In situ architecture of the ER-mitochondria encounter structure.

The endoplasmic reticulum and mitochondria are main hubs of eukaryotic membrane biogenesis that rely on lipid exchange via membrane contact sites1-3, but the underpinning mechanisms remain poorly understood. In yeast, tethering and lipid transfer between the two organelles is mediated by the endoplasmic reticulum-mitochondria encounter structure (ERMES), a four-subunit complex of unresolved stoichiometry and architecture4-6. Here we determined the molecular organization of ERMES within Saccharomyces cerevisiae cells using integrative structural biology by combining quantitative live imaging, cryo-correlative microscopy, subtomogram averaging and molecular modelling. We found that ERMES assembles into approximately 25 discrete bridge-like complexes distributed irregularly across a contact site. Each bridge consists of three synaptotagmin-like mitochondrial lipid binding protein domains oriented in a zig-zag arrangement. Our molecular model of ERMES reveals a pathway for lipids. These findings resolve the in situ supramolecular architecture of a major inter-organelle lipid transfer machinery and provide a basis for the mechanistic understanding of lipid fluxes in eukaryotic cells.

Comprehensive analysis on the expression profile and prognostic values of Synaptotagmins (SYTs) family members and their methylation levels in gastric cancer.

Synaptotagmins (SYTs), constitute a family of 17 membrane-trafficking protein, palying crucial roles in the development and progression of human cancers. However, only very few studies have investigated the expression profile and prognostic values of SYTs family members in gastric cancer (GC). Therefore, we comprehensively evaluated the expression, methylation, prognosis and immune significance of SYTs family members through bioinformatics analysis from the online databases in GC. The expressions of SYT4, SYT9, and SYT14 were up-regulated, and negatively associated with their methylation levels in GC. Both the over-expression of SYT4, SYT9 and SYT14 and their hypomethylation levels contributed to an unsatisfactory overall survival (OS) and progression-free survival (PFS) in GC. Moreover, the low expressions of several methylation cg sites (cg02795029, cg07581146, cg15149095, cg19922137, cg25371503, cg26158959, cg02269161, cg03226737, cg08185661, cg16437728, cg22723056 and cg24678137) were significantly correlated with an unfavorable OS and PFS in GC. Furthermore, the expression of SYT4, SYT9 and SYT14 played a pivotal role in immune cells infiltration in GC. Collectively, our current finding suggested that SYT4, SYT9 and SYT14 might be potent prognostic indictors and promising immunotherapeutic targets for GC patients.

Function of Drosophila Synaptotagmins in membrane trafficking at synapses.

The Synaptotagmin (SYT) family of proteins play key roles in regulating membrane trafficking at neuronal synapses. Using both Ca2+-dependent and Ca2+-independent interactions, several SYT isoforms participate in synchronous and asynchronous fusion of synaptic vesicles (SVs) while preventing spontaneous release that occurs in the absence of stimulation. Changes in the function or abundance of the SYT1 and SYT7 isoforms alter the number and route by which SVs fuse at nerve terminals. Several SYT family members also regulate trafficking of other subcellular organelles at synapses, including dense core vesicles (DCV), exosomes, and postsynaptic vesicles. Although SYTs are linked to trafficking of multiple classes of synaptic membrane compartments, how and when they interact with lipids, the SNARE machinery and other release effectors are still being elucidated. Given mutations in the SYT family cause disorders in both the central and peripheral nervous system in humans, ongoing efforts are defining how these proteins regulate vesicle trafficking within distinct neuronal compartments. Here, we review the Drosophila SYT family and examine their role in synaptic communication. Studies in this invertebrate model have revealed key similarities and several differences with the predicted activity of their mammalian counterparts. In addition, we highlight the remaining areas of uncertainty in the field and describe outstanding questions on how the SYT family regulates membrane trafficking at nerve terminals.

The C2A domain of synaptotagmin is an essential component of the calcium sensor for synaptic transmission.

