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Background: Tetraspanin-7 (Tspan7) is an islet autoantigen involved in autoimmune type 1 diabetes and known to regulate beta-cell L-type Ca-2(+) channel activity. However, the role of Tspan7 in pancreatic beta-cell function is not yet fully understood.Methods: Histological analyses were conducted using immunostaining. Whole-body metabolism was tested using glucose tolerance test. Islet hormone secretion was quantified using static batch incubation or dynamic perifusion. beta-cell transmembrane currents, electrical activity and exocytosis were measured using whole-cell patch-clamping and capacitance measurements. Gene expression was studied using m RNA-sequencing and quantitative PCR.Results: Tspan7 is expressed in insulin-containing granules of pancreatic beta-cells and glucagon-producing alpha-cells. Tspan7 knockout mice (Tspan(gamma/-) mouse) exhibit reduced body weight and ad libitum plasma glucose but normal glucose tolerance. Tspan(gamma/- )islets have normal insulin content and glucose- or tolbutamide-stimulated insulin secretion. Depolarisation-triggered Ca2+ current was enhanced in Tspan(gamma/-) beta-cells, but beta-cell electrical activity and depolarisation-evoked exocytosis were unchanged suggesting that exocytosis was less sensitive to Ca2+. TSPAN7 knockdown (KD) in human pseudo-islets led to a significant reduction in insulin secretion stimulated by 20 mM Transcriptomic analyses show that TSPAN7 KD in human pseudo-islets correlated with changes in genes involved in hormone secretion, apoptosis and ER stress. Consistent with rodent beta-cells, exocytotic Ca2+ sensitivity was reduced in a human beta-cell line (EndoC-beta H1) following Tspan7 KD.Conclusion: Tspan7 is involved in the regulation of Ca2+-dependent exocytosis in beta-cells. Its function is more significant in human beta-cells than their rodent counterparts.

International audience; Botulinum neurotoxin serotype B (BoNT/B) uses two separate protein and polysialoglycolipid-binding pockets to interact with synaptotagmin 1/2 and gangliosides. However, an integrated model of BoNT/B bound to its neuronal receptors in a native membrane topology is still lacking. Using a panel of in silico and experimental approaches, we present here a new model for BoNT/B binding to neuronal membranes, in which the toxin binds to a preassembled synaptotagminganglioside GT1b complex and a free ganglioside allowing a lipid-binding loop of 2 BoNT/B to interact with the glycone part of the synaptotagmin-associated GT1b. Furthermore, our data provide molecular support for the decrease in BoNT/B sensitivity in Felidae that harbor the natural variant synaptotagmin2-N59Q. These results reveal multiple interactions of BoNT/B with gangliosides and support a novel paradigm in which a toxin recognizes a protein/ganglioside complex.

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.

Fusion pores serve as an effective mechanism to connect intracellular organelles and release vesicle contents during exocytosis. A complex lipid rearrangement takes place as membranes approximate, bend, fuse, and establish a traversing water channel to define the fusion pore, linking initially isolated chambers. Thermodynamically, the process is unfavorable and thought to be mediated by specialized proteins. In this work, we have developed a reaction coordinate to induce fusion pores from initially flat and parallel lipid bilayers and we have used it to describe the effects of the synaptotagmin-1 C2B domain during the process. We have obtained free-energy profiles of the whole lipid reorganization in biologically realistic membranes, going from planar and parallel bilayers through stalk hemifusion to water channel formation. Our results point to a lysine-rich polybasic region on synaptotagmin-1 C2B as the key to lipid reorganization control through the formation of phosphatidylinositol bisphosphate clusters that stabilize the fusion pore. Fil: Caparotta, Marcelo Rubén. Universidad Nacional de Cuyo; Argentina Fil: Tomes, Claudia Nora. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; Argentina Fil: Mayorga, Luis Segundo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; Argentina Fil: Masone, Diego Fernando. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; Argentina

Exocytosis is a Ca2+-regulated process that requires the participation of Ca2+ sensors. In the 1980s, two classes of Ca2+-binding proteins were proposed as putative Ca2+ sensors: EF-hand protein calmodulin, and the C2 domain protein synaptotagmin. In the next few decades, numerous studies determined that in the final stage of membrane fusion triggered by a micromolar boost in the level of Ca2+, the low affinity Ca2+-binding protein synaptotagmin, especially synaptotagmin 1 and 2, acts as the primary Ca2+ sensor, whereas calmodulin is unlikely to be functional due to its high Ca2+ affinity. However, in the meantime emerging evidence has revealed that calmodulin is involved in the earlier exocytotic steps prior to fusion, such as vesicle trafficking, docking and priming by acting as a high affinity Ca2+ sensor activated at submicromolar level of Ca2+. Calmodulin directly interacts with multiple regulatory proteins involved in the regulation of exocytosis, including VAMP, myosin V, Munc13, synapsin, GAP43 and Rab3, and switches on key kinases, such as type II Ca2+/calmodulin-dependent protein kinase, to phosphorylate a series of exocytosis regulators, including syntaxin, synapsin, RIM and Ca2+ channels. Moreover, calmodulin interacts with synaptotagmin through either direct binding or indirect phosphorylation. In summary, calmodulin and synaptotagmin are Ca2+ sensors that play complementary roles throughout the process of exocytosis. In this review, we discuss the complementary roles that calmodulin and synaptotagmin play as Ca2+ sensors during exocytosis.

Significance Synaptotagmin 1 (syt1) is a synaptic vesicle (SV) protein that is rapidly activated by Ca2+ influx into presynaptic nerve terminals, triggering SV exocytosis. Syt1 also inhibits exocytosis, prior to Ca2+ influx, and thus helps synchronize evoked exocytosis upon Ca2+ binding. Herein, we identified a cluster of lysine residues, in the oft-ignored juxtamembrane linker region of syt1, that governs homo-multimerization in an anionic lipid-dependent manner. Neutralization of this positively charged region abolished syt1 self-association on phospholipid bilayers in vitro. Subsequently, in neurons, we found mutations that disrupted syt1 self-association were correlated with defects in clamping spontaneous SV release and in triggering and synchronizing evoked exocytosis. Thus, syt1 regulates SV exocytosis as an oligomer via charged residues in the juxtamembrane linker., Synaptotagmin 1 (syt1) is a Ca2+ sensor that regulates synaptic vesicle exocytosis. Cell-based experiments suggest that syt1 functions as a multimer; however, biochemical and electron microscopy studies have yielded contradictory findings regarding putative self-association. Here, we performed dynamic light scattering on syt1 in solution, followed by electron microscopy, and we used atomic force microscopy to study syt1 self-association on supported lipid bilayers under aqueous conditions. Ring-like multimers were clearly observed. Multimerization was enhanced by Ca2+ and required anionic phospholipids. Large ring-like structures (∼180 nm) were reduced to smaller rings (∼30 nm) upon neutralization of a cluster of juxtamembrane lysine residues; further substitution of residues in the second C2-domain completely abolished self-association. When expressed in neurons, syt1 mutants with graded reductions in self-association activity exhibited concomitant reductions in 1) clamping spontaneous release and 2) triggering and synchronizing evoked release. Thus, the juxtamembrane linker of syt1 plays a crucial role in exocytosis by mediating multimerization.

During calcium-regulated exocytosis, the constitutive fusion machinery is 'clamped' in a partially assembled state until synchronously released by calcium. The protein machinery involved in this process is known, but the supra-molecular architecture and underlying mechanisms are unclear. Here, we use cryo-electron tomography analysis in nerve growth factor-differentiated neuro-endocrine (PC12) cells to delineate the organization of the release machinery under the docked vesicles. We find that exactly six exocytosis modules, each likely consisting of a single SNAREpin with its bound Synaptotagmins, Complexin, and Munc18 proteins, are symmetrically arranged at the vesicle-PM interface. Mutational analysis suggests that the symmetrical organization is templated by circular oligomers of Synaptotagmin. The observed arrangement, including its precise radial positioning, is in-line with the recently proposed 'buttressed ring hypothesis'.

