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Syntaxin 1 and synaptosome-associated protein of 25 kD (SNAP-25) are neuronal plasmalemma proteins that appear to be essential for exocytosis of synaptic vesicles (SVs). Both proteins form a complex with synaptobrevin, an intrinsic membrane protein of SVs. This binding is thought to be responsible for vesicle docking and apparently precedes membrane fusion. According to the current concept, syntaxin 1 and SNAP-25 are members of larger protein families, collectively designated as target-SNAP receptors (t-SNAREs), whose specific localization to subcellular membranes define where transport vesicles bind and fuse. Here we demonstrate that major pools of syntaxin 1 and SNAP-25 recycle with SVs. Both proteins cofractionate with SVs and clathrin-coated vesicles upon subcellular fractionation. Using recombinant proteins as standards for quantitation, we found that syntaxin 1 and SNAP-25 each comprise approximately 3% of the total protein in highly purified SVs. Thus, both proteins are significant components of SVs although less abundant than synaptobrevin (8.7% of the total protein). Immunoisolation of vesicles using synaptophysin and syntaxin specific antibodies revealed that most SVs contain syntaxin 1. The widespread distribution of both syntaxin 1 and SNAP-25 on SVs was further confirmed by immunogold electron microscopy. Botulinum neurotoxin C1, a toxin that blocks exocytosis by proteolyzing syntaxin 1, preferentially cleaves vesicular syntaxin 1. We conclude that t-SNAREs participate in SV recycling in what may be functionally distinct forms.

Exosomes play pivotal roles in intercellular communication and cancer progression. Recent findings have shown exosomes to incite proangiogenesis and prepare lymph nodes as remote niches for accumulation and migration of melanoma cells. Exosomes present a highly curved morphology with a size range of ∼30-100 nm that is distinct from other lipid vesicles. Furthermore, exosomes have the potential as a general metastasis biomarker since their increased secretion in the peripheral blood has been correlated with cancer metastasis. Since aqueous suspensions of exosomes cannot be seen by conventional optical methods, we hypothesized that a molecular probe that binds to exosomes could be a novel tool for detecting these vesicles. We aim to develop a minimally-invasive diagnostic tool to detect and measure secreted exosomes as biomarkers of cancer metastasis that may help clinicians and patients decide on an appropriate therapeutic action. Taking a cue from nature and using solid phase ‘Click’ chemistry as a technique for peptide modification, we created a fluorophore-tagged cyclic peptide based on the membrane penetrating C2B domain of Synaptotagmin I. We investigated the peptide-lipid interactions using synthetic liposomes as preliminary models of exosomes through a combination of spectroscopic techniques. We found that the peptide probe was selective for highly curved liposomes (d 100 nm). We tested blood plasma samples from rat models to find out if our preliminary findings would translate to the detection of exosomes. Our results showed that blood plasma treated with the peptide probe showed fluorescence intensity that was remarkably higher than the untreated peptide, which indicated binding to exosomes. Furthermore, we used a real time nanoparticle tracking analysis system to visualize the exosomes in a dynamic and physiologically-relevant medium. The peptide-treated exosomes were detected as fast-moving, fluorescent particles even in the complex matrix of blood plasma. This technique provided us with a platform to study the size distribution and concentration of exosomes in blood plasma. We have created a peptide probe that selectively binds to exosomes from an animal model. The investigation of the exosome binding property of this probe on clinically relevant samples is ongoing. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4743. doi:1538-7445.AM2012-4743

Neurotransmitter release is potently blocked by a group of structurally related toxin proteins produced by Clostridium botulinum. Botulinum neurotoxin type B (BoNT/B) and tetanus toxin (TeTx) are zinc-dependent proteases that specifically cleave synaptobrevin (VAMP), a membrane protein of synaptic vesicles. Here we report that inhibition of transmitter release from synaptosomes caused by botulinum neurotoxin A (BoNT/A) is associated with the selective proteolysis of the synaptic protein SNAP-25. Furthermore, isolated or recombinant L chain of BoNT/A cleaves SNAP-25 in vitro. Cleavage occurred near the carboxyterminus and was sensitive to divalent cation chelators. In addition, a glutamate residue in the BoNT/A L chain, presumably required to stabilize a water molecule in the zinc-containing catalytic centre, was required for proteolytic activity. These findings demonstrate that BoNT/A acts as a zinc-dependent protease that selectively cleaves SNAP-25. Thus, a second component of the putative fusion complex mediating synaptic vesicle exocytosis is targeted by a clostridial neurotoxin.

