Diacylglycerol-dependent hexamers of the SNARE-assembling chaperone Munc13-1 cooperatively bind vesicles.

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. Hexamers have also been observed in crystals of Munc13-1 that form between opposed phospholipid bilayers [K. Grushin, R. V. Kalyana Sundaram, C. V. Sindelar, J. E. Rothman, Proc. Natl. Acad. Sci. U.S.A. 119, e2121259119 (2022)]. Mutations targeting the contacts stabilizing the crystallographic hexagons also disrupt the isolated hexamers, suggesting they are identical. Additionally, these mutations also convert vesicle binding from a cooperative to progressive mode. Our study provides an independent approach showing that Munc13-1 can form mainly hexamers on lipid bilayers each capable of vesicle capture.

Turbocharging synaptic transmission.

Evidence from biochemistry, genetics, and electron microscopy strongly supports the idea that a ring of Synaptotagmin is central to the clamping and release of synaptic vesicles (SVs) for synchronous neurotransmission. Recent direct measurements in cell-free systems suggest there are 12 SNAREpins in each ready-release vesicle, consisting of six peripheral and six central SNAREpins. The six central SNAREpins are directly bound to the Synaptotagmin ring, are directly released by Ca++ , and they initially open the fusion pore. The six peripheral SNAREpins are indirectly bound to the ring, each linked to a central SNAREpin by a bridging molecule of Complexin. We suggest that the primary role of peripheral SNAREpins is to provide additional force to 'turbocharge' neurotransmitter release, explaining how it can occur much faster than other forms of membrane fusion. The SV protein Synaptophysin forms hexamers that bear two copies of the v-SNARE VAMP at each vertex, one likely assembling into a peripheral SNAREpin and the other into a central SNAREpin.

Roles for diacylglycerol in synaptic vesicle priming and release revealed by complete reconstitution of core protein machinery.

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. As expected, DAG also increases the number of docked, release-ready vesicles. Dynamic single-molecule imaging of Complexin binding to release-ready vesicles directly establishes that DAG accelerates the rate of SNAREpin assembly mediated by chaperones, Munc13 and Munc18. The selective effects of physiologically validated mutations confirmed that the Munc18-Syntaxin-VAMP2 "template" complex is a functional intermediate in the production of primed, release-ready vesicles, which requires the coordinated action of Munc13 and Munc18.

Two successive oligomeric Munc13 assemblies scaffold vesicle docking and SNARE assembly to support neurotransmitter release.

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. We suggest that a progression of oligomeric Munc13 complexes couples vesicle docking and assembly of a precise number of SNARE molecules to support rapid and high-fidelity vesicle priming.

Novel Roles for Diacylglycerol in Synaptic Vesicle Priming and Release Revealed by Complete Reconstitution of Core Protein Machinery.

Here we introduce the full functional reconstitution of genetically-validated core protein machinery (SNAREs, Munc13, Munc18, Synaptotagmin, 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 Ca 2+ . Using this novel setup, we discover new roles for diacylglycerol (DAG) in regulating vesicle priming and Ca 2+- triggered release involving the SNARE assembly chaperone Munc13. We find that low concentrations of DAG profoundly accelerate the rate of Ca 2+ -dependent release, and high concentrations reduce clamping and permit extensive spontaneous release. As expected, DAG also increases the number of ready-release vesicles. Dynamic single-molecule imaging of Complexin binding to ready-release vesicles directly establishes that DAG accelerates the rate of SNAREpin assembly mediated by Munc13 and Munc18 chaperones. The selective effects of physiologically validated mutations confirmed that the Munc18-Syntaxin-VAMP2 'template' complex is a functional intermediate in the production of primed, ready-release vesicles, which requires the coordinated action of Munc13 and Munc18. SIGNIFICANCE STATEMENT: Munc13 and Munc18 are SNARE-associated chaperones that act as "priming" factors, facilitating the formation of a pool of docked, release-ready vesicles and regulating Ca 2+ -evoked neurotransmitter release. Although important insights into Munc18/Munc13 function have been gained, how they assemble and operate together remains enigmatic. To address this, we developed a novel biochemically-defined fusion assay which enabled us to investigate the cooperative action of Munc13 and Munc18 in molecular terms. We find that Munc18 nucleates the SNARE complex, while Munc13 promotes and accelerates the SNARE assembly in a DAG-dependent manner. The concerted action of Munc13 and Munc18 stages the SNARE assembly process to ensure efficient 'clamping' and formation of stably docked vesicles, which can be triggered to fuse rapidly (∼10 msec) upon Ca 2+ influx.

