Dynamics and number of trans-SNARE complexes determine nascent fusion pore properties.

The fusion pore is the first crucial intermediate formed during exocytosis, yet little is known about the mechanisms that determine the size and kinetic properties of these transient structures. Here, we reduced the number of available SNAREs (proteins that mediate vesicle fusion) in neurons and observed changes in transmitter release that are suggestive of alterations in fusion pores. To investigate these changes, we employed reconstituted fusion assays using nanodiscs to trap pores in their initial open state. Optical measurements revealed that increasing the number of SNARE complexes enhanced the rate of release from single pores and enabled the escape of larger cargoes. To determine whether this effect was due to changes in nascent pore size or to changes in stability, we developed an approach that uses nanodiscs and planar lipid bilayer electrophysiology to afford microsecond resolution at the single event level. Both pore size and stability were affected by SNARE copy number. Increasing the number of vesicle (v)-SNAREs per nanodisc from three to five caused a twofold increase in pore size and decreased the rate of pore closure by more than three orders of magnitude. Moreover, pairing of v-SNAREs and target (t)-SNAREs to form trans-SNARE complexes was highly dynamic: flickering nascent pores closed upon addition of a v-SNARE fragment, revealing that the fully assembled, stable SNARE complex does not form at this stage of exocytosis. Finally, a deletion at the base of the SNARE complex, which mimics the action of botulinum neurotoxin A, markedly reduced fusion pore stability. In summary, trans-SNARE complexes are dynamic, and the number of SNAREs recruited to drive fusion determines fundamental properties of individual pores.

In situ structure determination at nanometer resolution using TYGRESS.

The resolution of subtomogram averages calculated from cryo-electron tomograms (cryo-ET) of crowded cellular environments is often limited owing to signal loss in, and misalignment of, the subtomograms. By contrast, single-particle cryo-electron microscopy (SP-cryo-EM) routinely reaches near-atomic resolution of isolated complexes. We report a method called 'tomography-guided 3D reconstruction of subcellular structures' (TYGRESS) that is a hybrid of cryo-ET and SP-cryo-EM, and is able to achieve close-to-nanometer resolution of complexes inside crowded cellular environments. TYGRESS combines the advantages of SP-cryo-EM (images with good signal-to-noise ratio and contrast, as well as minimal radiation damage) and subtomogram averaging (three-dimensional alignment of macromolecules in a complex sample). Using TYGRESS, we determined the structure of the intact ciliary axoneme with up to resolution of 12 Å. These results reveal many structural details that were not visible by cryo-ET alone. TYGRESS is generally applicable to cellular complexes that are amenable to subtomogram averaging.

Phosphatidylinositol 4,5-bisphosphate drives Ca2+-independent membrane penetration by the tandem C2 domain proteins synaptotagmin-1 and Doc2ß.

Exocytosis mediates the release of neurotransmitters and hormones from neurons and neuroendocrine cells. Tandem C2 domain proteins in the synaptotagmin (syt) and double C2 domain (Doc2) families regulate exocytotic membrane fusion via direct interactions with Ca2+ and phospholipid bilayers. Syt1 is a fast-acting, low-affinity Ca2+ sensor that penetrates membranes upon binding Ca2+ to trigger synchronous vesicle fusion. The closely related Doc2ß is a slow-acting, high-affinity Ca2+ sensor that triggers spontaneous and asynchronous vesicle fusion, but whether it also penetrates membranes is unknown. Both syt1 and Doc2ß bind the dynamically regulated plasma membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP2), but it is unclear whether PIP2 serves only as a membrane contact or enables specialized membrane-binding modes by these Ca2+ sensors. Furthermore, it has been shown that PIP2 uncaging can trigger rapid, syt1-dependent exocytosis in the absence of Ca2+ influx, suggesting that current models for the action of these Ca2+ sensors are incomplete. Here, using a series of steady-state and time-resolved fluorescence measurements, we show that Doc2ß, like syt1, penetrates membranes in a Ca2+-dependent manner. Unexpectedly, we observed that PIP2 can drive membrane penetration by both syt1 and Doc2ß in the absence of Ca2+, providing a plausible mechanism for Ca2+-independent, PIP2-dependent exocytosis. Quantitative measurements of penetration depth revealed that, in the presence of Ca2+, PIP2 drives Doc2ß, but not syt1, substantially deeper into the membrane, defining a biophysical regulatory mechanism specific to this high-affinity Ca2+ sensor. Our results provide evidence of a novel role for PIP2 in regulating, and under some circumstances triggering, exocytosis.

