The Role of α-Synuclein in SNARE-mediated Synaptic Vesicle Fusion.

Neuronal communication depends on exquisitely regulated membrane fusion between synaptic vesicles and presynaptic neurons, which results in neurotransmitter release in precisely timed patterns. Presynaptic dysfunctions are known to occur prior to the onset of neurodegenerative diseases, including Parkinson's disease. Synaptic accumulation of α-synuclein (α-Syn) oligomers has been implicated in the pathway leading to such outcomes. α-Syn oligomers exert aberrant effects on presynaptic fusion machinery through their interactions with synaptic vesicles and proteins. Here, we summarize in vitro bulk and single-vesicle assays for investigating the functions of α-Syn monomers and oligomers in synaptic vesicle fusion and then discuss the current understanding of the roles of α-Syn monomers and oligomers in synaptic vesicle fusion. Finally, we suggest a new therapeutic avenue specifically targeting the mechanisms of α-Syn oligomer toxicity rather than the oligomer itself.

Stabilization of the SNARE Core by Complexin-1 Facilitates Fusion Pore Expansion.

In the neuron, neurotransmitter release is an essential function that must be both consistent and tightly regulated. The continuity of neurotransmitter release is dependent in large part on vesicle recycling. However, the protein factors that dictate the vesicle recycling pathway are elusive. Here, we use a single vesicle-to-supported bilayer fusion assay to investigate complexin-1 (cpx1)'s influence on SNARE-dependent fusion pore expansion. With total internal reflection (TIR) microscopy using a 10 kDa polymer fluorescence probe, we are able to detect the presence of large fusion pores. With cpx1, however, we observe a significant increase of the probability of the formation of large fusion pores. The domain deletion analysis reveals that the SNARE-binding core domain of cpx1 is mainly responsible for its ability to promote the fusion pore expansion. In addition, the results show that cpx1 helps the pore to expand larger, which results in faster release of the polymer probe. Thus, the results demonstrate a reciprocal relationship between event duration and the size of the fusion pore. Based on the data, a hypothetical mechanistic model can be deduced. In this mechanistic model, the cpx1 binding stabilizes the four-helix bundle structure of the SNARE core throughout the fusion pore expansion, whereby the highly curved bilayer within the fusion pore is stabilized by the SNARE pins.

Membrane Binding of α-Synuclein Stimulates Expansion of SNARE-Dependent Fusion Pore.

SNARE-dependent membrane fusion is essential for neurotransmitter release at the synapse. Recently, α-synuclein has emerged as an important regulator for membrane fusion. Misfolded α-synuclein oligomers are potent fusion inhibitors. However, the function of normal α-synuclein has been elusive. Here, we use the single vesicle-to-supported bilayer fusion assay to dissect the role of α-synuclein in membrane fusion. The assay employs 10 kD Rhodamine B-dextran as the content probe that can detect fusion pores larger than ∼6 nm. We find that the SNARE complex alone is inefficient at dilating fusion pores. However, α-synuclein dramatically increases the probability as well as the duration of large pores. When the SNARE-interacting C-terminal region of α-synuclein was truncated, the mutant behaves the same as the wild-type. However, the double proline mutants compromising membrane-binding show significantly reduced effects on fusion pore expansion. Thus, our results suggest that α-synuclein stimulates fusion pore expansion specifically through its membrane binding.

Cooperative inhibition of SNARE-mediated vesicle fusion by α-synuclein monomers and oligomers.

The primary hallmark of Parkinson's disease (PD) is the generation of Lewy bodies of which major component is α-synuclein (α-Syn). Because of increasing evidence of the fundamental roles of α-Syn oligomers in disease progression, α-Syn oligomers have become potential targets for therapeutic interventions for PD. One of the potential toxicities of α-Syn oligomers is their inhibition of SNARE-mediated vesicle fusion by specifically interacting with vesicle-SNARE protein synaptobrevin-2 (Syb2), which hampers dopamine release. Here, we show that α-Syn monomers and oligomers cooperatively inhibit neuronal SNARE-mediated vesicle fusion. α-Syn monomers at submicromolar concentrations increase the fusion inhibition by α-Syn oligomers. This cooperative pathological effect stems from the synergically enhanced vesicle clustering. Based on this cooperative inhibition mechanism, we reverse the fusion inhibitory effect of α-Syn oligomers using small peptide fragments. The small peptide fragments, derivatives of α-Syn, block the binding of α-Syn oligomers to Syb2 and dramatically reverse the toxicity of α-Syn oligomers in vesicle fusion. Our findings demonstrate a new strategy for therapeutic intervention in PD and related diseases based on this specific interaction of α-Syn.