The synaptic vesicle protein, synaptotagmin, is the principle Ca2+ sensor for synaptic transmission. Ca2+ influx into active nerve terminals is translated into neurotransmitter release by Ca2+ binding to synaptotagmin's tandem C2 domains, triggering the fast, synchronous fusion of multiple synaptic vesicles. Two hydrophobic residues, shown to mediate Ca2+-dependent membrane insertion of these C2 domains, are required for this process. Previous research suggested that one of its tandem C2 domains (C2B) is critical for fusion, while the other domain (C2A) plays only a facilitatory role. However, the function of the two hydrophobic residues in C2A have not been adequately tested in vivo. Here we show that these two hydrophobic residues are absolutely required for synaptotagmin to trigger vesicle fusion. Using in vivo electrophysiological recording at the Drosophila larval neuromuscular junction, we found that mutation of these two key C2A hydrophobic residues almost completely abolished neurotransmitter release. Significantly, mutation of both hydrophobic residues resulted in more severe deficits than those seen in synaptotagmin null mutants. Thus, we report the most severe phenotype of a C2A mutation to date, demonstrating that the C2A domain is absolutely essential for synaptotagmin's function as the electrostatic switch.

Synaptotagmin Binding to Botulinum Neurotoxins.

Botulinum neurotoxins (BoNTs) are exceptionally toxic proteins that cause paralysis but are also extensively used as treatment for various medical conditions. Most BoNTs bind two receptors on neuronal cells, namely, a ganglioside and a protein receptor. Differences in the sequence between the protein receptors from different species can impact the binding affinity and toxicity of the BoNTs. Here we have investigated how BoNT/B, /DC, and /G, all three toxins that utilize synaptotagmin I and II (Syt-I and Syt-II, respectively) as their protein receptors, bind to Syt-I and -II of mouse/rat, bovine, and human origin by isothermal titration calorimetry analysis. BoNT/G had the highest affinity for human Syt-I, and BoNT/DC had the highest affinity for bovine Syt-II. As expected, BoNT/B, /DC, and /G showed very low levels of binding to human Syt-II. Furthermore, we carried out saturation transfer difference (STD) and STD-TOCSY NMR experiments that revealed the region of the Syt peptide in direct contact with BoNT/G, which demonstrate that BoNT/G recognizes the Syt peptide in a model similar to that in the established BoNT/B-Syt-II complex. Our analyses also revealed that regions outside the Syt peptide's toxin-binding region are important for the helicity of the peptide and, therefore, the binding affinity.

In Vitro Assays to Measure the Membrane Tethering and Lipid Transport Activities of the Extended Synaptotagmins.

The three extended synaptotagmins (E-Syts) are endoplasmic reticulum (ER)-localized membrane proteins that mediate tethering of the ER to the plasma membrane (PM) via C2 domain-dependent interactions regulated by Ca2+ and/or PI(4,5)P2. The E-Syts also contains a Synaptotagmin-like Mitochondrial lipid-binding Protein (SMP) domain, a lipid-harboring module through which they mediate lipid transport between the two adjacent membranes. Here, we describe in vitro liposome-based methods to study the membrane tethering and lipid transport functions of E-Syt1. Its membrane tethering activity is monitored through a turbidity-based assay, and its lipid transport property is analyzed via fluorescence resonance energy transfer (FRET)-based assay. These in vitro methods have enabled us to gain insight into the mechanism of action and regulation of E-Syt1, such as the role of Ca2+ in releasing E-Syt1 from an autoinhibitory conformation. The same methods could be adapted to the study of other lipid transport proteins that function at membrane contact sites.

Membrane-Binding Cooperativity and Coinsertion by C2AB Tandem Domains of Synaptotagmins 1 and 7.