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.

Synaptotagmin-1 is an antagonist for Munc18-1 in SNARE zippering.

In neuroexocytosis, SNAREs and Munc18-1 may consist of the minimal membrane fusion machinery. Consistent with this notion, we observed, using single molecule fluorescence assays, that Munc18-1 stimulates SNARE zippering and SNARE-dependent lipid mixing in the absence of a major Ca(2+) sensor synaptotagmin-1 (Syt1), providing the structural basis for the conserved function of Sec1/Munc18 proteins in exocytosis. However, when full-length Syt1 is present, no enhancement of SNARE zippering and no acceleration of Ca(2+)-triggered content mixing by Munc18-1 are observed. Thus, our results show that Syt1 acts as an antagonist for Munc18-1 in SNARE zippering and fusion pore opening. Although the Sec1/Munc18 family may serve as part of the fusion machinery in other exocytotic pathways, Munc18-1 may have evolved to play a different role, such as regulating syntaxin-1a in neuroexocytosis.

Lipid molecules influence early stages of yeast SNARE-mediated membrane fusion.

Lipid molecules, structural components of biomembranes, have been proposed for an important role in membrane fusion. Through various techniques based on a protein-reconstituted vesicle-vesicle fusion system, we investigated the influence of several lipid molecules on different stages of a yeast soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-mediated membrane fusion process. Lipid compositions played a significant role in the early stages, docking and lipid mixing, while only exhibiting a minor effect on fusion pore formation and dilation phases, indicated by both small and large content mixing.

Multiple conformations of a single SNAREpin between two nanodisc membranes reveal diverse pre-fusion states.

SNAREpins must be formed between two membranes to allow vesicle fusion, a required process for neurotransmitter release. Although its post-fusion structure has been well characterized, pre-fusion conformations have been elusive. We used single-molecule FRET and EPR to investigate the SNAREpin assembled between two nanodisc membranes. The SNAREpin shows at least three distinct dynamic states, which might represent pre-fusion intermediates. Although the N-terminal half above the conserved ionic layer maintains a robust helical bundle structure, the membrane-proximal C-terminal half shows high FRET, representing a helical bundle (45%), low FRET, reflecting a frayed conformation (39%) or mid FRET revealing an as-yet unidentified structure (16%). It is generally thought that SNAREpins are trapped at a partially zipped conformation in the pre-fusion state, and complete SNARE (soluble N-ethylmaleimide-sensitive factor-attachment protein receptor) assembly happens concomitantly with membrane fusion. However, our results show that the complete SNARE complex can be formed without membrane fusion, which suggests that the complete SNAREpin formation could precede membrane fusion, providing an ideal access to the fusion regulators such as complexins and synaptotagmin 1.

Nonaggregated α-synuclein influences SNARE-dependent vesicle docking via membrane binding.

α-Synuclein (α-Syn), a major component of Lewy body that is considered as the hallmark of Parkinson's disease (PD), has been implicated in neuroexocytosis. Overexpression of α-Syn decreases the neurotransmitter release. However, the mechanism by which α-Syn buildup inhibits the neurotransmitter release is still unclear. Here, we investigated the effect of nonaggregated α-Syn on SNARE-dependent liposome fusion using fluorescence methods. In ensemble in vitro assays, α-Syn reduces lipid mixing mediated by SNAREs. Furthermore, with the more advanced single-vesicle assay that can distinguish vesicle docking from fusion, we found that α-Syn specifically inhibits vesicle docking, without interfering with the fusion. The inhibition in vesicle docking requires α-Syn binding to acidic lipid containing membranes. Thus, these results imply the existence of at least two mechanisms of inhibition of SNARE-dependent membrane fusion: at high concentrations, nonaggregated α-Syn inhibits docking by binding acidic lipids but not v-SNARE; on the other hand, at much lower concentrations, large α-Syn oligomers inhibit via a mechanism that requires v-SNARE interaction [ Choi et al. Proc. Natl. Acad. Sci. U. S. A. 2013 , 110 ( 10 ), 4087 - 4092 ].

The synaptotagmin 1 linker may function as an electrostatic zipper that opens for docking but closes for fusion pore opening.