Insulin secretion from β-cells is reduced at the onset of type-1 and during type-2 diabetes. Although inflammation and metabolic dysfunction of β-cells elicit secretory defects associated with type-1 or type-2 diabetes, accompanying changes to insulin granules have not been established. To address this, we performed detailed functional analyses of insulin granules purified from cells subjected to model treatments that mimic type-1 and type-2 diabetic conditions and discovered striking shifts in calcium affinities and fusion characteristics. We show that this behavior is correlated with two subpopulations of insulin granules whose relative abundance is differentially shifted depending on diabetic model condition. The two types of granules have different release characteristics, distinct lipid and protein compositions, and package different secretory contents alongside insulin. This complexity of β-cell secretory physiology establishes a direct link between granule subpopulation and type of diabetes and leads to a revised model of secretory changes in the diabetogenic process., eLife digest Diabetes is a disease that occurs when sugar levels in the blood can no longer be controlled by a hormone called insulin. People with type 1 diabetes lose the ability to produce insulin after their immune system attacks the β-cells in their pancreas that make this hormone. People with type 2 diabetes develop the disease when β-cells become exhausted from increased insulin demand and stop producing insulin. β-cells store insulin in small compartments called granules. When blood sugar levels rise, these granules fuse with the cell membrane allowing β-cells to release large quantities of insulin at once. This fusion is disrupted early in type 1 diabetes, but later in type 2: the underlying causes of these disruptions are unclear. In the laboratory, signals that trigger inflammation and molecules called fatty acids can mimic type 1 or type 2 diabetes respectively when applied to insulin-producing cells. Kreutzberger, Kiessling et al. wanted to know whether pro-inflammatory molecules and fatty acids affect insulin granules differently at the molecular level. To do this, insulin-producing cells were grown in the lab and treated with either fatty acids or pro-inflammatory molecules. The insulin granules of these cells were then isolated. Next, the composition of the granules and how they fused to lab-made membranes that mimic the cell membrane was examined. The experiments revealed that healthy β-cells have two types of granules, each with a different version of a protein called synaptotagmin. Cells treated with molecules mimicking type 1 diabetes lost granules with synaptotagmin-7, while granules with synaptotagmin-9 were lost in cells treated with fatty acids to imitate type 2 diabetes. Each type of granule responded differently to calcium levels in the cell and secreted different molecules, indicating that each elicits a different diabetic response in the body. These findings suggest that understanding how insulin granules are formed and regulated may help find treatments for type 1 and 2 diabetes, possibly leading to therapies that reverse the loss of different types of granules. Additionally, the molecules of these granules may also be used as markers to determine the stage of diabetes. More broadly, these results show how understanding how molecule release changes with disease in different cell types may help diagnose or stage a disease.

To infect their hosts and cause disease, plant viruses must replicate within cells and move throughout the plant both locally and systemically. RNA virus replication occurs on the surface of various cellular membranes, whose shape and composition become extensively modified in the process. Membrane contact sites (MCS) can mediate non-vesicular lipid-shuttling between different membranes and viruses co-opt components of these structures to make their membrane environment suitable for replication. Whereas animal viruses exit and enter cells when moving throughout their host, the rigid wall of plant cells obstructs this pathway and plant viruses therefore move between cells symplastically through plasmodesmata (PD). PD are membranous channels connecting nearly all plant cells and are now viewed to constitute a specialized type of endoplasmic reticulum (ER)-plasma membrane (PM) MCS themselves. Thus, both replication and movement of plant viruses rely on MCS. However, recent work also suggests that for some viruses, replication and movement are closely coupled at ER-PM MCS at the entrances of PD. Movement-coupled replication at PD may be distinct from the main bulk of replication and virus accumulation, which produces progeny virions for plant-to-plant transmission. Thus, MCS play a central role in plant virus infections, and may provide a link between two essential steps in the viral life cycle, replication and movement. Here, we provide an overview of plant virus-MCS interactions identified to date, and place these in the context of the connection between viral replication and cell-to-cell movement.

Granules of cytotoxic T lymphocytes (CTL) are derived from the lysosomal compartment. Synaptotagmin7 (Syt7) appears to be the calcium sensor triggering fusion of lysosomes in fibroblasts. Syt7 has been proposed to control cytotoxic granule (CG) fusion in lymphocytes and mice lacking Syt7 have reduced ability to clear infections. However, fusion of CG persists in the absence of Syt7. To clarify the role of Syt7 in CTL function, we have examined the fusion of cytotoxic granules of CD8+ T-lymphocytes from Syt7 knock-out mice. We have recorded granule fusion in living CTL, using total internal reflection microscopy. Since Syt7 is considered a high affinity calcium-sensor specialized for fusion under low calcium conditions, we have compared cytotoxic granule fusion under low and high calcium conditions in the same CTL. There was no difference in latencies or numbers of fusion events per CTL under low-calcium conditions, indicating that Syt7 is not required for cytotoxic granule fusion. A deficit of fusion in Syt7 KO CTL was seen when a high-calcium solution was introduced. Expressing wild type Syt7 in Syt7 KO lymphocytes reversed this deficit, confirming its Syt7-dependence. Mutations of Syt7 which disrupt calcium binding to its C2A domain reduced the efficacy of this rescue. We counted the cytotoxic granules present at the plasma membrane to determine if the lack of fusion events in the Syt7 KO CTL was due to a lack of granules. In low calcium there were no differences in fusion events per CTL, and granule numbers were similar. In high calcium, granule number was similar though wild type CTL exhibited significantly more fusion than Syt7 KO CTL. The modest differences in granule counts do not account for the lack of fusion in high calcium in Syt7 KO CTL. In Syt7 KO CTL expressing wild type Syt7, delivery of cytotoxic granules to the plasma membrane was comparable to that of wild type CTL. Syt7 KO CTL expressing Syt7 with deficient calcium binding in the C2A domain had significantly less fusion and fewer CG at the plasma membrane. These results indicate that Syt7 is involved in trafficking of CG to the plasma membrane.

Calcium (Ca2+)-evoked release of neurotransmitters from synaptic vesicles requires mechanisms both to prevent un-initiated fusion of vesicles (clamping) and to trigger fusion following Ca2+-influx. The principal components involved in these processes are the vesicular fusion machinery (SNARE proteins) and the regulatory proteins, Synaptotagmin-1 and Complexin. Here, we use a reconstituted single-vesicle fusion assay under physiologically-relevant conditions to delineate a novel mechanism by which Synaptotagmin-1 and Complexin act synergistically to establish Ca2+-regulated fusion. We find that under each vesicle, Synaptotagmin-1 oligomers bind and clamp a limited number of ‘central’ SNARE complexes via the primary interface and introduce a kinetic delay in vesicle fusion mediated by the excess of free SNAREpins. This in turn enables Complexin to arrest the remaining free ‘peripheral’ SNAREpins to produce a stably clamped vesicle. Activation of the central SNAREpins associated with Synaptotagmin-1 by Ca2+ is sufficient to trigger rapid (

The transport of LDL-derived cholesterol from lysosomes to peroxisomes is facilitated by membrane contacts formed between the lysosomal protein synaptotagmin VII and the peroxisomal lipid phosphatidylinositol 4, 5-bisphosphate [PI(4,5)P2]. Here, we used RNA interference to search for regulators of PI(4,5)P2 and to study the effects of altered PI(4,5)P2 homeostasis on cholesterol transport. We found that knockdown of phosphatidylinositol 5-phosphate 4-kinase type-2 α (PIP4K2A) reduced peroxisomal PI(4,5)P2 levels, decreased lysosome-peroxisome membrane contacts, and increased accumulation of lysosomal cholesterol in human SV-589 fibroblasts. Forced expression of peroxisome-localized, kinase-active PIP4K2A in the knockdown cells reduced cholesterol accumulation, and in vitro addition of recombinant PIP4K2A restored membrane contacts. These results suggest that PIP4K2A plays a critical role in intracellular cholesterol transport by upregulating PI(4,5)P2 levels in the peroxisomal membrane. Further research into PIP4K2A activity may inform future therapeutic interventions for managing lysosomal storage disorders.

