Manfred Lindau

Synaptobrevin (Syb/VAMP) is the vesicular SNARE that drives synaptic vesicle fusion. Before fusion, Syb is located in the vesicle membrane. After fusion, Syb is translocated to the presynaptic plasma membrane and exposed to a different lipid environment. How its membrane-proximal juxtamembrane and transmembrane domains (JMD–TMDs) organize these different surrounding membrane environments remains incompletely understood. Here, we used MARTINI coarse-grained molecular dynamics simulations in planar membrane models to investigate Syb1/2 membrane organization and collective behavior. Syb1 and Syb2 induced similar and robust local lipid disorder, whereas the spatial organization of lipid disorder and membrane mismatch depended more strongly on membrane lipid composition. In ten-copy systems, mean cluster sizes over the final 100 ns were approximately 30% lower for wild-type Syb1 and 39% lower for wild-type Syb2 in PIP₂-depleted than in the corresponding PIP₂-containing plasma membranes. Collective Syb assembly was accompanied by pronounced PIP₂ redistribution and strong local enrichment, whereas PS showed substantially weaker local enrichment when both lipid species were present. Clustering spatially overlapped otherwise localized lipid-disordered regions without proportionally amplifying their magnitude. The applicability of these findings to curved vesicles and fusion or endocytic intermediates remains to be established. Together, these results distinguish robust local membrane disorder from composition-dependent geometric remodeling and identify PIP₂ as a prominent correlate of higher-order Syb organization that may contribute to post-fusion Syb reorganization before endocytic retrieval.

Synaptobrevin (Syb/VAMP) is the vesicular SNARE that drives synaptic vesicle fusion. Before fusion, Syb is located in the vesicle membrane. After fusion, Syb is translocated to the presynaptic plasma membrane and exposed to a different lipid environment. How its membrane-proximal juxtamembrane and transmembrane domains (JMD–TMDs) organ-ize these different surrounding membrane environments remains incompletely under-stood. Here, we used MARTINI coarse-grained molecular dynamics simulations to in-vestigate Syb1/2 membrane organization and collective behavior. Syb1 and Syb2 induced similar and robust local lipid disorder, whereas the spatial organization of lipid disorder and membrane mismatch depended more strongly on membrane lipid composition. Higher order Syb clustering was promoted by the presence of PIP₂ in the membrane, which is decreased in its absence and insensitive to PS depletion. Consistently, Syb cluster formation was associated with pronounced PIP₂ redistribution and enrichment, while PS showed substantially weaker enrichment and decreased association with Syb in the presence of PIP₂. Clustering spatially overlapped otherwise localized lipid-disordered regions without proportionally amplifying their magnitude. Together, these results dis-tinguish robust local membrane disorder from composition-dependent geometric re-modeling and identify PIP₂ as a prominent correlate of higher-order Syb organization that may contribute to post-fusion Syb reorganization before endocytic retrieval.

Syntaxin-1 (Stx1), a core neuronal SNARE protein, regulates membrane docking and fusion during neurotransmitter release. Although Stx1 clustering is widely observed at presynaptic active zones and in neuroendocrine cells, how Stx1 isoforms and lipid interactions organize the plasma membrane prior to fusion remains unclear. Here, we performed MARTINI coarse-grained molecular dynamics (cgMD) simulations of 1–10 copies of the juxtamembrane and transmembrane domains (JMD–TMDs) of Stx1A and Stx1B embedded in plasma membrane models to investigate membrane lipid disorder, Stx1 JMD–TMD clustering dynamics, and regulation by Stx1 TMD palmitoylation and phosphatidylinositol 4,5-bisphosphate (PIP₂). Stx1 JMD–TMD regions induced local lipid disorder associated with hydrophobic mismatch, with Stx1A producing stronger and more spatially extended perturbations than Stx1B. The magnitude of local lipid disorder remained largely independent of clustering and TMD palmitoylation, whereas palmitoylation spatially restricted and PIP₂ depletion broadened the radial extent of membrane perturbation. During clustering, Stx1 JMD–TMDs progressively recruited and redistributed PIP₂ into local membrane domains, resulting in ~7-fold local enrichment relative to the bulk membrane. Together, these findings support a model where hydrophobic mismatch primarily governs membrane disorder, whereas multivalent PIP₂ recruitment and redistribution stabilize higher-order Stx1 oligomers, thereby promoting membrane environments permissive for SNARE-mediated fusion.