Syt1 (synaptotagmin 1), a major Ca2+ sensor for fast neurotransmitter release, contains tandem Ca2+-binding C2 domains (C2AB), a single transmembrane α-helix and a highly charged 60-residue-long linker in between. Using single-vesicle-docking and content-mixing assays we found that the linker region of Syt1 is essential for its two signature functions: Ca2+-independent vesicle docking and Ca2+-dependent fusion pore opening. The linker contains the basic-amino-acid-rich N-terminal region and the acidic-amino-acid-rich C-terminal region. When the charge segregation was disrupted, fusion pore opening was slowed, whereas docking was unchanged. Intramolecular disulfide cross-linking between N- and C-terminal regions of the linker or deletion of 40 residues from the linker reduced docking while enhancing pore opening, although the changes were subtle. EPR analysis showed Ca2+-induced line broadening reflecting a conformational change in the linker region. Thus the results of the present study suggest that the electrostatically bipartite linker region may extend for docking and fold to facilitate pore opening.

Structural and kinetic analysis of Saccharomyces cerevisiae thioredoxin Trx1: implications for the catalytic mechanism of GSSG reduced by the thioredoxin system.

Thioredoxin (Trx) and glutathione/glutaredoxin (GSH/Grx) systems play the dominant role in cellular redox homeostasis. Recently the Trx system has been shown to be responsible to control the balance of GSH/GSSG once the glutathione reductase system is not available. To decipher the structural basis of electron transfer from the Trx system to GSSG, we solved the crystal structures of oxidized Trx1 and glutathionylated Trx1Cys33Ser mutant at 1.76 and 1.80 A, respectively. Comparative structural analysis revealed a key residue Met35 involved in the Trx-GSSG recognition. Subsequent mutagenesis and kinetic studies proved that Met35Arg mutation could alter the apparent K(m) and V(max) values of the reaction. These findings gave us the structural insights into GSSG reduction catalyzed by the Trx system.

Chlamydomonas PKD2 organizes mastigonemes, hair-like glycoprotein polymers on cilia.

Mutations in the channel protein PKD2 cause autosomal dominant polycystic kidney disease, but the function of PKD2 in cilia remains unclear. Here, we show that PKD2 targets and anchors mastigonemes, filamentous polymers of the glycoprotein MST1, to the extracellular surface of Chlamydomonas cilia. PKD2-mastigoneme complexes physically connect to the axonemal doublets 4 and 8, positioning them perpendicular to the plane of ciliary beating. pkd2 mutant cilia lack mastigonemes, and mutant cells swim with reduced velocity, indicating a motility-related function of the PKD2-mastigoneme complex. Association with both the axoneme and extracellular structures supports a mechanosensory role of Chlamydomonas PKD2. We propose that PKD2-mastigoneme arrays, on opposing sides of the cilium, could perceive forces during ciliary beating and transfer these signals to locally regulate the response of the axoneme.

Structure of the thioredoxin-fold domain of human phosducin-like protein 2.

Human phosducin-like protein 2 (hPDCL2) has been identified as belonging to subgroup II of the phosducin (Pdc) family. The members of this family share an N-terminal helix domain and a C-terminal thioredoxin-fold (Trx-fold) domain. The X-ray crystal structure of the Trx-fold domain of hPDCL2 was solved at 2.70 A resolution and resembled the Trx-fold domain of rat phosducin. Comparative structural analysis revealed the structural basis of their putative functional divergence.

Purification, crystallization and preliminary X-ray diffraction analysis of glutathionylated Trx1 C33S mutant from yeast.

Thioredoxins (Trxs) are a family of small redox-active proteins that are found in all living organisms. In Saccharomyces cerevisiae, two cytosolic Trxs (Trx1 and Trx2) and one mitochondrial Trx (Trx3) have previously been identified. In this work, cytosolic Trx1 containing a C33S mutant was overexpressed, purified, glutathionylated and crystallized using the hanging-drop vapour-diffusion method. A set of X-ray diffraction data was collected to 1.80 A resolution. The crystal belonged to space group P1, with unit-cell parameters a = 38.53, b = 38.81, c = 41.70 A, alpha = 72.91, beta = 87.51, gamma = 60.58 degrees.

Catalytic metalloporphyrin protects against paraquat neurotoxicity in vivo.