Amyloid-β (Aβ), Aβ40, Aβ42, and, recently, Aβ25-35 have been directly implicated in the pathogenesis of Alzheimer’s disease. We have studied the effects of Aβ on neuronal death, reactive oxygen species (ROS) production, and synaptic assembling in neurons in primary culture. Aβ25-35, Aβ40, and Aβ42 significantly decreased neuronal viability, although Aβ25-35 showed a higher effect. Aβ25-35 showed a more penetrating ability to reach mitochondria while Aβ40 did not enter the neuronal cytosol and Aβ42 was scarcely internalized. We did not observe a direct correlation between ROS production and cell death because both Aβ40 and Aβ42 decreased neuronal viability but Aβ40 did not change ROS production. Rather, ROS production seems to correlate with the penetrating ability of each Aβ. No significant differences were found between Aβ40 and Aβ42 regarding the extent of the deleterious effects of both peptides on neuronal viability or synaptophysin expression. However, Aβ40 elicited a clear delocalization of PSD-95 and synaptotagmin from prospective synapsis to the neuronal soma, suggesting the occurrence of a crucial effect of Aβ40 on synaptic disassembling. The formation of Aβ40- or Aβ42-serum albumin complexes avoided the effects of these peptides on neuronal viability, synaptophysin expression, and PSD-95/synaptotagmin disarrangement suggesting that sequestration of Aβ by albumin prevents deleterious effects of these peptides. We can conclude that Aβ borne by albumin can be safely transported through body fluids, a fact that may be compulsory for Aβ disposal by peripheral tissues.

The activities of mitochondrial enzymes, which are essential for neural function, decline with age and in age-related disease. In particular, the activity of cytochrome c oxidase (COX/complex IV) decreases in patients with Alzheimer's disease (AD). COX, a mitochondrial inner membrane protein complex that contains heme, plays an essential role in the electron transport chain that generates ATP. Heme synthesis begins with 5-aminolevulinic acid (5-ALA) in mitochondria. 5-ALA synthetase is the rate-limiting enzyme in heme synthesis, suggesting that supplementation with 5-ALA might help preserve mitochondrial activity in the aged brain. We administered a diet containing 5-ALA to triple-transgenic AD (3xTg-AD) model mice for 6 months, starting at 3 months of age. COX activity and protein expression, as well as mitochondrial membrane potential, were significantly higher in brains of 5-ALA-fed mice than in controls. Synaptotagmin protein levels were also significantly higher in 5-ALA-fed mice, suggesting improved preservation of synapses. Although brain Aβ levels tended to decrease in 5-ALA-fed mice, we observed no other significant changes in other biochemical and pathological hallmarks of AD. Nevertheless, our study suggests that daily oral administration of 5-ALA could preserve mitochondrial enzyme activities in the brains of aged individuals, thereby contributing to the preservation of neural activity.

Mutations in the PINK1 gene are responsible for typical symptoms of Parkinson's disease. Using Drosophila melanogaster mutant PINK1B9 and after PINK1 silencing with RNAi using transgenic lines, we observed defects in synapses and behavior. The lack or reduced expression of PINK1 prolonged sleep during the day (nap) and decreased the total locomotor activity during 24 h, in addition to a decrease in climbing ability and a reduced lifespan. In the brain, PINK1 mutants had a lower level of Bruchpilot (BRP), a presynaptic scaffolding protein that is crucial for neurotransmission in all type of synapses in Drosophila. In addition, other proteins that are involved in synaptic transmission; Rab5, Syntaxin and Wishful Thinking were also decreased in abundance in mutants, except Synaptotagmin. Transmission electron microscopy (TEM) also confirmed less and abnormal synaptic vesicles at tetrad synapses in the visual system of PINK1 mutants. The lower level of BRP and longer day sleep observed was also detected in white mutants, which were examined to test the effect of the\r\nwhite background on the PINK1B9 strain. The reduced locomotor activity and longer day sleep in PINK1 mutants and after decreasing the PINK1 level in neurons seem to be correlated with a decrease in mitochondria number during the day, when they normally peak, and with impaired synaptic transmission.

Recent studies have revealed that SNARE proteins are involved in exocytotic release in mast cells. Previously, we reported that mast cell SNARE proteins induce membrane fusion between liposomes. Moreover, we found that synaptotagmin 2, a candidate Ca 2 + sensor for mast cell exocytosis, enhanced SNARE-mediated membrane fusion via Ca 2 + and phosphatidylserine. Phosphatidylinositol 4,5-bisphosphate (PIP2) is an acidic phospholipid like phosphatidylserine. In the present study, we investigated whether PIP2 is involved in the enhancement effect of synaptotagmin 2 on SNARE-mediated membrane fusion. PIP2 did not show any significant effect on SNARE-mediated membrane fusion by itself. In the presence of Ca 2 + , synaptotagmin 2 enhanced SNARE-mediated membrane fusion between liposomes containing PIP2. However, even in the presence of Ca 2 + , when we used 100% PC liposomes, synaptotagmin 2 did not show any significant effect on SNARE-mediated membrane fusion. These results indicated that PIP2 is involved in the enhancement effect of synaptotagmin 2 on membrane fusion between liposomes containing mast cell SNARE proteins.

Despite intensive research, it is still unclear how an immediate and profound acceleration of exocytosis is triggered by appropriate Ca(2+)-stimuli in presynaptic terminals. This is due to the fact that the molecular mechanisms of "docking" and "priming" reactions, which set up secretory vesicles to fuse at millisecond time scale, are extremely hard to study. Yet, driven by a fruitful combination of in vitro and in vivo analyses, our mechanistic understanding of Ca(2+)-triggered vesicle fusion has certainly advanced in the past few years. In this review, we aim to highlight recent progress and emerging views on the molecular mechanisms, by which constitutively forming SNAREpins are organized in functional, tightly regulated units for synchronized release. In particular, we will focus on the role of the small regulatory factor complexin whose function in Ca(2+)-dependent exocytosis has been controversially discussed for more than a decade. Special emphasis will also be laid on the functional relationship of complexin and synaptotagmin, as both proteins possibly act as allies and/or antagonists to govern SNARE-mediated exocytosis.

Background: Munc18-1 is an important factor for synaptic transmitter release, but its molecular mechanism remains an enigma. Results: Munc18a does not affect fusion kinetics. It can sequester syntaxin-1A molecules from the syntaxin-1A·SNAP-25 t-SNARE complex. Conclusion: Munc18a has no effect on complete fusion in conjunction with synaptotagmin-1, complexin-1, and neuronal SNAREs. Significance: This work provides new insights into Munc18-1 and its interactions with other synaptic proteins., Sec1/Munc18 (SM) proteins are essential for membrane trafficking, but their molecular mechanism remains unclear. Using a single vesicle-vesicle content-mixing assay with reconstituted neuronal SNAREs, synaptotagmin-1, and complexin-1, we show that the neuronal SM protein Munc18a/nSec1 has no effect on the intrinsic kinetics of both spontaneous fusion and Ca2+-triggered fusion between vesicles that mimic synaptic vesicles and the plasma membrane. However, wild type Munc18a reduced vesicle association ∼50% when the vesicles bearing the t-SNAREs syntaxin-1A and SNAP-25 were preincubated with Munc18 for 30 min. Single molecule experiments with labeled SNAP-25 indicate that the reduction of vesicle association is a consequence of sequestration of syntaxin-1A by Munc18a and subsequent release of SNAP-25 (i.e. Munc18a captures syntaxin-1A via its high affinity interaction). Moreover, a phosphorylation mimic mutant of Munc18a with reduced affinity to syntaxin-1A results in less reduction of vesicle association. In summary, Munc18a does not directly affect fusion, although it has an effect on the t-SNARE complex, depending on the presence of other factors and experimental conditions. Our results suggest that Munc18a primarily acts at the prefusion stage.

Proteoliposomes have been widely used for in vitro studies of membrane fusion mediated by synaptic proteins. Initially, such studies were made with large unsynchronized ensembles of vesicles. Such ensemble assays limited the insights into the SNARE-mediated fusion mechanism that could be obtained from them. Single particle microscopy experiments can alleviate many of these limitations but they pose significant technical challenges. Here we summarize various approaches that have enabled studies of fusion mediated by SNAREs and other synaptic proteins at a single-particle level. Currently available methods are described and their advantages and limitations are discussed.

