Quantitative single-molecule analysis of assembly and Ca-dependent disassembly of synaptotagmin oligomers on lipid bilayers.

Synaptotagmin-1 (Syt-1) self-assembles into ring-like oligomers, and genetic and biochemical evidence suggest that oligomerization is needed to clamp synaptic vesicles and stabilize them for Ca-evoked release. However, oligomerization has not yet been demonstrated on lipid bilayers or studied in quantitative biophysical terms. Here we utilize single-molecule imaging methods to monitor the assembly and disassembly of oligomeric clusters of Syt-1 on lipid bilayers in real-time.

A proteome-wide quantitative platform for nanoscale spatially resolved extraction of membrane proteins into native nanodiscs.

The native membrane environment profoundly influences every aspect of membrane protein (MP) biology. Despite this, the most prevalent method of studying MPs uses detergents to disrupt and remove this vital membrane context, impeding our ability to decipher the local molecular context and its effect. Here we develop a membrane proteome-wide platform that enables rapid spatially resolved extraction of target MPs directly from cellular membranes into native nanodiscs that maintain the local membrane context, using a library of membrane-active polymers.

Unraveling cellular complexity with transient adapters in highly multiplexed super-resolution imaging.

Mapping the intricate spatial relationships between the many different molecules inside a cell is essential to understanding cellular functions in all their complexity. Super-resolution fluorescence microscopy offers the required spatial resolution but struggles to reveal more than four different targets simultaneously. Exchanging labels in subsequent imaging rounds for multiplexed imaging extends this number but is limited by its low throughput.

Biallelic NAA60 variants with impaired n-terminal acetylation capacity cause autosomal recessive primary familial brain calcifications.

Primary familial brain calcification (PFBC) is characterized by calcium deposition in the brain, causing progressive movement disorders, psychiatric symptoms, and cognitive decline. PFBC is a heterogeneous disorder currently linked to variants in six different genes, but most patients remain genetically undiagnosed. Here, we identify biallelic NAA60 variants in ten individuals from seven families with autosomal recessive PFBC.

Synaptophysin chaperones the assembly of 12 SNAREpins under each ready-release vesicle.

The synaptic vesicle protein Synaptophysin (Syp) has long been known to form a complex with the Vesicle associated soluble N-ethylmaleimide sensitive fusion protein attachment receptor (v-SNARE) Vesicle associated membrane protein (VAMP), but a more specific molecular function or mechanism of action in exocytosis has been lacking because gene knockouts have minimal effects. Utilizing fully defined reconstitution and single-molecule measurements, we now report that Syp functions as a chaperone that determines the number of SNAREpins assembling between a ready-release vesicle and its target membrane bilayer. Specifically, Syp directs the assembly of 12 ± 1 SNAREpins under each docked vesicle, even in the face of an excess of SNARE proteins.

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.

Starting at Go: Protein structure prediction succumbs to machine learning.

This year's Lasker Basic Science Award recognizes the invention of AlphaFold, a revolutionary advance in the history of protein research which for the first time offers the practical ability to accurately predict the three-dimensional arrangement of amino acids in the vast majority of proteins on a genomic scale on the basis of sequence alone [J. Jumper , , 583-589 (2021) and K. Tunyasuvunakool , 590-596 (2021)].

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.

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 Ca. Using this setup, we identify new roles for diacylglycerol (DAG) in regulating vesicle priming and Ca-triggered release involving the SNARE assembly chaperone Munc13. We find that low concentrations of DAG profoundly accelerate the rate of Ca-dependent release, and high concentrations reduce clamping and permit extensive spontaneous release.

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 -triggered vesicle fusion in vitro and impaired neurotransmitter secretion and motor nervous system function in vivo.

Synaptophysin Chaperones the Assembly of 12 SNAREpins under each Ready-Release Vesicle.

The synaptic vesicle protein Synaptophysin has long been known to form a complex with the v-SNARE VAMP, but a more specific molecular function or mechanism of action in exocytosis has been lacking because gene knockouts have minimal effects. Utilizing fully-defined reconstitution and single-molecule measurements, we now report that Synaptophysin functions as a chaperone that determines the number of SNAREpins assembling between a ready-release vesicle and its target membrane bilayer. Specifically, Synaptophysin directs the assembly of 12 ± 1 SNAREpins under each docked vesicle, even in the face of an excess of SNARE proteins.