Munc18-1 induces conformational changes of syntaxin-1 in multiple intermediates for SNARE assembly.

In neuronal exocytosis, SNARE assembly into a stable four-helix bundle drives membrane fusion. Previous studies have revealed that the SM protein Munc18-1 plays a critical role for precise SNARE assembly with the help of Munc13-1, but the underlying mechanism remains unclear. Here, we used single-molecule FRET assays with a nanodisc membrane reconstitution system to investigate the conformational dynamics of SNARE/Munc18-1 complexes in multiple intermediate steps towards the SNARE complex. We found that single Munc18-1 proteins induce the closed conformation of syntaxin-1 not only in the free syntaxin-1 but also in the t-SNARE (syntaxin-1/SNAP-25) complex. These results implicate that Munc18-1 may act as a gatekeeper for both binary and ternary SNARE complex formation by locking the syntaxin-1 in a cleft of Munc18-1. Furthermore, the kinetic analysis of the opening/closing transition reveals that the closed syntaxin-1 in the syntaxin-1/SNAP-25/Munc18-1 complex is less stable than that in the closed syntaxin-1/Munc18-1 complex, which is manifested by the infrequent closing transition, indicating that the conformational equilibrium of the ternary complex is biased toward the open conformation of syntaxin-1 compared with the binary complex.

Alpha-Synuclein Continues to Enhance SNARE-Dependent Vesicle Docking at Exorbitant Concentrations.

Recently, Parkinson's disease-associated α-synuclein (αS) has emerged as an important regulator for SNARE-dependent vesicle fusion. However, it is controversial if excessive accumulation of αS, even in the absence of aggregation, impairs neurotransmission. Here we use a single vesicle fusion assay with ms time resolution capable of dissecting the impact of αS on each step of membrane fusion. Unlike the previous results from various in vitro, cellular, and in vivo studies, we find that non-aggregated αS promotes vesicle merger even at exorbitant concentrations. The enhancement has been seen as much as 13 fold. Delving into the kinetics of the intermediate states for vesicle fusion reveals that αS stimulates vesicle docking without altering the dynamics of bilayer merger (lipid mixing). However, minute amounts of soluble aggregated species abolish SNARE-dependent bilayer merger completely. Thus, the results show that excessive accumulation of non-aggregated αS may not be toxic for neurotransmitter release.

Virucidal nano-perforator of viral membrane trapping viral RNAs in the endosome.

Membrane-disrupting agents that selectively target virus versus host membranes could potentially inhibit a broad-spectrum of enveloped viruses, but currently such antivirals are lacking. Here, we develop a nanodisc incorporated with a decoy virus receptor that inhibits virus infection. Mechanistically, nanodiscs carrying the viral receptor sialic acid bind to influenza virions and are co-endocytosed into host cells. At low pH in the endosome, the nanodiscs rupture the viral envelope, trapping viral RNAs inside the endolysosome for enzymatic decomposition. In contrast, liposomes containing a decoy receptor show weak antiviral activity due to the lack of membrane disruption. The nanodiscs inhibit influenza virus infection and reduce morbidity and mortality in a mouse model. Our results suggest a new class of antivirals applicable to other enveloped viruses that cause irreversible physical damage specifically to virus envelope by viruses' own fusion machine. In conclusion, the lipid nanostructure provides another dimension for antiviral activity of decoy molecules.

Search for a minimal machinery for Ca2+-triggered millisecond neuroexocytosis.