Munc13 structural transitions and oligomers that may choreograph successive stages in vesicle priming for neurotransmitter release.

How can exactly six SNARE complexes be assembled under each synaptic vesicle? Here we report cryo-EM crystal structures of the core domain of Munc13, the key chaperone that initiates SNAREpin assembly. The functional core of Munc13, consisting of C1-C2B-MUN-C2C (Munc13C) spontaneously crystallizes between phosphatidylserine-rich bilayers in two distinct conformations, each in a radically different oligomeric state. In the open conformation (state 1), Munc13C forms upright trimers that link the two bilayers, separating them by ∼21 nm. In the closed conformation, six copies of Munc13C interact to form a lateral hexamer elevated ∼14 nm above the bilayer. Open and closed conformations differ only by a rigid body rotation around a flexible hinge, which when performed cooperatively assembles Munc13 into a lateral hexamer (state 2) in which the key SNARE assembly-activating site of Munc13 is autoinhibited by its neighbor. We propose that each Munc13 in the lateral hexamer ultimately assembles a single SNAREpin, explaining how only and exactly six SNARE complexes are templated. We suggest that state 1 and state 2 may represent two successive states in the synaptic vesicle supply chain leading to "primed" ready-release vesicles in which SNAREpins are clamped and ready to release (state 3).

Symmetrical arrangement of proteins under release-ready vesicles in presynaptic terminals.

Controlled release of neurotransmitters stored in synaptic vesicles (SVs) is a fundamental process that is central to all information processing in the brain. This relies on tight coupling of the SV fusion to action potential-evoked presynaptic Ca2+ influx. This Ca2+-evoked release occurs from a readily releasable pool (RRP) of SVs docked to the plasma membrane (PM). The protein components involved in initial SV docking/tethering and the subsequent priming reactions which make the SV release ready are known. Yet, the supramolecular architecture and sequence of molecular events underlying SV release are unclear. Here, we use cryoelectron tomography analysis in cultured hippocampal neurons to delineate the arrangement of the exocytosis machinery under docked SVs. Under native conditions, we find that vesicles are initially "tethered" to the PM by a variable number of protein densities (∼10 to 20 nm long) with no discernible organization. In contrast, we observe exactly six protein masses, each likely consisting of a single SNAREpin with its bound Synaptotagmins and Complexin, arranged symmetrically connecting the "primed" vesicles to the PM. Our data indicate that the fusion machinery is likely organized into a highly cooperative framework during the priming process which enables rapid SV fusion and neurotransmitter release following Ca2+ influx.

Structural basis for the clamping and Ca2+ activation of SNARE-mediated fusion by synaptotagmin.

Synapotagmin-1 (Syt1) interacts with both SNARE proteins and lipid membranes to synchronize neurotransmitter release to calcium (Ca2+) influx. Here we report the cryo-electron microscopy structure of the Syt1-SNARE complex on anionic-lipid containing membranes. Under resting conditions, the Syt1 C2 domains bind the membrane with a magnesium (Mg2+)-mediated partial insertion of the aliphatic loops, alongside weak interactions with the anionic lipid headgroups. The C2B domain concurrently interacts the SNARE bundle via the 'primary' interface and is positioned between the SNAREpins and the membrane. In this configuration, Syt1 is projected to sterically delay the complete assembly of the associated SNAREpins and thus, contribute to clamping fusion. This Syt1-SNARE organization is disrupted upon Ca2+-influx as Syt1 reorients into the membrane, likely displacing the attached SNAREpins and reversing the fusion clamp. We thus conclude that the cation (Mg2+/Ca2+) dependent membrane interaction is a key determinant of the dual clamp/activator function of Synaptotagmin-1.

Symmetrical organization of proteins under docked synaptic vesicles.