The synaptic vesicle Ca2+ sensor Synaptotagmin binds Ca2+ through its two C2 domains to trigger membrane interactions. Beyond membrane insertion by the C2 domains, other requirements for Synaptotagmin activity are still being elucidated. To identify key residues within Synaptotagmin required for vesicle cycling, we took advantage of observations that mutations in the C2B domain Ca2+-binding pocket dominantly disrupt release from invertebrates to humans. We performed an intragenic screen for suppressors of lethality induced by expression of Synaptotagmin C2B Ca2+-binding mutants in Drosophila. This screen uncovered essential residues within Synaptotagmin that suggest a structural basis for several activities required for fusion, including a C2B surface implicated in SNARE complex interaction that is required for rapid synchronization and Ca2+ cooperativity of vesicle release. Using electrophysiological, morphological and computational characterization of these mutants, we propose a sequence of molecular interactions mediated by Synaptotagmin that promote Ca2+ activation of the synaptic vesicle fusion machinery.

Summary Heterozygous mutations in proline-rich transmembrane protein 2 (PRRT2) underlie a group of paroxysmal disorders, including epilepsy, kinesigenic dyskinesia, and migraine. Most of the mutations lead to impaired PRRT2 expression, suggesting that loss of PRRT2 function may contribute to pathogenesis. We show that PRRT2 is enriched in presynaptic terminals and that its silencing decreases the number of synapses and increases the number of docked synaptic vesicles at rest. PRRT2-silenced neurons exhibit a severe impairment of synchronous release, attributable to a sharp decrease in release probability and Ca2+ sensitivity and associated with a marked increase of the asynchronous/synchronous release ratio. PRRT2 interacts with the synaptic proteins SNAP-25 and synaptotagmin 1/2. The results indicate that PRRT2 is intimately connected with the Ca2+-sensing machinery and that it plays an important role in the final steps of neurotransmitter release., Graphical Abstract, Highlights • PRRT2 is a presynaptic protein • PRRT2 is required for synchronous neurotransmitter release • PRRT2 silencing decreases synaptic density and release probability • PRRT2 interacts with the fast Ca2+ sensors synaptotagmin 1/2, Valente et al. show that PRRT2, a single causative gene for a group of paroxysmal neurological diseases, is a key component of regulated exocytosis. Silencing PRRT2 dramatically impairs neurotransmitter release by markedly reducing release probability. PRRT2 interacts with the fast Ca2+ sensors synaptotagmin 1/2 and endows the SNARE complex with Ca2+ sensitivity.

Phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2] is essential for exocytosis. Classical ways of manipulating PI(4,5)P2 levels are slower than its metabolism, making it difficult to distinguish effects of PI(4,5)P2 from those of its metabolites. We developed a membrane-permeant, photoactivatable PI(4,5)P2, which is loaded into cells in an inactive form and activated by light, allowing sub-second increases in PI(4,5)P2 levels. By combining this compound with electrophysiological measurements in mouse adrenal chromaffin cells, we show that PI(4,5)P2 uncaging potentiates exocytosis and identify synaptotagmin-1 (the Ca2+ sensor for exocytosis) and Munc13-2 (a vesicle priming protein) as the relevant effector proteins. PI(4,5)P2 activation of exocytosis did not depend on the PI(4,5)P2-binding CAPS-proteins, suggesting that PI(4,5)P2 uncaging may bypass CAPS-function. Finally, PI(4,5)P2 uncaging triggered the rapid fusion of a subset of readily-releasable vesicles, revealing a rapid role of PI(4,5)P2 in fusion triggering. Thus, optical uncaging of signaling lipids can uncover their rapid effects on cellular processes and identify lipid effectors., eLife digest Cells in our body communicate by releasing compounds called transmitters that carry signals from one cell to the next. Packages called vesicles store transmitters within the signaling cell. When the cell needs to send a signal, the vesicles fuse with the cell's membrane and release their cargo. For many signaling processes, such as those used by neurons, this fusion is regulated, fast, and coupled to the signal that the cell receives to activate release. Specialized molecular machines made up of proteins and fatty acid molecules called signaling lipids enable this to happen. One signaling lipid called PI(4,5)P2 (short for phosphatidylinositol 4,5-bisphosphate) is essential for vesicle fusion as well as for other processes in cells. It interacts with several proteins that help it control fusion and the release of transmitter. While it is possible to study the role of these proteins using genetic tools to inactivate them, the signaling lipids are more difficult to manipulate. Existing methods result in slow changes in PI(4,5)P2 levels, making it hard to directly attribute later changes to PI(4,5)P2. Walter, Müller, Tawfik et al. developed a new method to measure how PI(4,5)P2 affects transmitter release in living mammalian cells, which causes a rapid increase in PI(4,5)P2 levels. The method uses a chemical compound called “caged PI(4,5)P2” that can be loaded into cells but remains undetected until ultraviolet light is shone on it. The ultraviolet light uncages the compound, generating active PI(4,5)P2 in less than one second. Walter et al. found that when they uncaged PI(4,5)P2 in this way, the amount of transmitter released by cells increased. Combining this with genetic tools, it was possible to investigate which proteins of the release machinery were required for this effect. The results suggest that two different types of proteins that interact with PI(4,5)P2 are needed: one must bind PI(4,5)P2 to carry out its role and the other helps PI(4,5)P2 accumulate at the site of vesicle fusion. The new method also allowed Walter et al. to show that a fast increase in PI(4,5)P2 triggers a subset of vesicles to fuse very rapidly. This shows that PI(4,5)P2 rapidly regulates the release of transmitter. Caged PI(4,5)P2 will be useful to study other processes in cells that need PI(4,5)P2, helping scientists understand more about how signaling lipids control many different events at cellular membranes.

The synaptic vesicle Ca2+ sensor Synaptotagmin binds Ca2+ through its two C2 domains to trigger membrane interactions. Beyond membrane insertion by the C2 domains, other requirements for Synaptotagmin activity are still being elucidated. To identify key residues within Synaptotagmin required for vesicle cycling, we took advantage of observations that mutations in the C2B domain Ca2+-binding pocket dominantly disrupt release from invertebrates to humans. We performed an intragenic screen for suppressors of lethality induced by expression of Synaptotagmin C2B Ca2+-binding mutants in Drosophila. This screen uncovered essential residues within Synaptotagmin that suggest a structural basis for several activities required for fusion, including a C2B surface implicated in SNARE complex interaction that is required for rapid synchronization and Ca2+ cooperativity of vesicle release. Using electrophysiological, morphological and computational characterization of these mutants, we propose a sequence of molecular interactions mediated by Synaptotagmin that promote Ca2+ activation of the synaptic vesicle fusion machinery.

Neuronal communication and brain function mainly depend on the fundamental biological events of neurotransmission, including the exocytosis of presynaptic vesicles (SVs) for neurotransmitter release and the subsequent endocytosis for SV retrieval. Neurotransmitters are released through the Ca2+- and SNARE-dependent fusion of SVs with the presynaptic plasma membrane. Following exocytosis, endocytosis occurs immediately to retrieve SV membrane and fusion machinery for local recycling and thus maintain the homeostasis of synaptic structure and sustained neurotransmission. Apart from the general endocytic machinery, recent studies have also revealed the involvement of SNARE proteins (synaptobrevin, SNAP25, and syntaxin), synaptophysin, Ca2+/calmodulin, and members of the synaptotagmin protein family (Syt1, Syt4, Syt7, and Syt11) in the balance and tight coupling of exo-endocytosis in neurons. Here, we provide an overview of recent progress in understanding how these neuron-specific adaptors coordinate to ensure precise and efficient endocytosis during neurotransmission.