Neurons have the remarkable ability to release a batch of neurotransmitters into the synapse immediately after an action potential. This signature event is made possible by the simultaneous fusion of a number of synaptic vesicles to the plasma membrane upon Ca2+ entry into the active zone. The outcomes of both cellular and in vitro studies suggest that soluble N-ethylmaleimide-sensitive-factor attachment protein receptors (SNAREs) and synaptotagmin 1 (Syt1) constitute the minimal fast exocytosis machinery in the neuron. Syt1 is the major Ca2+-sensor and orchestrates the synchronous start of individual vesicle fusion events while SNAREs are the membrane fusion machinery that dictates the kinetics of each single fusion event. The data also suggest that Ca2+-bound Syt1 is involved in the upstream docking step which leads to an increase in the number of fusion events or the size of the release, leaving the SNARE complex alone to carry out membrane fusion by themselves.

EPR Lineshape Analysis to Investigate the SNARE Folding Intermediates.

SNARE complex formation, which is believed to drive intracellular membrane fusion, transits through multiple conformational states along the membrane fusion pathway. The SNARE intermediates are biologically important because they serve as targets for fusion regulators and clostridial neurotoxins. Spin-labeling EPR has contributed significantly to the understanding of the structures and the dynamics of SNARE intermediates. In particular, the EPR lineshape analysis, which is highly sensitive to protein conformational changes such as the local coil-to-helix transition, has revealed the sequential compacting steps leading to formation of the highly stable four-helix bundle.

Real-Time Observation of Target Search by the CRISPR Surveillance Complex Cascade.

CRISPR-Cas systems defend bacteria and archaea against infection by bacteriophage and other threats. The central component of these systems are surveillance complexes that use guide RNAs to bind specific regions of foreign nucleic acids, marking them for destruction. Surveillance complexes must locate targets rapidly to ensure timely immune response, but the mechanism of this search process remains unclear. Here, we used single-molecule FRET to visualize how the type I-E surveillance complex Cascade searches DNA in real time. Cascade rapidly and randomly samples DNA through nonspecific electrostatic contacts, pausing at short PAM recognition sites that may be adjacent to the target. We identify Cascade motifs that are essential for either nonspecific sampling or positioning and readout of the PAM. Our findings provide a comprehensive structural and kinetic model for the Cascade target-search mechanism, revealing how CRISPR surveillance complexes can rapidly search large amounts of genetic material en route to target recognition.

Productive and Non-productive Pathways for Synaptotagmin 1 to Support Ca2+-Triggered Fast Exocytosis.

Ca2+-triggered SNARE-mediated membrane fusion is essential for neuronal communication. The speed of this process is of particular importance because it sets a time limit to cognitive and physical activities. In this work, we expand the proteoliposome-to-supported bilayer (SBL) fusion assay by successfully incorporating synaptotagmin 1 (Syt1), a major Ca2+ sensor. We report that Syt1 and Ca2+ together can elicit more than a 50-fold increase in the number of membrane fusion events when compared with membrane fusion mediated by SNAREs only. What is remarkable is that ~55% of all vesicle fusion events occurs within 20 ms upon vesicle docking. Furthermore, pre-binding of Syt1 to SNAREs prior to Ca2+ inhibits spontaneous fusion, but intriguingly, this leads to a complete loss of the Ca2+ responsiveness. Thus, our results suggest that there is a productive and a non-productive pathway for Syt1, depending on whether there is a premature interaction between Syt1 and SNAREs. Our results show that Ca2+ binding to Syt1 prior to Syt1's binding to SNAREs may be a prerequisite for the productive pathway. The successful reconstitution of Syt1 activities in the physiological time scale provides new opportunities to test the current mechanistic models for Ca2+-triggered exocytosis.

Botulinum Toxins A and E Inflict Dynamic Destabilization on t-SNARE to Impair SNARE Assembly and Membrane Fusion.

Botulinum toxins (BoNTs) A and E block neurotransmitter release by specifically cleaving the C- terminal ends of SNAP-25, a plasma membrane SNARE protein. Here, we find that SNAP-25A and E, the cleavage products of BoNT A and E, respectively, terminate membrane fusion via completely different mechanisms. Combined studies of single-molecule FRET and single-vesicle fusion assays reveal that SNAP-25E is incapable of supporting SNARE pairing and thus, vesicle docking. In contrast, SNAP-25A facilitates robust SNARE pairing and vesicle docking with somewhat reduced SNARE zippering, which leads to severe impairment of fusion pore opening. The electron paramagnetic resonance results show that the discrepancy between SNAP-25A and E might stem from the extent of the dynamic destabilization of the t-SNARE core at the N-terminal half, which plays a pivotal role in nucleating SNARE complex formation. Thus, the results provide insights into the structure/dynamics-based mechanism by which BoNT A and E impair membrane fusion.

