trimer or lateral hexamer assemblies of Munc13C disrupted vesicle docking, trans-SNARE formation, and Ca 2+ -triggered vesicle fusion in vitro and impaired neurotransmitter secretion and motor nervous system function in vivo...