Ca(2+)-dependent transmitter release occurs in a fast and in a slow phase, but the differential roles of Ca(2+) buffers and Ca(2+) sensors in shaping release kinetics are still controversial. Replacing extracellular Ca(2+) by Sr(2+) causes decreased fast release but enhanced slow release at many synapses. Here, we established presynaptic Sr(2+) uncaging and made quantitative Sr(2+)- and Ca(2+)-imaging experiments at the mouse calyx of Held synapse, to reveal the interplay between Ca(2+) sensors and Ca(2+) buffers in the control of fast and slow release. We show that Sr(2+) activates the fast, Synaptotagmin-2 (Syt2) sensor for vesicle fusion with sixfold lower affinity but unchanged high cooperativity. Surprisingly, Sr(2+) also activates the slow sensor that remains in Syt2 knock-out synapses with a lower efficiency, and Sr(2+) was less efficient than Ca(2+) in the limit of low concentrations in wild-type synapses. Quantitative imaging experiments show that the buffering capacity of the nerve terminal is markedly lower for Sr(2+) than for Ca(2+) (~5-fold). This, together with an enhanced Sr(2+) permeation through presynaptic Ca(2+) channels (~2-fold), admits a drastically higher spatially averaged Sr(2+) transient compared with Ca(2+). Together, despite the lower affinity of Sr(2+) at the fast and slow sensors, the massively higher amplitudes of spatially averaged Sr(2+) transients explain the enhanced late release. This also allows us to conclude that Ca(2+) buffering normally controls late release and prevents the activation of the fast release sensor by residual Ca(2+).

How clathrin-mediated endocytosis (CME) retrieves vesicle proteins into newly formed synaptic vesicles (SVs) remains a major puzzle. Besides its roles in stimulating clathrin-coated vesicle formation and regulating SV size, the clathrin assembly protein AP180 has been identified as a key player in retrieving SV proteins. The mechanisms by which AP180 recruits SV proteins are not fully understood. Here, we show that following acute inactivation of AP180 in Drosophila, SV recycling is severely impaired at the larval neuromuscular synapse based on analyses of FM 1-43 uptake and synaptic ultrastructure. More dramatically, AP180 activity is important to maintain the integrity of SV protein complexes at the plasma membrane during endocytosis. These observations suggest that AP180 normally clusters SV proteins together during recycling. Consistent with this notion, SV protein composition and distribution are altered in AP180 mutant flies. Finally, AP180 co-immunoprecipitates with SV proteins, including the vesicular glutamate transporter and neuronal synaptobrevin. These results reveal a new mode by which AP180 couples protein retrieval to CME of SVs. AP180 is also genetically linked to Alzheimer's disease. Hence, the findings of this study may provide new mechanistic insight into the role of AP180 dysfunction in Alzheimer's disease.

International audience; Neurotransmission is achieved by soluble NSF attachment protein receptor (SNARE)-driven fusion of readily releasable vesicles that are docked and primed at the presynaptic plasma membrane. After neurotransmission, the readily releasable pool of vesicles must be refilled in less than 100 ms for subsequent release. Here we show that the initial association of SNARE complexes, SNAREpins, is far too slow to support this rapid refilling owing to an inherently high activation energy barrier. Our data suggest that acceleration of this process, i.e., lowering of the barrier, is physiologically necessary and can be achieved by molecular factors. Furthermore, under zero force, a low second energy barrier transiently traps SNAREpins in a half-zippered state similar to the partial assembly that engages calcium-sensitive regulatory machinery. This result suggests that the barrier must be actively raised in vivo to generate a sufficient pause in the zippering process for the regulators to set in place. We show that the heights of the activation energy barriers can be selectively changed by molecular factors. Thus, it is possible to modify, both in vitro and in vivo, the lifespan of each metastable state. This controllability provides a simple model in which vesicle docking/priming, an intrinsically slow process, can be substantially accelerated. It also explains how the machinery that regulates vesicle fusion can be set in place while SNAREpins are trapped in a half-zippered state.

Background Synaptic degeneration is a central pathogenic event in Alzheimer’s disease that occurs early during the course of disease and correlates with cognitive symptoms. The pre-synaptic vesicle protein synaptotagmin-1 appears to be essential for the maintenance of an intact synaptic transmission and cognitive function. Synaptotagmin-1 in cerebrospinal fluid is a candidate Alzheimer biomarker for synaptic dysfunction that also may correlate with cognitive decline. Methods In this study, a novel mass spectrometry-based assay for measurement of cerebrospinal fluid synaptotagmin-1 was developed, and was evaluated in two independent sample sets of patients and controls. Sample set I included cerebrospinal fluid samples from patients with dementia due to Alzheimer’s disease (N = 17, age 52–86 years), patients with mild cognitive impairment due to Alzheimer’s disease (N = 5, age 62–88 years), and controls (N = 17, age 41–82 years). Sample set II included cerebrospinal fluid samples from patients with dementia due to Alzheimer’s disease (N = 24, age 52–84 years), patients with mild cognitive impairment due to Alzheimer’s disease (N = 18, age 58–83 years), and controls (N = 36, age 43–80 years). Results The reproducibility of the novel method showed coefficients of variation of the measured synaptotagmin-1 peptide 215–223 (VPYSELGGK) and peptide 238–245 (HDIIGEFK) of 14 % or below. In both investigated sample sets, the CSF levels of synaptotagmin-1 were significantly increased in patients with dementia due to Alzheimer’s disease (P ≤ 0.0001) and in patients with mild cognitive impairment due to Alzheimer’s disease (P

Spinal cord injury causes neuronal death, limiting subsequent regeneration and recovery. Thus, there is a need to develop strategies for improving neuronal survival after injury. Relative to our understanding of axon regeneration, comparatively little is known about the mechanisms that promote the survival of damaged neurons. To address this, we took advantage of lamprey giant reticulospinal neurons whose large size permits detailed examination of post-injury molecular responses at the level of individual, identified cells. We report here that spinal cord injury caused a select subset of giant reticulospinal neurons to accumulate synuclein, a synaptic vesicle-associated protein best known for its atypical aggregation and causal role in neurodegeneration in Parkinson's and other diseases. Post-injury synuclein accumulation took the form of punctate aggregates throughout the somata and occurred selectively in dying neurons, but not in those that survived. In contrast, another synaptic vesicle protein, synaptotagmin, did not accumulate in response to injury. We further show that the post-injury synuclein accumulation was greatly attenuated after single dose application of either the "molecular tweezer" inhibitor, CLR01, or a translation-blocking synuclein morpholino. Consequently, reduction of synuclein accumulation not only improved neuronal survival, but also increased the number of axons in the spinal cord proximal and distal to the lesion. This study is the first to reveal that reducing synuclein accumulation is a novel strategy for improving neuronal survival after spinal cord injury.

Cold acclimation is the capacity of certain plants to increase their freezing tolerance in response to a period of low non-freezing temperatures. Cold acclimation involves a series of biochemical and physiological adaptations, including a deep transcriptional reprogramming and drastic changes in the lipid composition of cellular membranes in order to prevent the freeze-induced damage (1). While a profound knowledge has been acquired on the regulation of gene expression triggered by cold-acclimation, very little is known about the mechanisms governing the cold-induced changes in membranes’ lipid composition. In this study we report that in Arabidopsis, the constitutively expressed Synaptotagmin 1 (SYT1) and the cold-induced homolog Synaptotagmin 3 (SYT3) are essential for cold- acclimated freezing tolerance and for the lipid remodelling of the plasma membrane during cold-acclimation. SYT1 and SYT3 are phospholipid-binding proteins located in Endoplasmic Reticulum-Plasma Membrane contact sites (ER-PMcs), conserved structures defined as regions of the cortical ER in close apposition to the PM (2). ER-PMcs facilitate the non-vesicular lipid transport between ER and PM in yeast and mammals, and are essential for lipid homeostasis (3). In contrast to the high and ubiquitous SYT1 expression, SYT3 expression is low and mainly restricted to meristemoids, young stomata, and old primary root. TIRF microscopy analyses show that during cold acclimation there is an increase of SYT1::SYT1:GFP and SYT3::SYT3:GFP signals as spots at the PM. High-resolution lipidome analyses show the over-accumulation of phosphatidylinositols phosphate (PIPs) and glycerolipids in vivo in syt1 and specially syt1/syt3 mutant plants compared to WT in one-week cold-acclimated plants. Interestingly, protein-lipid overlay assays (membrane-strips and PIP-strips) reveal PIPs and glycerolipids as major interactors for both, SYT1 and SYT3. Here we show that 1) Arabidopsis SYT1 and SYT3 are induced by cold, 2) SYT1 and SYT3 localize to ER-PMcs, 3) the specific lipids that directly interact with SYT1 and SYT3 accumulate in syt1/syt3 mutant after cold acclimation, and 4) syt1/syt3 show reduced cold acclimated freezing tolerance. We propose that SYT1 and SYT3 have essential roles in ER-PMcs mediated lipid remodelling during cold acclimation, which in turn leads to freezing tolerance. Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech.