Structures and transport dynamics of a Campylobacter jejuni multidrug efflux pump.

Resistance-nodulation-cell division efflux pumps are integral membrane proteins that catalyze the export of substrates across cell membranes. Within the hydrophobe-amphiphile efflux subfamily, these resistance-nodulation-cell division proteins largely form trimeric efflux pumps. The drug efflux process has been proposed to entail a synchronized motion between subunits of the trimer to advance the transport cycle, leading to the extrusion of drug molecules. Here we use X-ray crystallography and single-molecule fluorescence resonance energy transfer imaging to elucidate the structures and functional dynamics of the Campylobacter jejuni CmeB multidrug efflux pump. We find that the CmeB trimer displays a very unique conformation. A direct observation of transport dynamics in individual CmeB trimers embedded in membrane vesicles indicates that each CmeB subunit undergoes conformational transitions uncoordinated and independent of each other. On the basis of our findings and analyses, we propose a model for transport mechanism where CmeB protomers function independently within the trimer.Multidrug efflux pumps significantly contribute for bacteria resistance to antibiotics. Here the authors present the structure of Campylobacter jejuni CmeB pump combined with functional FRET assays to propose a transport mechanism where each CmeB protomers is functionally independent from the trimer.

α-Synuclein may cross-bridge v-SNARE and acidic phospholipids to facilitate SNARE-dependent vesicle docking.

Misfolded α-synuclein (A-syn) is widely recognized as the primal cause of neurodegenerative diseases including Parkinson's disease and dementia with Lewy bodies. The normal cellular function of A-syn has, however, been elusive. There is evidence that A-syn plays multiple roles in the exocytotic pathway in the neuron, but the underlying molecular mechanisms are unclear. A-syn has been known to interact with negatively charged phospholipids and with vesicle SNARE protein VAMP2. Using single-vesicle docking/fusion assays, we find that A-syn promotes SNARE-dependent vesicles docking significantly at 2.5 µM. When phosphatidylserine (PS) is removed from t-SNARE-bearing vesicles, the docking enhancement by A-syn disappears and A-syn instead acts as an inhibitor for docking. In contrast, subtraction of PS from the v-SNARE-carrying vesicles enhances vesicle docking even further. Moreover, when we truncate the C-terminal 45 residues of A-syn that participates in interacting with VAMP2, the promotion of vesicle docking is abrogated. Thus, the results suggest that the A-syn's interaction with v-SNARE through its C-terminal tail and its concurrent interaction with PS in trans through its amphipathic N-terminal domain facilitate SNARE complex formation, whereby A-syn aids SNARE-dependent vesicle docking.

Hemifusion in Synaptic Vesicle Cycle.

In the neuron, early neurotransmitters are released through the fusion pore prior to the complete vesicle fusion. It has been thought that the fusion pore is a gap junction-like structure made of transmembrane domains (TMDs) of soluble N-ethylmaleimide-sensitive-factor attachment protein receptor (SNARE) proteins. However, evidence has accumulated that lipid mixing occurs prior to the neurotransmitter release through the fusion pore lined predominantly with lipids. To explain these observations, the hemifusion, a membrane structure in which two bilayers are partially merged, has emerged as a key step preceding the formation of the fusion pore. Furthermore, the hemifusion appears to be the bona fide intermediate step not only for the synaptic vesicle cycle, but for a wide range of membrane remodeling processes, such as viral membrane fusion and endocytotic membrane fission.

Visualization of SNARE-Mediated Hemifusion between Giant Unilamellar Vesicles Arrested by Myricetin.