During calcium-regulated exocytosis, the constitutive fusion machinery is 'clamped' in a partially assembled state until synchronously released by calcium. The protein machinery involved in this process is known, but the supra-molecular architecture and underlying mechanisms are unclear. Here, we use cryo-electron tomography analysis in nerve growth factor-differentiated neuro-endocrine (PC12) cells to delineate the organization of the release machinery under the docked vesicles. We find that exactly six exocytosis modules, each likely consisting of a single SNAREpin with its bound Synaptotagmins, Complexin, and Munc18 proteins, are symmetrically arranged at the vesicle-PM interface. Mutational analysis suggests that the symmetrical organization is templated by circular oligomers of Synaptotagmin. The observed arrangement, including its precise radial positioning, is in-line with the recently proposed 'buttressed ring hypothesis'.

Hypothesis - buttressed rings assemble, clamp, and release SNAREpins for synaptic transmission.

Neural networks are optimized to detect temporal coincidence on the millisecond timescale. Here, we offer a synthetic hypothesis based on recent structural insights into SNAREs and the C2 domain proteins to explain how synaptic transmission can keep this pace. We suggest that an outer ring of up to six curved Munc13 'MUN' domains transiently anchored to the plasma membrane via its flanking domains surrounds a stable inner ring comprised of synaptotagmin C2 domains to serve as a work-bench on which SNAREpins are templated. This 'buttressed-ring hypothesis' affords straightforward answers to many principal and long-standing questions concerning how SNAREpins can be assembled, clamped, and then released synchronously with an action potential.

Conformational Response of Influenza A M2 Transmembrane Domain to Amantadine Drug Binding at Low pH (pH 5.5).

The M2 protein from influenza A plays important roles in its viral cycle. It contains a single transmembrane helix, which oligomerizes into a homotetrameric proton channel that conducts in the low-pH environment of the host-cell endosome and Golgi apparatus, leading to virion uncoating at an early stage of infection. We studied conformational rearrangements that occur in the M2 core transmembrane domain residing on the lipid bilayer, flanked by juxtamembrane residues (M2TMD21-49 fragment), upon its interaction with amantadine drug at pH 5.5 when M2 is conductive. We also tested the role of specific mutation and lipid chain length. Electron spin resonance (ESR) spectroscopy and electron microscopy were applied to M2TMD21-49, labeled at the residue L46C with either nitroxide spin-label or Nanogold® reagent, respectively. Electron microscopy confirmed that M2TMD21-49 reconstituted into DOPC/POPS at 1:10,000 peptide-to-lipid molar ratio (P/L) either with or without amantadine, is an admixture of monomers, dimers, and tetramers, confirming our model based on a dimer intermediate in the assembly of M2TMD21-49. As reported by double electron-electron resonance (DEER), in DOPC/POPS membranes amantadine shifts oligomer equilibrium to favor tetramers, as evidenced by an increase in DEER modulation depth for P/L's ranging from 1:18,000 to 1:160. Furthermore, amantadine binding shortens the inter-spin distances (for nitroxide labels) by 5-8 Å, indicating drug induced channel closure on the C-terminal side. No such effect was observed for the thinner membrane of DLPC/DLPS, emphasizing the role of bilayer thickness. The analysis of continuous wave (cw) ESR spectra of spin-labeled L46C residue provides additional support to a more compact helix bundle in amantadine-bound M2TMD 21-49 through increased motional ordering. In contrast to wild-type M2TMD21-49, the amantadine-bound form does not exhibit noticeable conformational changes in the case of G34A mutation found in certain drug-resistant influenza strains. Thus, the inhibited M2TMD21-49 channel is a stable tetramer with a closed C-terminal exit pore. This work is aimed at contributing to the development of structure-based anti-influenza pharmaceuticals.

Dimeric Organization of Blood Coagulation Factor VIII bound to Lipid Nanotubes.

Membrane-bound Factor VIII (FVIII) has a critical function in blood coagulation as the pro-cofactor to the serine-protease Factor IXa (FIXa) in the FVIIIa-FIXa complex assembled on the activated platelet membrane. Defects or deficiency of FVIII cause Hemophilia A, a mild to severe bleeding disorder. Despite existing crystal structures for FVIII, its membrane-bound organization has not been resolved. Here we present the dimeric FVIII membrane-bound structure when bound to lipid nanotubes, as determined by cryo-electron microscopy. By combining the structural information obtained from helical reconstruction and single particle subtomogram averaging at intermediate resolution (15-20 Å), we show unambiguously that FVIII forms dimers on lipid nanotubes. We also demonstrate that the organization of the FVIII membrane-bound domains is consistently different from the crystal structure in solution. The presented results are a critical step towards understanding the mechanism of the FVIIIa-FIXa complex assembly on the activated platelet surface in the propagation phase of blood coagulation.