Brain function depends on a crucial feature: The ability of individual neurons to share packets of information, known as quantal transmission. Given the sheer number of tasks the brain has to deal with, this information sharing must be extremely rapid. Synapses are specialized points of contact between neurons, where fast transmission takes place. Though the basic elements and functions of the synapse had been established since the 1950s, the molecular basis for regulation of fast synaptic transmitter release was not known 20 years ago. However, around 1990, crucial discoveries were made by Richard Scheller, James Rothman, and Thomas Sudhof, leading a few years later to the formulation of the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) hypothesis and a new understanding of the molecular events controlling vesicular release of transmitter in synapses. The 2010 Kavli Prize in neuroscience was awarded to these three researchers, “for their work to reveal the precise molecular basis of the transfer of signals between nerve cells in the brain.”

Synaptotagmins are vesicular proteins implicated in many membrane trafficking events. They are highly conserved in evolution and the mammalian family contains 16 isoforms. We now show that the tandem C2 domains of several calcium-sensitive synaptotagmin isoforms tested, including Drosophila synaptotagmin, rapidly cross-link phospholipid membranes. In contrast to the tandem structure, individual C2 domains failed to trigger membrane cross-linking in several novel assays. Large-scale liposomal aggregation driven by tandem C2 domains in response to calcium was confirmed by the following techniques: turbidity assay, dynamic light-scattering and both confocal and negative stain electron microscopy. Firm cross-linking of membranes was evident from laser trap experiments. High-resolution cryo-electron microscopy revealed that membrane cross-linking by tandem C2 domains results in a constant distance of similar to 9 rum between the apposed membranes. Our findings show the conserved nature of this important property of synaptotagmin, demonstrate the significance of the tandem C2 domain structure and provide a plausible explanation for the accelerating effect of synaptotagmins on membrane fusion. (C) 2008 Elsevier Ltd. All rights reserved.

Background Accumulation of the β-amyloid peptide (Aβ) is a major pathological hallmark of Alzheimer’s disease (AD). Recent studies have shown that synaptic Aβ toxicity may directly impair synaptic function. However, proteins regulating Aβ generation at the synapse have not been characterized. Here, we sought to identify synaptic proteins that interact with the extracellular domain of APP and regulate Aβ generation. Results Affinity purification-coupled mass spectrometry identified members of the Synaptotagmin (Syt) family as novel interacting proteins with the APP ectodomain in mouse brains. Syt-1, −2 and −9 interacted with APP in cells and in mouse brains in vivo. Using a GST pull-down approach, we have further demonstrated that the Syt interaction site lies in the 108 amino acids linker region between the E1 and KPI domains of APP. Stable overexpression of Syt-1 or Syt-9 with APP in CHO and rat pheochromocytoma cells (PC12) significantly increased APP-CTF and sAPP levels, with a 2 to 3 fold increase in secreted Aβ levels in PC12 cells. Moreover, using a stable knockdown approach to reduce the expression of endogenous Syt-1 in PC12 cells, we have observed a ~ 50 % reduction in secreted Aβ generation. APP processing also decreased in these cells, shown by lower CTF levels. Lentiviral-mediated knock down of endogenous Syt-1 in mouse primary neurons also led to a significant reduction in both Aβ40 and Aβ42 generation. As secreted sAPPβ levels were significantly reduced in PC12 cells lacking Syt-1 expression, our results suggest that Syt-1 regulates Aβ generation by modulating BACE1-mediated cleavage of APP. Conclusion Altogether, our data identify the synaptic vesicle proteins Syt-1 and 9 as novel APP-interacting proteins that promote Aβ generation and thus may play an important role in the pathogenesis of AD. Electronic supplementary material The online version of this article (doi:10.1186/s13024-015-0028-5) contains supplementary material, which is available to authorized users.

Botulinum neurotoxins (BoNTs) cause botulism by entering neurons and cleaving proteins that mediate neurotransmitter release; disruption of exocytosis results in paralysis and death. The receptors for BoNTs are thought to be composed of both proteins and gangliosides; however, protein components that mediate toxin entry have not been identified. Using gain-of-function and loss-of-function approaches, we report here that the secretory vesicle proteins, synaptotagmins (syts) I and II, mediate the entry of BoNT/B (but not BoNT/A or E) into PC12 cells. Further, we demonstrate that BoNT/B entry into PC12 cells and rat diaphragm motor nerve terminals was activity dependent and can be blocked using fragments of syt II that contain the BoNT/B-binding domain. Finally, we show that syt II fragments, in conjunction with gangliosides, neutralized BoNT/B in intact mice. These findings establish that syts I and II can function as protein receptors for BoNT/B.

SNAREs (soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptors) are membrane-associated proteins that participate in the fusion of internal membranes in eukaryotic cells. SNAREs comprise three distinct and well-conserved families of molecules that act directly as membrane fusogens or, at the least, as elements that bring membranes into close apposition and allow for subsequent fusion events to occur. While the molecular events leading to fusion are still under debate, it is clear that a number of additional factors are required to bring about SNARE-mediated membrane fusion in vivo. Many of these factors, which collectively can be called SNARE regulators (e.g. Sec1/Munc18, synaptotagmin, GATE-16, LMA1, Munc13/UNC-13, synaptophysin, tomosyn, Vsm1, etc.), bind directly to SNAREs and are involved in the regulation of SNARE assembly as well as the ability of SNAREs to participate in trafficking events. In addition, recent studies have suggested a role for posttranslational modification (e.g., phosphorylation) in the regulation of SNARE functions. In this review the possible role of SNARE regulators in SNARE assembly and the involvement of SNARE phosphorylation in the regulation of intracellular membrane trafficking will be discussed.

T he synaptotagmins (syts) are a family of membrane proteins proposed to regulate membrane traffic in neuronal and nonneuronal cells. In neurons, the Ca2+-sensing ability of syt I is critical for fusion of docked synaptic vesicles with the plasma membrane in response to stimulation. Several putative Ca2+–syt effectors have been identified, but in most cases the functional significance of these interactions remains unknown. Here, we have used recombinant C2 domains derived from the cytoplasmic domains of syts I–XI to interfere with endogenous syt–effector interactions during Ca2+-triggered exocytosis from cracked PC12 cells. Inhibition was closely correlated with syntaxin–SNAP-25 and phosphatidylinositol 4,5-bisphosphate (PIP2)–binding activity. Moreover, we measured the expression levels of endogenous syts in PC12 cells; the major isoforms are I and IX, with trace levels of VII. As expected, if syts I and IX function as Ca2+ sensors, fragments from these isoforms blocked secretion. These data suggest that syts trigger fusion via their Ca2+-regulated interactions with t-SNAREs and PIP2, target molecules known to play critical roles in exocytosis.

To evaluate the relationship between inflammatory markers and severity of asthma in children, the amount of interleukin-8 (IL-8) and granulocyte/macrophage colony-stimulating factor (GM-CSF) released by peripheral blood mononuclear cells, exhaled nitric oxide (FE NO) levels, p65 nuclear factor-kappaB subunit, and phosphorylated IkBalpha expression by peripheral blood mononuclear cells were assessed in six control subjects, 12 steroid-naives subjects with intermittent asthma, and 17 children with moderate asthma. To investigate their predictive value, biomarker levels were correlated with the number of exacerbations during a 18-month follow-up period. We found that GM-CSF release was higher in moderate and intermittent asthmatics than in control subjects, whereas IL-8 release was higher in moderate than in intermittent asthmatics and control subjects. FE NO levels were similar among study groups. In moderate asthmatics, IL-8, GM-CSF, and FE NO significantly correlated with the exacerbation numbers. Moreover, p65 and phosphorylated IkBalpha levels were greater in moderate than in intermittent asthmatics and control subjects. According to GM-CSF, IL-8, and FE NO levels, two distinct subgroups of moderate asthmatics (low and high producers) were identified. High producers experienced more exacerbations than low producers. This study shows ongoing inflammation associated with biological and clinical heterogeneity in moderate asthmatics despite regular treatment and proposes that large prospective studies confirm the importance of biomarkers to assess inflammation and asthma control in children with asthma.