Synaptotagmin-1 (Syt-1) and synaptotagmin-7 (Syt-7) contain analogous tandem C2 domains, C2A and C2B, which together sense Ca2+ to bind membranes and promote the stabilization of exocytotic fusion pores. Syt-1 triggers fast release of neurotransmitters, whereas Syt-7 functions in processes that involve lower Ca2+ concentrations such as hormone secretion. Syt-1 C2 domains are reported to bind membranes cooperatively, based on the observation that they penetrate farther into membranes as the C2AB tandem than as individual C2 domains. In contrast, we previously suggested that the two C2 domains of Syt-7 bind membranes independently, based in part on measurements of their liposome dissociation kinetics. Here, we investigated C2A-C2B interdomain cooperativity with Syt-1 and Syt-7 using directly comparable measurements. Equilibrium Ca2+ titrations demonstrate that the Syt-7 C2AB tandem binds liposomes lacking phosphatidylinositol-4,5-bisphosphate (PIP2) with greater Ca2+ sensitivity than either of its individual domains and binds to membranes containing PIP2 even in the absence of Ca2+. Stopped-flow kinetic measurements show differences in cooperativity between Syt-1 and Syt-7: Syt-1 C2AB dissociates from PIP2-free liposomes much more slowly than either of its individual C2 domains, indicating cooperativity, whereas the major population of Syt-7 C2AB has a dissociation rate comparable to its C2A domain, suggesting a lack of cooperativity. A minor subpopulation of Syt-7 C2AB dissociates at a slower rate, which could be due to a small cooperative component and/or liposome clustering. Measurements using an environment-sensitive fluorescent probe indicate that the Syt-7 C2B domain inserts deeply into membranes as part of the C2AB tandem, similar to the coinsertion previously reported for Syt-1. Overall, coinsertion of C2A and C2B domains is coupled to cooperative energetic effects in Syt-1 to a much greater extent than in Syt-7. The difference can be understood in terms of the relative contributions of C2A and C2B domains toward membrane binding in the two proteins.

Multiple factors maintain assembled trans-SNARE complexes in the presence of NSF and αSNAP.

Neurotransmitter release requires formation of trans-SNARE complexes between the synaptic vesicle and plasma membranes, which likely underlies synaptic vesicle priming to a release-ready state. It is unknown whether Munc18-1, Munc13-1, complexin-1 and synaptotagmin-1 are important for priming because they mediate trans-SNARE complex assembly and/or because they prevent trans-SNARE complex disassembly by NSF-αSNAP, which can lead to de-priming. Here we show that trans-SNARE complex formation in the presence of NSF-αSNAP requires both Munc18-1 and Munc13-1, as proposed previously, and is facilitated by synaptotagmin-1. Our data also show that Munc18-1, Munc13-1, complexin-1 and likely synaptotagmin-1 contribute to maintaining assembled trans-SNARE complexes in the presence of NSF-αSNAP. We propose a model whereby Munc18-1 and Munc13-1 are critical not only for mediating vesicle priming but also for precluding de-priming by preventing trans-SNARE complex disassembly; in this model, complexin-1 also impairs de-priming, while synaptotagmin-1 may assist in priming and hinder de-priming.

A molecular mechanism for calcium-mediated synaptotagmin-triggered exocytosis.

The regulated exocytotic release of neurotransmitter and hormones is accomplished by a complex protein machinery whose core consists of SNARE proteins and the calcium sensor synaptotagmin-1. We propose a mechanism in which the lipid membrane is intimately involved in coupling calcium sensing to release. We found that fusion of dense core vesicles, derived from rat PC12 cells, was strongly linked to the angle between the cytoplasmic domain of the SNARE complex and the plane of the target membrane. We propose that, as this tilt angle increases, force is exerted on the SNARE transmembrane domains to drive the merger of the two bilayers. The tilt angle markedly increased following calcium-mediated binding of synaptotagmin to membranes, strongly depended on the surface electrostatics of the membrane, and was strictly coupled to the lipid order of the target membrane.

The high-affinity calcium sensor synaptotagmin-7 serves multiple roles in regulated exocytosis.

Synaptotagmin (Syt) proteins comprise a 17-member family, many of which trigger exocytosis in response to calcium. Historically, most studies have focused on the isoform Syt-1, which serves as the primary calcium sensor in synchronous neurotransmitter release. Recently, Syt-7 has become a topic of broad interest because of its extreme calcium sensitivity and diversity of roles in a wide range of cell types. Here, we review the known and emerging roles of Syt-7 in various contexts and stress the importance of its actions. Unique functions of Syt-7 are discussed in light of recent imaging, electrophysiological, and computational studies. Particular emphasis is placed on Syt-7-dependent regulation of synaptic transmission and neuroendocrine cell secretion. Finally, based on biochemical and structural data, we propose a mechanism to link Syt-7's role in membrane fusion with its role in subsequent fusion pore expansion via strong calcium-dependent phospholipid binding.