Factor VIII organisation on nanodiscs with different lipid composition.

Nanodiscs (ND) are lipid bilayer membrane patches held by amphiphilic scaffolding proteins (MSP) of ~10 nm in diameter. Nanodiscs have been developed as lipid nanoplatforms for structural and functional studies of membrane and membrane associated proteins. Their size and monodispersity have rendered them unique for electron microscopy (EM) and single particle analysis studies of proteins and complexes either spanning or associated to the ND membrane. Binding of blood coagulation factors and complexes, such as the Factor VIII (FVIII) and the Factor VIIIa - Factor IXa (intrinsic tenase) complex to the negatively charged activated platelet membrane is required for normal haemostasis. In this study we present our work on optimising ND, specifically designed to bind FVIII at close to physiological conditions. The binding of FVIII to the negatively charged ND rich in phosphatidylserine (PS) was followed by electron microscopy at three different PS compositions and two different membrane scaffolding protein (MSP1D1) to lipid ratios. Our results show that the ND with highest PS content (80 %) and lowest MSP1D1 to lipid ratio (1:47) are the most suitable for structure determination of the membrane-bound FVIII by single particle EM. Our preliminary FVIII 3D reconstruction as bound to PS containing ND demonstrates the suitability of the optimised ND for structural studies by EM. Further assembly of the activated FVIII form (FVIIIa) and the whole FVIIIa-FIXa complex on ND, followed by EM and single particle reconstruction will help to identify the protein-protein and protein-membrane interfaces critical for the intrinsic tenase complex assembly and function.

Helical organization of blood coagulation factor VIII on lipid nanotubes.

Cryo-electron microscopy (Cryo-EM)(1) is a powerful approach to investigate the functional structure of proteins and complexes in a hydrated state and membrane environment(2). Coagulation Factor VIII (FVIII)(3) is a multi-domain blood plasma glycoprotein. Defect or deficiency of FVIII is the cause for Hemophilia type A - a severe bleeding disorder. Upon proteolytic activation, FVIII binds to the serine protease Factor IXa on the negatively charged platelet membrane, which is critical for normal blood clotting(4). Despite the pivotal role FVIII plays in coagulation, structural information for its membrane-bound state is incomplete(5). Recombinant FVIII concentrate is the most effective drug against Hemophilia type A and commercially available FVIII can be expressed as human or porcine, both forming functional complexes with human Factor IXa(6,7). In this study we present a combination of Cryo-electron microscopy (Cryo-EM), lipid nanotechnology and structure analysis applied to resolve the membrane-bound structure of two highly homologous FVIII forms: human and porcine. The methodology developed in our laboratory to helically organize the two functional recombinant FVIII forms on negatively charged lipid nanotubes (LNT) is described. The representative results demonstrate that our approach is sufficiently sensitive to define the differences in the helical organization between the two highly homologous in sequence (86% sequence identity) proteins. Detailed protocols for the helical organization, Cryo-EM and electron tomography (ET) data acquisition are given. The two-dimensional (2D) and three-dimensional (3D) structure analysis applied to obtain the 3D reconstructions of human and porcine FVIII-LNT is discussed. The presented human and porcine FVIII-LNT structures show the potential of the proposed methodology to calculate the functional, membrane-bound organization of blood coagulation Factor VIII at high resolution.

Lipid nanotechnologies for structural studies of membrane-associated proteins.

