Synergistic control of neurotransmitter release by different members of the synaptotagmin family

Quantal neurotransmitter release at nerve terminals is tightly regulated by the presynaptic Ca2+ concentration. Here, we summarise current advances in understanding how the interplay between presynaptic Ca2+ dynamics and different Ca2+ release sensors shapes action potential-evoked release on a timescale from hundreds of microseconds to hundreds of milliseconds. In particular, we review recent studies that reveal the synergistic roles of the low Ca2+ affinity/fast release sensors synaptotagmins 1, 2 and 9 and the high affinity/slow release sensor synaptotagmin 7 in the regulation of synchronous and asynchronous release and of short-term synaptic plasticity. We also examine new biochemical and structural data and outline a working model that could potentially explain the cooperative roles of different synaptotagmins in molecular terms

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

Ring-like Oligomers of Synaptotagmins and Related C2 Domain Proteins

We recently reported that the C2AB portion of Synaptotagmin 1 (Syt1) could self-assemble into Ca2+-sensitive ring-like oligomers on membranes, which could potentially regulate neurotransmitter release. Here we report that analogous ring-like oligomers assemble from the C2AB domains of other Syt isoforms (Syt2, Syt7, Syt9) as well as related C2 domain containing protein, Doc2B and extended Synaptotagmins (E-Syts). Evidently, circular oligomerization is a general and conserved structural aspect of many C2 domain proteins, including Synaptotagmins. Further, using electron microscopy combined with targeted mutations, we show that under physiologically relevant conditions, both the Syt1 ring assembly and its rapid disruption by Ca2+ involve the well-established functional surfaces on the C2B domain that are important for synaptic transmission. Our data suggests that ring formation may be triggered at an early step in synaptic vesicle docking and positions Syt1 to synchronize neurotransmitter release to Ca2+ influx

Synaptotagmin-1 membrane binding is driven by the C2B domain and assisted cooperatively by the C2A domain

Synaptotagmin interaction with anionic lipid (phosphatidylserine/phosphatidylinositol) containing membranes, both in the absence and presence of calcium ions (Ca2+), is critical to its central role in orchestrating neurotransmitter release. The molecular surfaces involved, namely the conserved polylysine motif in the C2B domain and Ca2+-binding aliphatic loops on both C2A and C2B domains, are known. Here we use surface force apparatus combined with systematic mutational analysis of the functional surfaces to directly measure Syt1-membrane interaction and fully map the site-binding energetics of Syt1 both in the absence and presence of Ca2+. By correlating energetics data with the molecular rearrangements measured during confinement, we find that both C2 domains cooperate in membrane binding, with the C2B domain functioning as the main energetic driver, and the C2A domain acting as a facilitator

Munc13 binds and recruits SNAP25 to chaperone SNARE complex assembly

Synaptic vesicle fusion is mediated by SNARE proteins—VAMP2 on the vesicle and Syntaxin‐1/SNAP25 on the presynaptic membrane. Chaperones Munc18‐1 and Munc13‐1 cooperatively catalyze SNARE assembly via an intermediate ‘template’ complex containing Syntaxin‐1 and VAMP2. How SNAP25 enters this reaction remains a mystery. Here, we report that Munc13‐1 recruits SNAP25 to initiate the ternary SNARE complex assembly by direct binding, as judged by bulk FRET spectroscopy and single‐molecule optical tweezer studies. Detailed structure–function analyses show that the binding is mediated by the Munc13‐1 MUN domain and is specific for the SNAP25 ‘linker’ region that connects the two SNARE motifs. Consequently, freely diffusing SNAP25 molecules on phospholipid bilayers are concentrated and bound in ~ 1 : 1 stoichiometry by the self‐assembled Munc13‐1 nanoclusters

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

Mutations in Membrin/GOSR2 Reveal Stringent Secretory Pathway Demands of Dendritic Growth and Synaptic Integrity.

Mutations in the Golgi SNARE (SNAP [soluble NSF attachment protein] receptor) protein Membrin (encoded by the GOSR2 gene) cause progressive myoclonus epilepsy (PME). Membrin is a ubiquitous and essential protein mediating ER-to-Golgi membrane fusion. Thus, it is unclear how mutations in Membrin result in a disorder restricted to the nervous system. Here, we use a multi-layered strategy to elucidate the consequences of Membrin mutations from protein to neuron. We show that the pathogenic mutations cause partial reductions in SNARE-mediated membrane fusion. Importantly, these alterations were sufficient to profoundly impair dendritic growth in Drosophila models of GOSR2-PME. Furthermore, we show that Membrin mutations cause fragmentation of the presynaptic cytoskeleton coupled with transsynaptic instability and hyperactive neurotransmission. Our study highlights how dendritic growth is vulnerable even to subtle secretory pathway deficits, uncovers a role for Membrin in synaptic function, and provides a comprehensive explanatory basis for genotype-phenotype relationships in GOSR2-PME

Rearrangements under confinement lead to increased binding energy of Synaptotagmin-1 with anionic membranes in Mg2+ and Ca2+

Synaptotagmin‐1 (Syt1) is the primary calcium sensor (Ca2+) that mediates neurotransmitter release at the synapse. The tandem C2 domains (C2A and C2B) of Syt1 exhibit functionally critical, Ca2+‐dependent interactions with the plasma membrane. With the surface forces apparatus, we directly measure the binding energy of membrane‐anchored Syt1 to an anionic membrane and find that Syt1 binds with ~6 kBT in EGTA, ~10 kBT in Mg2+ and ~18 kBT in Ca2+. Molecular rearrangements measured during confinement are more prevalent in Ca2+ and Mg2+ and suggest that Syt1 initially binds through C2B, then reorients the C2 domains into the preferred binding configuration. These results provide energetic and mechanistic details of the Syt1 Ca2+‐activation process in synaptic transmission

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’