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

Unlabelled: 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 . Using this novel setup, we discover new roles for diacylglycerol (DAG) in regulating vesicle priming and Ca triggered release involving the SNARE assembly chaperone Munc13. We find that low concentrations of DAG profoundly accelerate the rate of Ca -dependent release, and high concentrations reduce clamping and permit extensive spontaneous release.

Direct determination of oligomeric organization of integral membrane proteins and lipids from intact customizable bilayer.

Hierarchical organization of integral membrane proteins (IMP) and lipids at the membrane is essential for regulating myriad downstream signaling. A quantitative understanding of these processes requires both detections of oligomeric organization of IMPs and lipids directly from intact membranes and determination of key membrane components and properties that regulate them. Addressing this, we have developed a platform that enables native mass spectrometry (nMS) analysis of IMP-lipid complexes directly from intact and customizable lipid membranes.

The gripping story of integrins.

The foundational research recognized by this year's Lasker Basic Science Research Award "for discoveries concerning the integrins-key mediators of cell-matrix and cell-cell adhesion in physiology and disease" reaches back to the 1970s.

Synaptotagmin rings as high-sensitivity regulators of synaptic vesicle docking and fusion.

Synchronous release at neuronal synapses is accomplished by a machinery that senses calcium influx and fuses the synaptic vesicle and plasma membranes to release neurotransmitters. Previous studies suggested the calcium sensor synaptotagmin (Syt) is a facilitator of vesicle docking and both a facilitator and inhibitor of fusion. On phospholipid monolayers, the Syt C2AB domain spontaneously oligomerized into rings that are disassembled by Ca, suggesting Syt rings may clamp fusion as membrane-separating "washers" until Ca-mediated disassembly triggers fusion and release [J.

Molecular determinants of complexin clamping and activation function.

Previously we reported that Synaptotagmin-1 and Complexin synergistically clamp the SNARE assembly process to generate and maintain a pool of docked vesicles that fuse rapidly and synchronously upon Ca influx (Ramakrishnan et al., 2020). Here, using the same in vitro single-vesicle fusion assay, we determine the molecular details of the Complexin-mediated fusion clamp and its role in Ca-activation.

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.

Cooperation of Conical and Polyunsaturated Lipids to Regulate Initiation and Processing of Membrane Fusion.

The shape of lipids has long been suspected to be a critical determinant for the control of membrane fusion. To experimentally test this assertion, we used conical and malleable lipids and measured their influence on the fusion kinetics. We found that, as previously suspected, both types of lipids accelerate fusion.

Vesicle capture by membrane-bound Munc13-1 requires self-assembly into discrete clusters.

Munc13-1 is a large banana-shaped soluble protein that is involved in the regulation of synaptic vesicle docking and fusion. Recent studies suggest that multiple copies of Munc13-1 form nano-assemblies in active zones of neurons. However, it is not known whether such clustering of Munc13-1 is correlated with multivalent binding to synaptic vesicles or specific plasma membrane domains at docking sites in the active zone.

Three-dimensional adaptive optical nanoscopy for thick specimen imaging at sub-50-nm resolution.

Understanding cellular organization demands the best possible spatial resolution in all three dimensions. In fluorescence microscopy, this is achieved by 4Pi nanoscopy methods that combine the concepts of using two opposing objectives for optimal diffraction-limited 3D resolution with switching fluorescent molecules between bright and dark states to break the diffraction limit. However, optical aberrations have limited these nanoscopes to thin samples and prevented their application in thick specimens.

Nascent fusion pore opening monitored at single-SNAREpin resolution.

Vesicle fusion with a target membrane is a key event in cellular trafficking and ensures cargo transport within the cell and between cells. The formation of a protein complex, called SNAREpin, provides the energy necessary for the fusion process. In a three-dimensional microfluidic chip, we monitored the fusion of small vesicles with a suspended asymmetric lipid bilayer.

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 Ca influx. This Ca-evoked release occurs from a readily releasable pool (RRP) of SVs docked to the plasma membrane (PM).

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.

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 (Ca), is critical to its central role in orchestrating neurotransmitter release. The molecular surfaces involved, namely the conserved polylysine motif in the C2B domain and Ca-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 Ca.