Formation of G-wires, bimolecular and tetramolecular quadruplex: Cation-induced structural polymorphs of G-rich DNA sequence of human SYTX gene.

An exceptional property of auto-folding into a range of intra- as well as intermolecular quadruplexes by guanine-rich oligomers (GROs) of promoters, telomeres and various other genomic locations is still one of the most attractive areas of research at present times. The main reason for this attention is due to their established in vivo existence and biological relevance. Herein, the structural status of a 20-nt long G-rich sequence with two G5 stretches (SG20) is investigated using various biophysical and biochemical techniques. Bioinformatics analysis suggested the presence of a 17-nt stretch of this SG20 sequence in the intronic region of human SYTX (Synaptotagmin 10) gene. The SYTX gene helps in sensing out the Ca2+ ion, causing its intake in the pre-synaptic neuron. A range of various topologies like bimolecular, tetramolecular and guanine-wires (nano-wires) was exhibited by the studied sequence, as a function of cations (Na+ /K+ ) concentration. UV-thermal denaturation, gel electrophoresis, and circular dichroism (CD) spectroscopy showed correlations and established a cation-dependent structural switch. The G-wire formation, in the presence of K+ , may further be explored for its possible relevance in nano-biotechnological applications.

Structural Impact of Phosphorylation and Dielectric Constant Variation on Synaptotagmin's IDR.

We used time-resolved Förster resonance energy transfer, circular dichroism, and molecular dynamics simulation to investigate the structural dependence of synaptotagmin 1's intrinsically disordered region (IDR) on phosphorylation and dielectric constant. We found that a peptide corresponding to the full-length IDR sequence, a ∼60-residue strong polyampholyte, can sample structurally collapsed states in aqueous solution, consistent with its κ-predicted behavior, where κ is a sequence-dependent parameter that is used to predict IDR compaction. In implicit solvent simulations of this same sequence, lowering the dielectric constant to more closely mimic the environment near a lipid bilayer surface promoted further sampling of collapsed structures. We then examined the structural tendencies of central region residues of the IDR in isolation. We found that the exocytosis-modulating phosphorylation of Thr112 disrupts a local disorder-to-order transition induced by trifluoroethanol/water mixtures that decrease the solution dielectric constant and stabilize helical structure. Implicit solvent simulations on these same central region residues testing the impact of dielectric constant alone converge on a similar result, showing that helical structure is formed with higher probability at a reduced dielectric. In these helical conformers, lysine-aspartic acid salt bridges contribute to stabilization of transient secondary structure. In contrast, phosphorylation results in formation of salt bridges unsuitable for helix formation. Collectively, these results suggest a model in which phosphorylation and compaction of the IDR sequence regulate structural transitions that in turn modulate neuronal exocytosis.

The Krebs Cycle Enzyme Isocitrate Dehydrogenase 3A Couples Mitochondrial Metabolism to Synaptic Transmission.

Neurotransmission is a tightly regulated Ca2+-dependent process. Upon Ca2+ influx, Synaptotagmin1 (Syt1) promotes fusion of synaptic vesicles (SVs) with the plasma membrane. This requires regulation at multiple levels, but the role of metabolites in SV release is unclear. Here, we uncover a role for isocitrate dehydrogenase 3a (idh3a), a Krebs cycle enzyme, in neurotransmission. Loss of idh3a leads to a reduction of the metabolite, alpha-ketoglutarate (αKG), causing defects in synaptic transmission similar to the loss of syt1. Supplementing idh3a flies with αKG suppresses these defects through an ATP or neurotransmitter-independent mechanism. Indeed, αKG, but not glutamate, enhances Syt1-dependent fusion in a reconstitution assay. αKG promotes interaction between the C2-domains of Syt1 and phospholipids. The data reveal conserved metabolic regulation of synaptic transmission via αKG. Our studies provide a synaptic role for αKG, a metabolite that has been proposed as a treatment for aging and neurodegenerative disorders.