Synaptotagmins (Syts) are Ca²⁺-sensing exocytosis regulators whose tandem C2 domains interact with phosphoinositides and membranes to trigger neurotransmitter and hormone release. Although Ca²⁺ binding is known to enhance C2 domain-membrane interactions, the sequence determinants governing lipid binding and membrane penetration across Syt isoforms remain incompletely understood. Here, we performed MARTINI coarse-grained molecular dynamics simulations of isolated C2A and C2B domains from eight Ca²⁺-sensing Syt isoforms (Syt1, Syt2, Syt3, Syt5, Syt6, Syt7, Syt9, and Syt10) interacting with phosphatidylinositol 4,5-bisphosphate (PIP₂)-containing plasma membranes. To systematically modulate electrostatic properties, we introduced partial and full charge-flip mutations at conserved acidic residues within the calcium-binding loops (CBLs). By integrating simulations across multiple isoforms and charge states, we sought to identify the dominant sequence determinants governing membrane interactions. We found that PIP₂ binding to both, CBLs and polybasic patches (PBs), is associated with loop net charge, yielding correlations > 0.95 across all isoforms. However, membrane penetration is not sufficiently explained by loop net charge alone. The local phenylalanines additionally increase membrane penetration independent of loop net charge. Together, these findings establish a comprehensive electrostatic–aromatic framework where loop net charge governs PIP₂ binding, whereas loop net charge and local phenylalanine enrichment jointly govern membrane penetration across Syt C2 domains.

Syntaxin-1 (Stx1), a core neuronal SNARE protein, regulates membrane docking and fusion during neurotransmitter release. Although Stx1 clustering is widely observed at presynaptic active zones and in neuroendocrine cells, how Stx1 isoforms and lipid interactions organize the plasma membrane prior to fusion remains unclear. Here, we performed MARTINI coarse-grained molecular dynamics (cgMD) simulations of 1–10 copies of the juxtamembrane and transmembrane domains (JMD–TMDs) of Stx1A and Stx1B embedded in plasma membrane models to investigate membrane lipid disorder, Stx1 JMD–TMD clustering dynamics, and regulation by Stx1 TMD palmitoylation and phosphatidylinositol 4,5-bisphosphate (PIP2). Stx1 JMD–TMD regions induced local lipid disorder primarily through hydrophobic mismatch, with Stx1A producing stronger and more spatially extended perturbations than Stx1B. The magnitude of local lipid disorder remained largely independent of clustering and TMD palmitoylation, whereas palmitoylation spatially restricted and PIP2 depletion broadened the radial extent of membrane perturbation. Stx1 proteins spontaneously formed protein-density-dependent oligomers, with PIP2 depletion and palmitoylation suppressing higher-order oligomerization. Together, these findings support a model in which hydrophobic mismatch, palmitoylation, and PIP2 cooperatively regulate membrane organization and Stx1 clustering, thereby modulating transitions toward membrane environments permissive for SNARE-mediated fusion.

Neurotransmitter release via synaptic vesicle fusion with the plasma membrane is driven by SNARE proteins (Synaptobrevin, Syntaxin, and SNAP-25) and accessory proteins (Synaptotagmin, Complexin, Munc13, and Munc18). While extensively studied experimentally, the precise mechanisms and dynamics remain elusive due to spatiotemporal limitations. Molecular dynamics (MD) simulations—both all-atom (AA) and coarse-grained (CG)—bridge these gaps by capturing fusion dynamics beyond experimental resolution. This review explores the use of these simulations in understanding SNARE-mediated membrane fusion and its regulation by Synaptotagmin and Complexin. We first examine two competing hypotheses regarding the driving force of fusion: (1) SNARE zippering transducing energy through rigid juxtamembrane domains (JMDs) and (2) SNAREs generating entropic forces via flexible JMDs. Despite different origins of forces, the conserved fusion pathway – from membrane adhesion to stalk and fusion pore (FP) formation – emerges across models. We also highlight the critical role of SNARE transmembrane domains (TMDs) and their regulation by post-translational modifications like palmitoylation in fast fusion. Further, we review Ca²⁺-dependent interactions of Synaptotagmin’s C2 domains with lipids and SNAREs at the primary and tripartite interfaces, and how these interactions regulate fusion timing. Complexin’s role in clamping spontaneous fusion while facilitating evoked release via its central and accessory helices is also discussed. We present a case study leveraging AA and CG simulations to investigate ion selectivity in FPs, balancing timescale and accuracy. We conclude with the limitations in current simulations and using AI tools to construct complete fusion machinery and explore isoform-specific functions in fusion machinery.