The synaptic vesicle protein synaptotagmin I has been proposed to serve as a Ca2+ sensor for rapid exocytosis. In the present work, two fragments of the large cytoplasmic domain of synaptotagmin I, C2A and C2AB, were compared by combining surface plasmon resonance with circular dichroism and fluorescence techniques. C2AB and C2A had almost identical membrane binding constants, indicating that C2A is the predominate domain to bind to negatively charged phospholipids. After reacting with inositol hexakisphosphate (InsP6) a conformational change of C2AB was detected in the presence of liposome. The InsP6 binding notably weakened the Ca2+-dependent C2AB-membrane interaction, which suggests that InsP6 may act as a modulator of neurotransmitter release by altering the state of synaptotagmin–phospholipid interaction.

The complex manner in which patterns of presynaptic neural activity are translated into short-term plasticity (STP) suggests the existence of multiple presynaptic calcium (Ca(2+)) sensors, which regulate the amplitude and time-course of STP and are the focus of this review. We describe two canonical Ca(2+)-binding protein domains (C2 domains and EF-hands) and define criteria that need to be met for a protein to qualify as a Ca(2+) sensor mediating STP. With these criteria in mind, we discuss various forms of STP and identify established and putative Ca(2+) sensors. We find that despite the multitude of proposed sensors, only three are well established in STP: Munc13, protein kinase C (PKC) and synaptotagmin-7. For putative sensors, we pinpoint open questions and potential pitfalls. Finally, we discuss how the molecular properties and modes of action of Ca(2+) sensors can explain their differential involvement in STP and shape net synaptic output.

Investigations over the last two decades have made major inroads in clarifying the cellular and molecular events that underlie the fast, synchronous release of neurotransmitter at nerve endings. Thus, appreciable progress has been made in establishing the structural features and biophysical properties of the calcium (Ca2+) channels that mediate the entry into nerve endings of the Ca2+ ions that trigger neurotransmitter release. It is now clear that presynaptic Ca2+ channels are regulated at many levels and the interplay of these regulatory mechanisms is just beginning to be understood. At the same time, many lines of research have converged on the conclusion that members of the synaptotagmin family serve as the primary Ca2+ sensors for the action potential-dependent release of neurotransmitter. This identification of synaptotagmins as the proteins which bind Ca2+ and initiate the exocytotic fusion of synaptic vesicles with the plasma membrane has spurred widespread efforts to reveal molecular details of synaptotagmin's action. Currently, most models propose that synaptotagmin interfaces directly or indirectly with SNARE (soluble, N-ethylmaleimide sensitive factor attachment receptors) proteins to trigger membrane fusion. However, in spite of intensive efforts, the field has not achieved consensus on the mechanism by which synaptotagmins act. Concurrently, the precise sequence of steps underlying SNARE-dependent membrane fusion remains controversial. This review considers the pros and cons of the different models of SNARE-mediated membrane fusion and concludes by discussing a novel proposal in which synaptotagmins might directly elicit membrane fusion without the intervention of SNARE proteins in this final fusion step.

Synaptotagmin (syt) 7 is one of three syt isoforms found in all metazoans; it is ubiquitously expressed, yet its function in neurons remains obscure. Here, we resolved Ca2+-dependent and Ca2+-independent synaptic vesicle (SV) replenishment pathways, and found that syt 7 plays a selective and critical role in the Ca2+-dependent pathway. Mutations that disrupt Ca2+-binding to syt 7 abolish this function, suggesting that syt 7 functions as a Ca2+-sensor for replenishment. The Ca2+-binding protein calmodulin (CaM) has also been implicated in SV replenishment, and we found that loss of syt 7 was phenocopied by a CaM antagonist. Moreover, we discovered that syt 7 binds to CaM in a highly specific and Ca2+-dependent manner; this interaction requires intact Ca2+-binding sites within syt 7. Together, these data indicate that a complex of two conserved Ca2+-binding proteins, syt 7 and CaM, serve as a key regulator of SV replenishment in presynaptic nerve terminals. DOI: http://dx.doi.org/10.7554/eLife.01524.001, eLife digest Neurons communicate with one another at junctions called synapses. The arrival of an electrical signal called an action potential at the first neuron triggers the release of chemicals called neurotransmitters into the synapse. These chemicals then diffuse across the gap between the neurons and bind to receptors on the second cell. The neurotransmitter molecules are stored in the first cell in packages known as vesicles, which release their contents by fusing with the cell membrane. Following a fusion event, neurons must replenish their vesicle stocks to ensure that they are ready for the arrival of the next action potential. This replenishment process is known to involve a calcium-dependent pathway and a calcium-independent pathway. A protein called calmodulin, that binds calcium ions, has an important role in the first of these pathways. Now, Liu et al. have shown that another protein, synaptotagmin 7, also has a key role in the replenishment of synaptic vesicles, possibly as a sensor for calcium ions. Moreover, Liu et al. found that synaptotagmin 7 and calmodulin bind to each other to form a complex, which suggests that the calcium-dependent replenishment pathway is regulated by this complex. The synaptotagmins are a family of 17 proteins, three of which are present in all animals. Two of these were known to have roles in synapses, but the role of the third—synaptotagmin 7—had been unclear. In addition to providing a more complete understanding of the replenishment of synaptic vesicles, the work of Liu et al. also supplies the final piece of the jigsaw regarding the role of the synaptotagmins that are present in all animals. DOI: http://dx.doi.org/10.7554/eLife.01524.002

We examined the fine structure of the type I signal-anchor sequence of synaptotagmin II, which has a 60-residue N-terminal domain followed by a hydrophobic region (H-region), focusing on the hinge region between the N-terminal and the H-regions. It was found that the charged or highly polar residues support the translocation of the N-terminal domain through the endoplasmic reticulum membrane at specific positions in the hinge. The residue requirement correlated with the turn propensity scale for transmembranes. It is suggested that a certain conformation, likely helical hairpin, in the hinge is critical for N-terminal domain translocation.

Real-time voltammetry measurements from cracked PC12 cells were used to analyze the role of synaptotagmin–SNARE interactions during Ca2+-triggered exocytosis. The isolated C2A domain of synaptotagmin I neither binds SNAREs nor inhibits norepinephrine secretion. In contrast, two C2 domains in tandem (either C2A-C2B or C2A-C2A) bind strongly to SNAREs, displace native synaptotagmin from SNARE complexes, and rapidly inhibit exocytosis. The tandem C2 domains of synaptotagmin cooperate via a novel mechanism in which the disruptive effects of Ca2+ ligand mutations in one C2 domain can be partially alleviated by the presence of an adjacent C2 domain. Complete disruption of Ca2+-triggered membrane and target membrane SNARE interactions required simultaneous neutralization of Ca2+ ligands in both C2 domains of the protein. We conclude that synaptotagmin–SNARE interactions regulate membrane fusion and that cooperation between synaptotagmin's C2 domains is crucial to its function.

It is currently a widely accepted opinion that adaptive, plastic changes in the molecular and cellular components of neuronal signaling systems correlate with the effects on mood and cognition observed after long-term treatment with antidepressant drugs. Protein phosphorylation represents a key step for most signaling systems, and it is involved in the regulation of virtually all cellular functions. Two serine/threonine kinases, Ca2+/calmodulin-dependent protein kinase II and cyclic AMP-dependent protein kinase, have been shown to be activated in the brain following antidepressant treatment. The changes in kinase activity are mirrored by changes in the phosphorylation of selected protein substrates in subcellular compartments (presynaptic terminals and microtubules), which, in turn, may contribute to the modulation of synaptic transmission observed with antidepressants. The molecular consequences of protein kinase activation may account for some of the alterations in neural function induced by antidepressants, and may suggest novel possible strategies of pharmacological intervention.