OBJECTIVE: To examine the neuroprotective effects of a novel manganese porphyrin, manganese (III) meso-tetrakis (N,N'-diethylimidazolium-2-yl) porphyrin (MnTDM), in the mouse model of Parkinson's disease (PD) induced by paraquat (PQ). METHODS: Male C57BL/6 mice were subcutaneously injected with either saline or PQ at 2-day intervals for a total of 10 doses, MnTDM was subcutaneously injected with the PQ 2 h before treatment. Performance on the pole and swim test were measured 7 days after the last injection and animals were sacrificed one day later. Levels of dopamine (DA) and its metabolites in the striatum were measured by high-performance liquid chromatography with an electrochemical detector (HPLC-ECD). Thiobarbituric acid (TBA) method was used to assay the lipid peroxidation product, malondialdehyde (MDA), and the number of tyrosine hydroxylase (TH) positive neurons was estimated using immunohistochemistry. RESULTS: Pretreatment with MnTDM significantly attenuated PQ-impaired behavioral performance, depleted dopamine content in striata, increased MDA, and dopaminergic neuron loss in the substantia nigra. CONCLUSIONS: Oxidative stress plays an important role in PQ-induced neurotoxicity which can be potentially prevented by manganese porphyrin. These findings also propose a possible therapeutical strategy for neurodegenerative disorders associated with oxidative stress such as PD.

SNARE zippering.

SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins are a highly conserved set of membrane-associated proteins that mediate intracellular membrane fusion. Cognate SNAREs from two separate membranes zipper to facilitate membrane apposition and fusion. Though the stable post-fusion conformation of SNARE complex has been extensively studied with biochemical and biophysical means, the pathway of SNARE zippering has been elusive. In this review, we describe some recent progress in understanding the pathway of SNARE zippering. We particularly focus on the half-zippered intermediate, which is most likely to serve as the main point of regulation by the auxiliary factors.

Functional analysis of the interface between the tandem C2 domains of synaptotagmin-1.

C2 domains are widespread motifs that often serve as Ca(2+)-binding modules; some proteins have more than one copy. An open issue is whether these domains, when duplicated within the same parent protein, interact with one another to regulate function. In the present study, we address the functional significance of interfacial residues between the tandem C2 domains of synaptotagmin (syt)-1, a Ca(2+) sensor for neuronal exocytosis. Substitution of four residues, YHRD, at the domain interface, disrupted the interaction between the tandem C2 domains, altered the intrinsic affinity of syt-1 for Ca(2+), and shifted the Ca(2+) dependency for binding to membranes and driving membrane fusion in vitro. When expressed in syt-1 knockout neurons, the YHRD mutant yielded reductions in synaptic transmission, as compared with the wild-type protein. These results indicate that physical interactions between the tandem C2 domains of syt-1 contribute to excitation-secretion coupling.

Molecular origins of synaptotagmin 1 activities on vesicle docking and fusion pore opening.

Synaptotagmin 1 (Syt1), a major Ca(2+) sensor in neuroexocytosis, utilizes SNARE- and membrane-binding to regulate vesicle fusion, a required process for neurotransmitter release at the synapse. However, the mechanism by which Syt1 orchestrates SNARE- and membrane- binding to control individual vesicle fusion steps is still unclear. In this study, we used a number of single vesicle assays that can differentiate intermediates of neuroexocytosis, to focus on Syt1 mutants that might impair Syt1-SNARE/PIP2 interaction, Ca(2+)-binding, or membrane penetration. Our results show that, although putative Syt1-SNARE/PIP2 coupling through the polybasic region of the C2B domain is critical for vesicle docking, its disruption does not affect content release. In contrast, Ca(2+)-binding and membrane-penetration mutants significantly reduce content release. Our results thus delineate multiple functions of Syt1 along the pathway of Ca(2+)-triggered exocytosis in unprecedented detail.

Synaptotagmin 1 and Ca2+ drive trans SNARE zippering.

Synaptotagmin 1 (Syt1) is a major Ca(2+)-sensor that evokes neurotransmitter release. Here we used site-specific fluorescence resonance energy transfer (FRET) assay to investigate the effects of Syt1 on SNAREpin assembly. C2AB, a soluble version of Syt1, had virtually no stimulatory effect on the rate of the FRET at N-terminus of SNARE complex both with and without Ca(2+), indicating C2AB does not interfere with the initial nucleation of SNARE assembly. However, C2AB-Ca(2+) accelerated the FRET rate significantly at membrane proximal region, indicating C2AB-Ca(2+) promotes the transition from a partially assembled SNARE complex to the fusion-competent SNAREpin. Similar enhancement was also observed at the end of the transmembrane domain of SNARE proteins. The stimulatory effect disappeared if there was no membrane or only neutral membrane present.