A SNAP25 based FRET construct named SCORE (SNARE COmplex REporter) has revealed a transient FRET increase that specifically occurred at fusion sites preceding fusion events by tens of milliseconds and presumably reflects vesicle priming. The FRET increase lasts for a few seconds until it is reversed. In those experiments, the FRET increase was found to be localized to areas 2 at sites of transmitter release as detected amperometrically using electrochemical detector arrays. Due to the localization to such small areas, it was unknown if the reversal of the FRET increase is due to local dispersion of high-FRET SCORE copies leaving the site after fusion and exchange with surrounding low-FRET copies, or if it reflects disassembly of the high-FRET complexes. To resolve this question, we performed whole-cell patch clamp pulse stimulation experiments, imaging the entire footprint of the cells in Total Internal Reflection Fluorescence (TIRF) excitation mode such that diffusional exchange between high-FRET and low-FRET copies does not produce a net FRET change. We show here that pulse stimulation of calcium currents results in FRET ratio transients with a time course very similar that related to fusion events. By comparing the kinetics of the FRET ratio decay with analytical and numerical diffusion simulation results, we show that the experimentally observed kinetics cannot be explained by diffusional exchange and conclude that the SCORE FRET ratio transients reflect incorporation of SCORE in SNARE complexes followed by SNARE complex disassembly. Experiments using Synaptobrevin 2/Cellubrevin double knock-out mouse embryonal chromaffin cells showed no pulse induced FRET change, indicating that the vSNARE is required for the incorporation of SCORE (or SNAP25 in wild type cells) in the SNARE complex during priming. Statement of Significance: In chromaffin cells, SNAP25-based FRET constructs (SCORE) revealed transient FRET increases within 2 areas, preceding individual fusion events that reversed within seconds. It remained unknown whether this reversal stems from high-FRET complex disassembly or diffusion-mediated exchange with low-FRET complexes. Here, we performed whole-cell patch-clamp pulse stimulation with TIRF microscopy, imaging large ∼30 µm ² areas of the cell footprint. Calcium currents induced FRET transients with the same decay time constant of ∼1.5 s, significantly shorter than the time scale of diffusion. The SCORE FRET ratio thus reports in real time the dynamics of SNRE complex assembly and disassembly in live cells. Using Synaptobrevin 2/Cellubrevin double knock-out mouse chromaffin cells we show that vSNAREs are required for SNAP25 incorporation into SNARE complexes during priming.

Neurotransmitter release is triggered by the fusion of synaptic vesicles with the plasma membrane, orchestrated by SNARE proteins Synaptobrevin 2 (Syb2), Syntaxin 1A (Stx1A), and SNAP25. Recent experimental studies showed that Stx1A palmitoylation of C271/C272 promotes spontaneous neurotransmitter release. However, the mechanistic role of SNARE transmembrane domain (TMD) palmitoylation in membrane fusion remains unclear. To investigate the structural and functional implications of TMD palmitoylation, we employed coarse-grained molecular dynamics simulations with the MARTINI force field. In simulations of individual SNAREs and of SNAP-25/Stx1A (t-SNARE) complexes in a membrane the palmitoyl chains of Syb2 and Stx1A localize to the membrane midplane, with Stx1A palmitoyl chains bending toward the extracellular leaflet. Non-palmitoylated Stx1A assumed a conformation where the SNARE domain was lying flat, adhering to the intracellular surface of the membrane. Stx1A dual palmitoylation induced dramatic changes, reducing the tilt of its TMD and stabilizing a more upright conformation of its SND. This conformation resembles the Stx1A conformation in a s Stx1A-SNAP25 t-SNARE complex, providing a potential mechanistic explanation of how Stx1A TMD palmitoylation facilitates early steps in SNARE complex formation and thus promotes spontaneous release. In simulations of the late steps of layers 5 to 8 SNARE complex zippering in a system of 4 SNARE complexes bridging a 10-nm nanodisc and a planar membrane, FPs spontaneously opened after a few hundred nanoseconds, preceded by distal leaflet lipid transfer and followed by FP flickering conductance before FP closure. At this stage, Stx1A TMD palmitoylation delayed lipid transfer and FP formation and decreased FP flicker open times, whereas the palmitoylation of Syb2 did not affect fusion pore dynamics. These findings suggest that after facilitation of priming before FP opening, Stx1A TMD palmitoylation, directly affects FP dynamics. These results highlight the essential role of SNARE TMD palmitoylation at multiple stages of neurotransmitter release. Statement of Significance Synaptic vesicle fusion is critical for neurotransmitter release, enabling neuron-to-neuron communication at synapses. Post-translational modifications, such as palmitoylation, are known to influence this process. Using MARTINI coarse-grained molecular dynamics simulations, we examined the impact of SNARE transmembrane domain (TMD) palmitoylation on SNARE protein conformation and fusion dynamics. Stx1A palmitoylation reduces its TMD tilting and changes its SNARE domain conformation, facilitating SNARE complex formation. In fusion pore (FP) simulations, Stx1A palmitoylation delayed FP opening, decreased FP flicker open times, and shortened FP conductance flicker durations by direct interactions with the FP. Interestingly, dual palmitoylation of Stx1A and Syb2 restored flickering duration but decreased FP opening probability within 4 μs, suggesting a nuanced role of TMD palmitoylation in modulating neurotransmitter release.