The synaptotagmin gene family currently includes 12 members. Analysis of the three known genomic synaptotagmin sequences reveals conserved exon-intron patterns which delineate the synaptotagmin structural domains. We used expressed sequence tag, reverse transcription PCR and RNAse protection assay analysis of synaptotagmin messenger RNAs to demonstrate the occurrence of alternative splicing events involving a number of exons. Exon-skipped messages where transmembrane sequences have been removed or altered were found to be abundantly expressed by synaptotagmins 1, 4, 6 and 7. Although the expression of most synaptotagmins predominates in neural tissue, we find that by contrast, synaptotagmin 6 is more abundant in thymus.

Secretory epithelial cells of the proximal airways synthesize and secrete gel-forming polymeric mucins. The secreted mucins adsorb water to form mucus that is propelled by neighboring ciliated cells, providing a mobile barrier which removes inhaled particles and pathogens from the lungs. Several features of the intracellular trafficking of mucins make the airway secretory cell an interesting comparator for the cell biology of regulated exocytosis. Polymeric mucins are exceedingly large molecules (up to 3 × 106 Da per monomer) whose folding and initial polymerization in the ER requires the protein disulfide isomerase Agr2. In the Golgi, mucins further polymerize to form chains and possibly branched networks comprising more than 20 monomers. The large size of mucin polymers imposes constraints on their packaging into transport vesicles along the secretory pathway. Sugar side chains account for >70% of the mass of mucins, and their attachment to the protein core by O-glycosylation occurs in the Golgi. Mature polymeric mucins are stored in large secretory granules ∼1 μm in diameter. These are translocated to the apical membrane to be positioned for exocytosis by cooperative interactions among myristoylated alanine-rich C kinase substrate, cysteine string protein, heat shock protein 70, and the cytoskeleton. Mucin granules undergo exocytic fusion with the plasma membrane at a low basal rate and a high stimulated rate. Both rates are mediated by a regulated exocytic mechanism as indicated by phenotypes in both basal and stimulated secretion in mice lacking Munc13-2, a sensor of the second messengers calcium and diacylglycerol (DAG). Basal secretion is induced by low levels of activation of P2Y2 purinergic and A3 adenosine receptors by extracellular ATP released in paracrine fashion and its metabolite adenosine. Stimulated secretion is induced by high levels of the same ligands, and possibly by inflammatory mediators as well. Activated receptors are coupled to phospholipase C by Gq, resulting in the generation of DAG and of IP3 that releases calcium from apical ER. Stimulated secretion requires activation of the low affinity calcium sensor Synaptotagmin-2, while a corresponding high affinity calcium sensor in basal secretion is not known. The core exocytic machinery is comprised of the SNARE proteins VAMP8, SNAP23, and an unknown Syntaxin protein, together with the scaffolding protein Munc18b. Common and distinct features of this exocytic system in comparison to neuroendocrine cells and neurons are highlighted.

Background Neurons in sympathetic ganglia and neuroendocrine cells in the adrenal medulla share not only their embryonic origin from sympathoadrenal precursors in the neural crest but also a range of functional features. These include the capacity for noradrenaline biosynthesis, vesicular storage and regulated release. Yet the regulation of neuronal properties in early neuroendocrine differentiation is a matter of debate and the developmental expression of the vesicle fusion machinery, which includes components found in both neurons and neuroendocrine cells, is not resolved. Results Analysis of synaptic protein and pan-neuronal marker mRNA expression during mouse development uncovers profound differences between sympathetic neurons and adrenal chromaffin cells, which result in qualitatively similar but quantitatively divergent transcript profiles. In sympathetic neurons embryonic upregulation of synaptic protein mRNA follows early and persistent induction of pan-neuronal marker transcripts. In adrenal chromaffin cells pan-neuronal marker expression occurs only transiently and synaptic protein messages remain at distinctly low levels throughout embryogenesis. Embryonic induction of synaptotagmin I (Syt1) in sympathetic ganglia and postnatal upregulation of synaptotagmin VII (Syt7) in adrenal medulla results in a cell type-specific difference in isoform prevalence. Dicer 1 inactivation in catecholaminergic cells reduces high neuronal synaptic protein mRNA levels but not their neuroendocrine low level expression. Pan-neuronal marker mRNAs are induced in chromaffin cells to yield a more neuron-like transcript pattern, while ultrastructure is not altered. Conclusions Our study demonstrates that remarkably different gene regulatory programs govern the expression of synaptic proteins in the neuronal and neuroendocrine branch of the sympathoadrenal system. They result in overlapping but quantitatively divergent transcript profiles. Dicer 1-dependent regulation is required to establish high neuronal mRNA levels for synaptic proteins and to maintain repression of neurofilament messages in neuroendocrine cells.

Plants undergo continuous exposure to various abiotic stresses in their natural environment. For this reason they have evolved intricate mechanisms to perceive external signals and trigger the physiological changes necessary to adapt and survive under such conditions. The plasma membrane is a physical barrier that separates the intracellular and extracellular environments and its integrity is essential for stress tolerance. Here we present a role for plant synaptotagmins in plasma membrane integrity maintenance through Vesicle-PM fusions. We also evaluate the physiological consequences of the SYT depletion under several abiotic stress conditions Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech. This work has been supported by Spanish Ministry of Economy and competitivity FPI-BES 2012 -052324 and the Marie Curie FP7 People Action IIF-FP7 301419

125I-labeled botulinum type B neurotoxin was shown to bind specifically to recombinant rat synaptotagmins I and II. Binding required reconstitution of the recombinant proteins with gangliosides GT1b/GD1a. Scatchard plot analyses revealed a single class of binding site with dissociation constants of 0.23 and 2.3 nM for synaptotagmin II and synaptotagmin I, respectively, values very similar to those of the high- (0.4 nM) and low-affinity (4.1 nM) binding sites in synaptosomes. The high-affinity binding of neurotoxin to synaptosomes was specifically inhibited by a monoclonal antibody recognizing with the amino-terminal region of synaptotagmin II. These results suggest that this region of synaptotagmin II participates in the formation of the high-affinity toxin binding site by associating with specific gangliosides.

Leucine-rich repeat kinase 2 (LRRK2) is a gene that, upon mutation, causes autosomal-dominant familial Parkinson’s disease (PD). Yeast two-hybrid screening revealed that Snapin, a SNAP-25 (synaptosomal-associated protein-25) interacting protein, interacts with LRRK2. An in vitro kinase assay exhibited that Snapin is phosphorylated by LRRK2. A glutathione-S-transferase (GST) pull-down assay showed that LRRK2 may interact with Snapin via its Ras-of-complex (ROC) and N-terminal domains, with no significant difference on interaction of Snapin with LRRK2 wild type (WT) or its pathogenic mutants. Further analysis by mutation study revealed that Threonine 117 of Snapin is one of the sites phosphorylated by LRRK2. Furthermore, a Snapin T117D phosphomimetic mutant decreased its interaction with SNAP-25 in the GST pull-down assay. SNAP-25 is a component of the SNARE (Soluble NSF Attachment protein REceptor) complex and is critical for the exocytosis of synaptic vesicles. Incubation of rat brain lysate with recombinant Snapin T117D, but not WT, protein caused decreased interaction of synaptotagmin with the SNARE complex based on a co-immunoprecipitation assay. We further found that LRRK2-dependent phosphorylation of Snapin in the hippocampal neurons resulted in a decrease in the number of readily releasable vesicles and the extent of exocytotic release. Combined, these data suggest that LRRK2 may regulate neurotransmitter release via control of Snapin function by inhibitory phosphorylation. Researchers in Korea have found that a protein linked with Parkinson's disease (PD) may play a role in regulating neuronal signaling. Mutations in the gene encoding the leucine-rich repeat kinase 2 (LRRK2) protein, a kinase whose enhanced enzyme activity is related to PD pathogenesis, are hereditary risk factors for this neurodegenerative disorder. Researchers led by Wongi Seol of Wonkwang University searched for substrates for LRRK2 kinase that might explain this enzyme's involvement in PD. They determined that LRRK2 phosphorylates Snapin, a protein that ensures proper localization of neurotransmitter reserves and LRRK2-mediated phosphorylation inhibits Snapin, thus reducing the size of these reserves and the amount of neurotransmitter release. The extent to which LRRK2-Snapin interaction contributes to Parkinson's remains unclear, but this pathway nevertheless seems to play an important role in proper brain function.