Neurotransmitters are released within a millisecond after Ca2+ arrives at an active zone. However, the vesicle fusion pathway underlying this synchronous release is yet to be understood. At the center of controversy is whether hemifusion, in which outer leaflets are merged while inner leaflets are still separated, is an on-pathway or off-pathway product of Ca2+-triggered exocytosis. Using the single vesicle fusion assay, we recently demonstrated that hemifusion is an on-pathway intermediate that immediately proceeds to full fusion upon Ca2+ triggering. It has been shown that the flavonoid myricetin arrests soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptor (SNARE)-mediated vesicle fusion at hemifusion, but that the hemifused vesicles spontaneously convert to full fusion when the myricetin clamp is removed by the enzyme laccase. In the present study, we visualized SNARE-mediated hemifusion between two SNARE-reconstituted giant unilamellar vesicles (GUVs) arrested by myricetin. The large size of the GUVs enabled us to directly image the hemifusion between them. When two merging GUVs were labeled with different fluorescent dyes, GUV pairs showed asymmetric fluorescence intensities depending on the position on the GUV pair consistent with what is expected for hemifusion. The flow of lipids from one vesicle to the other was revealed with fluorescence recovery after photobleaching (FRAP), indicating that the two membranes had hemifused. These results support the hypothesis that hemifusion may be the molecular status that primes Ca2+-triggered millisecond exocytosis. This study represents the first imaging of SNARE-driven hemifusion between GUVs.

Inositol pyrophosphates inhibit synaptotagmin-dependent exocytosis.

Inositol pyrophosphates such as 5-diphosphoinositol pentakisphosphate (5-IP7) are highly energetic inositol metabolites containing phosphoanhydride bonds. Although inositol pyrophosphates are known to regulate various biological events, including growth, survival, and metabolism, the molecular sites of 5-IP7 action in vesicle trafficking have remained largely elusive. We report here that elevated 5-IP7 levels, caused by overexpression of inositol hexakisphosphate (IP6) kinase 1 (IP6K1), suppressed depolarization-induced neurotransmitter release from PC12 cells. Conversely, IP6K1 depletion decreased intracellular 5-IP7 concentrations, leading to increased neurotransmitter release. Consistently, knockdown of IP6K1 in cultured hippocampal neurons augmented action potential-driven synaptic vesicle exocytosis at synapses. Using a FRET-based in vitro vesicle fusion assay, we found that 5-IP7, but not 1-IP7, exhibited significantly higher inhibitory activity toward synaptic vesicle exocytosis than IP6 Synaptotagmin 1 (Syt1), a Ca(2+) sensor essential for synaptic membrane fusion, was identified as a molecular target of 5-IP7 Notably, 5-IP7 showed a 45-fold higher binding affinity for Syt1 compared with IP6 In addition, 5-IP7-dependent inhibition of synaptic vesicle fusion was abolished by increasing Ca(2+) levels. Thus, 5-IP7 appears to act through Syt1 binding to interfere with the fusogenic activity of Ca(2+) These findings reveal a role of 5-IP7 as a potent inhibitor of Syt1 in controlling the synaptic exocytotic pathway and expand our understanding of the signaling mechanisms of inositol pyrophosphates.

Biophysical characterization of the structural change of Nopp140, an intrinsically disordered protein, in the interaction with CK2α.

Nucleolar phosphoprotein 140 (Nopp140) is a nucleolar protein, more than 80% of which is disordered. Previous studies have shown that the C-terminal region of Nopp140 (residues 568-596) interacts with protein kinase CK2α, and inhibits the catalytic activity of CK2. Although the region of Nopp140 responsible for the interaction with CK2α was identified, the structural features and the effect of this interaction on the structure of Nopp140 have not been defined due to the difficulty of structural characterization of disordered protein. In this study, the disordered feature of Nopp140 and the effect of CK2α on the structure of Nopp140 were examined using single-molecule fluorescence resonance energy transfer (smFRET) and electron paramagnetic resonance (EPR). The interaction with CK2α was increased conformational rigidity of the CK2α-interacting region of Nopp140 (Nopp140C), suggesting that the disordered and flexible conformation of Nopp140C became more rigid conformation as it binds to CK2α. In addition, site specific spin labeling and EPR analysis confirmed that the residues 574-589 of Nopp140 are critical for binding to CK2α. Similar technical approaches can be applied to analyze the conformational changes in other IDPs during their interactions with binding partners.