Emerging Functional Differences between the Synaptotagmin and Ferlin Calcium Sensor Families.

The ferlin family proteins have emerged as multi-C2 domain regulators of calcium-triggered membrane fusion and fission events. While initially determined to share many of the features of members of the synaptotagmin family of calcium sensors, ferlins in more recent studies have been found to interact directly with non-neuronal voltage-gated calcium channels and nucleate the assembly of membrane-trafficking protein complexes, functions that distinguish them from the more well studied members of the synaptotagmin family. Here we highlight some of the recent findings that have advanced our understanding of ferlins and their functional differences with the synaptotagmin family.

Drosophila studies support a role for a presynaptic synaptotagmin mutation in a human congenital myasthenic syndrome.

During chemical transmission, the function of synaptic proteins must be coordinated to efficiently release neurotransmitter. Synaptotagmin 2, the Ca2+ sensor for fast, synchronized neurotransmitter release at the human neuromuscular junction, has recently been implicated in a dominantly inherited congenital myasthenic syndrome associated with a non-progressive motor neuropathy. In one family, a proline residue within the C2B Ca2+-binding pocket of synaptotagmin is replaced by a leucine. The functional significance of this residue has not been investigated previously. Here we show that in silico modeling predicts disruption of the C2B Ca2+-binding pocket, and we examine the in vivo effects of the homologous mutation in Drosophila. When expressed in the absence of native synaptotagmin, this mutation is lethal, demonstrating for the first time that this residue plays a critical role in synaptotagmin function. To achieve expression similar to human patients, the mutation is expressed in flies carrying one copy of the wild type synaptotagmin gene. We now show that Drosophila carrying this mutation developed neurological and behavioral manifestations similar to those of human patients and provide insight into the mechanisms underlying these deficits. Our Drosophila studies support a role for this synaptotagmin point mutation in disease etiology.

Exceptionally tight membrane-binding may explain the key role of the synaptotagmin-7 C2A domain in asynchronous neurotransmitter release.

Synaptotagmins (Syts) act as Ca2+ sensors in neurotransmitter release by virtue of Ca2+-binding to their two C2 domains, but their mechanisms of action remain unclear. Puzzlingly, Ca2+-binding to the C2B domain appears to dominate Syt1 function in synchronous release, whereas Ca2+-binding to the C2A domain mediates Syt7 function in asynchronous release. Here we show that crystal structures of the Syt7 C2A domain and C2AB region, and analyses of intrinsic Ca2+-binding to the Syt7 C2 domains using isothermal titration calorimetry, did not reveal major differences that could explain functional differentiation between Syt7 and Syt1. However, using liposome titrations under Ca2+ saturating conditions, we show that the Syt7 C2A domain has a very high membrane affinity and dominates phospholipid binding to Syt7 in the presence or absence of l-α-phosphatidylinositol 4,5-diphosphate (PIP2). For Syt1, the two Ca2+-saturated C2 domains have similar affinities for membranes lacking PIP2, but the C2B domain dominates binding to PIP2-containing membranes. Mutagenesis revealed that the dramatic differences in membrane affinity between the Syt1 and Syt7 C2A domains arise in part from apparently conservative residue substitutions, showing how striking biochemical and functional differences can result from the cumulative effects of subtle residue substitutions. Viewed together, our results suggest that membrane affinity may be a key determinant of the functions of Syt C2 domains in neurotransmitter release.

Endoplasmic Reticulum - Plasma Membrane Crosstalk Mediated by the Extended Synaptotagmins.