We present a methodology of lipid nanotubes (LNT) and nanodisks technologies optimized in our laboratory for structural studies of membrane-associated proteins at close to physiological conditions. The application of these lipid nanotechnologies for structure determination by cryo-electron microscopy (cryo-EM) is fundamental for understanding and modulating their function. The LNTs in our studies are single bilayer galactosylceramide based nanotubes of ∼20 nm inner diameter and a few microns in length, that self-assemble in aqueous solutions. The lipid nanodisks (NDs) are self-assembled discoid lipid bilayers of ∼10 nm diameter, which are stabilized in aqueous solutions by a belt of amphipathic helical scaffold proteins. By combining LNT and ND technologies, we can examine structurally how the membrane curvature and lipid composition modulates the function of the membrane-associated proteins. As proof of principle, we have engineered these lipid nanotechnologies to mimic the activated platelet's phosphtaidylserine rich membrane and have successfully assembled functional membrane-bound coagulation factor VIII in vitro for structure determination by cryo-EM. The macromolecular organization of the proteins bound to ND and LNT are further defined by fitting the known atomic structures within the calculated three-dimensional maps. The combination of LNT and ND technologies offers a means to control the design and assembly of a wide range of functional membrane-associated proteins and complexes for structural studies by cryo-EM. The presented results confirm the suitability of the developed methodology for studying the functional structure of membrane-associated proteins, such as the coagulation factors, at a close to physiological environment.

Circadian phase-dependent effect of nitric oxide on L-type voltage-gated calcium channels in avian cone photoreceptors.

Nitric oxide (NO) plays an important role in phase-shifting of circadian neuronal activities in the suprachiasmatic nucleus and circadian behavior activity rhythms. In the retina, NO production is increased in a light-dependent manner. While endogenous circadian oscillators in retinal photoreceptors regulate their physiological states, it is not clear whether NO also participates in the circadian regulation of photoreceptors. In this study, we demonstrate that NO is involved in the circadian phase-dependent regulation of L-type voltage-gated calcium channels (L-VGCCs). In chick cone photoreceptors, the L-VGCCα1 subunit expression and the maximal L-VGCC currents are higher at night, and both Ras-mitogen-activated protein kinase (MAPK)-extracellular signal-regulated kinase (Erk) and Ras-phosphatidylinositol 3 kinase (PI3K)-protein kinase B (Akt) are part of the circadian output pathways regulating L-VGCCs. The NO-cGMP-protein kinase G (PKG) pathway decreases L-VGCCα1 subunit expression and L-VGCC currents at night, but not during the day, and exogenous NO donor or cGMP decreases the phosphorylation of Erk and Akt at night. The protein expression of neural NO synthase (nNOS) is also under circadian control, with both nNOS and NO production being higher during the day. Taken together, NO/cGMP/PKG signaling is involved as part of the circadian output pathway to regulate L-VGCCs in cone photoreceptors. In cone photoreceptors, the protein expression of neural nitric oxide synthase (nNOS) and NO production are under circadian control. NO-cGMP-protein kinase G (PKG) signaling serves in the circadian output pathway to regulate the circadian rhythms of L-type voltage-gated calcium channels (L-VGCCs) in part through regulating the phosphorylation states of extracellular-signal-regulated kinase (Erk) and protein kinase B (Akt).

Circadian profiles in the embryonic chick heart: L-type voltage-gated calcium channels and signaling pathways.

Circadian clocks exist in the heart tissue and modulate multiple physiological events, from cardiac metabolism to contractile function and expression of circadian oscillator and metabolic-related genes. Ample evidence has demonstrated that there are endogenous circadian oscillators in adult mammalian cardiomyocytes. However, mammalian embryos cannot be entrained independently to light-dark (LD) cycles in vivo without any maternal influence, but circadian genes are well expressed and able to oscillate in embryonic stages. The authors took advantage of using chick embryos that are independent of maternal influences to investigate whether embryonic hearts could be entrained under LD cycles in ovo. The authors found circadian regulation of L-type voltage-gated calcium channels (L-VGCCs), the ion channels responsible for the production of cardiac muscle contraction in embryonic chick hearts. The mRNA levels and protein expression of VGCCα1C and VGCCα1D are under circadian control, and the average L-VGCC current density is significantly larger when cardiomyocytes are recorded during the night than day. The phosphorylation states of several kinases involved in insulin signaling and cardiac metabolism, including extracellular signal-regulated kinase (Erk), stress-activated protein kinase (p38), protein kinase B (Akt), and glycogen synthase kinase-3ß (GSK-3ß), are also under circadian control. Both Erk and p38 have been implicated in regulating cardiac contractility and in the development of various pathological states, such as cardiac hypertrophy and heart failure. Even though both Erk and phosphoinositide 3-kinase (PI3K)-Akt signaling pathways participate in complex cellular processes regarding physiological or pathological states of cardiomyocytes, the circadian oscillators in the heart regulate these pathways independently, and both pathways contribute to the circadian regulation of L-VGCCs.