The endoplasmic reticulum (ER) possesses multiplicity of functions including protein synthesis, membrane lipid biogenesis, and Ca2+ storage and has broad localization throughout the cell. While the ER and most other membranous organelles are highly interconnected via vesicular traffic that relies on membrane budding and fusion reactions, the ER forms direct contacts with virtually all other membranous organelles, including the plasma membrane (PM), without membrane fusion. Growing evidence suggests that these contacts play major roles in cellular physiology, including the regulation of Ca2+ homeostasis and signaling and control of cellular lipid homeostasis. Extended synaptotagmins (E-Syts) are evolutionarily conserved family of ER-anchored proteins that tether the ER to the PM in PM PI(4,5)P2-dependent and cytosolic Ca2+-regulated manner. In addition, E-Syts possess a cytosolically exposed lipid-harboring module that confers the ability to transfer/exchange glycerolipids between the ER and the PM at E-Syts-mediated ER-PM contacts. In this chapter, the functions of ER-PM contacts and their role in non-vesicular lipid transport with special emphasis on the crosstalk between the two bilayers mediated by E-Syts will be discussed.

The Extended-Synaptotagmins.

The extended-synaptotagmins (tricalbins in yeast) derive their name from their partial domain structure similarity to the synaptotagmins, which are characterized by an N-terminal membrane anchor and cytosolically exposed C2 domains. However, they differ from the synaptotagmins in localization and function. The synaptotagmins tether secretory vesicles, including synaptic vesicles, to the plasma membrane (PM) via their C2 domains and regulate their Ca2+ triggered exocytosis. In contrast, the extended-synaptotagmins are resident proteins of the endoplasmic reticulum (ER), which tether this organelle to the plasma membrane via their C2 domains, but not as a premise to fusion of the two membranes. They transport glycerolipids between the two bilayers via their lipid-harboring SMP domains and Ca2+ regulates their membrane tethering and lipid transport function. Additionally, the extended-synaptotagmins are more widely expressed in different organisms, as they are present not only in animal cells, but also in fungi and plants, which do not express the synaptotagmins. Thus, they have a more general function than the synaptotagmins, whose appearance in animal species correlated with the occurrence of Ca2+ triggered exocytosis. This article is part of a Special Issue entitled: Membrane Contact Sites edited by Christian Ungermann and Benoit Kornmann.

Structural analysis of Ca2+-binding pocket of synaptotagmin 5 C2A domain.

Synaptotagmins constitute a family of multifunctional integral membrane proteins found predominantly on vesicles in neural and endocrine tissues. 17 isoforms of synaptotagmin family in mammals have been identified, 7 isoforms among them are known to be able to bind Ca2+ via their C2 domains. This study presents the crystal structure of the first C2 domain (C2A domain) of synaptotagmin 5 complexed with Ca2+ at 1.90Å resolution. Comparison of the Ca2+-binding pocket of synaptotagmin 5 C2A domain with other synaptotagmin C2 domains demonstrated that a serine residue locating at Ca2+-binding loop probably responsible to the conformational variation of Ca2+-binding pocket, and thus impacts the Ca2+-binding mechanism of C2 domain, which is verified by structural analysis of the serine mutant and Ca2+-binding assays via isothermal titration calorimetry. Alteration of Ca2+-binding mechanism might be correlated with different Ca2+ response rates of synaptotagmins, which is the basis of the functions of synaptotagmins in regulating various types of Ca2+-triggered vesicle-membrane fusion processes.

Differential Membrane Binding Mechanics of Synaptotagmin Isoforms Observed in Atomic Detail.

Synaptotagmin (Syt) is a membrane-associated protein involved in vesicle fusion through the SNARE complex that is found throughout the human body in 17 different isoforms. These isoforms have two membrane-binding C2 domains, which sense Ca2+ and thereby promote anionic membrane binding and lead to vesicle fusion. Through molecular dynamics simulations using the highly mobile membrane mimetic acclerated bilayer model, we have investigated how small protein sequence changes in the Ca2+-binding loops of the C2 domains may give rise to the experimentally determined difference in binding kinetics between Syt-1 and Syt-7 isoforms. Syt-7 C2 domains are found to form more close contacts with anionic phospholipid headgroups, particularly in loop 1, where an additional positive charge in Syt-7 draws the loop closer to the membrane and causes the anchoring residue F167 to insert deeper into the bilayer than the corresponding methionine in Syt-1 (M173). By performing additional replica exchange umbrella sampling calculations, we demonstrate that these additional contacts increase the energetic cost of unbinding the Syt-7 C2 domains from the bilayer, causing them to unbind more slowly than their counterparts in Syt-1.