Tetanus neurotoxin causes the disease tetanus, which is characterized by rigid paralysis. The toxin acts by inhibiting the release of neurotransmitters from inhibitory neurons in the spinal cord that innervate motor neurons and is unique among the clostridial neurotoxins due to its ability to shuttle from the periphery to the central nervous system. Tetanus neurotoxin is thought to interact with a high affinity receptor complex that is composed of lipid and protein components; however, the identity of the protein receptor remains elusive. In the current study, we demonstrate that toxin binding, to dissociated hippocampal and spinal cord neurons, is greatly enhanced by driving synaptic vesicle exocytosis. Moreover, tetanus neurotoxin entry and subsequent cleavage of synaptobrevin II, the substrate for this toxin, was also dependent on synaptic vesicle recycling. Next, we identified the potential synaptic vesicle binding protein for the toxin and found that it corresponded to SV2; tetanus neurotoxin was unable to cleave synaptobrevin II in SV2 knockout neurons. Toxin entry into knockout neurons was rescued by infecting with viruses that express SV2A or SV2B. Tetanus toxin elicited the hyper excitability in dissociated spinal cord neurons - due to preferential loss of inhibitory transmission - that is characteristic of the disease. Surprisingly, in dissociated cortical cultures, low concentrations of the toxin preferentially acted on excitatory neurons. Further examination of the distribution of SV2A and SV2B in both spinal cord and cortical neurons revealed that SV2B is to a large extent localized to excitatory terminals, while SV2A is localized to inhibitory terminals. Therefore, the distinct effects of tetanus toxin on cortical and spinal cord neurons are not due to differential expression of SV2 isoforms. In summary, the findings reported here indicate that SV2A and SV2B mediate binding and entry of tetanus neurotoxin into central neurons., Author Summary Tetanus neurotoxin is one of the most deadly bacterial toxins known and is the causative agent for the disease tetanus, also known as lockjaw. Tetanus neurotoxin utilizes motor neurons as a means of transport in order to enter the spinal cord. Once in the spinal cord, the toxin leaves motor neurons and enters inhibitory neurons through a “Trojan-horse” strategy, thereby preventing the release of inhibitory neurotransmitters onto motor neurons. This causes hyper-excitability of the motor neuron and excessive release of acetylcholine at the neuromuscular junction, resulting in rigid paralysis. There is a major gap in our understanding of the mechanism by which tetanus neurotoxin enters neurons. In the current study we discovered that the “Trojan-horse”, utilized by tetanus neurotoxin to enter central neurons, corresponds to recycling synaptic vesicles. Furthermore, we discovered that SV2 is critical for the binding and entry of tetanus neurotoxin into these neurons. These findings will enable further development of drugs that antagonize the action of the toxin and will also aid in the development of drug delivery systems that target spinal cord neurons.

Mutations in otoferlin are linked to human hearing loss. New research defines a function for this C2 domain–containing protein in synaptic vesicle exocytosis in cochlear hair cells., Otoferlin is a large multi–C2 domain protein proposed to act as a calcium sensor that regulates synaptic vesicle exocytosis in cochlear hair cells. Although mutations in otoferlin have been associated with deafness, its contribution to neurotransmitter release is unresolved. Using recombinant proteins, we demonstrate that five of the six C2 domains of otoferlin sense calcium with apparent dissociation constants that ranged from 13–25 µM; in the presence of membranes, these apparent affinities increase by up to sevenfold. Using a reconstituted membrane fusion assay, we found that five of the six C2 domains of otoferlin stimulate membrane fusion in a calcium-dependent manner. We also demonstrate that a calcium binding–deficient form of the C2C domain is incapable of stimulating membrane fusion, further underscoring the importance of calcium for the protein’s function. These results demonstrate for the first time that otoferlin is a calcium sensor that can directly regulate soluble N-ethyl-maleimide sensitive fusion protein attachment protein receptor–mediated membrane fusion reactions.

In biological systems, membrane fusion is mediated by specialized proteins. Although soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptors (SNAREs) provide the minimal molecular machinery required to drive membrane fusion, the precise mechanism for SNARE-mediated fusion remains to be established. Here, we used atomic force microscope (AFM) spectroscopy to determine whether the pulling force generated by interacting SNAREs is directly coupled to membrane fusion. The mechanical strength of the SNARE binding interaction was determined by single molecule force measurements. It was revealed that the forced unbinding of the SNARE complex formed between opposing (trans) bilayers involves two activation barriers; where the steep inner barrier governs the transition from the bound to an intermediate state and the outer barrier governs the transition between the intermediate and the unbound state. Moreover, truncation of either SNAP-25 or VAMP 2 reduced the slope of the inner barrier significantly and, consequently, reduced the pulling strength of the SNARE complex; thus, suggesting that the inner barrier determines the binding strength of the SNARE complex. In parallel, AFM compression force measurements revealed that truncated SNAREs were less efficient than native SNAREs in facilitating hemifusion of the apposed bilayers. Together, these findings reveal a mechanism by which a pulling force generated by interacting trans-SNAREs reduces the slope of the hemifusion barrier and, subsequently, facilitates hemifusion and makes the membranes more prone to fusion.

Neuromodulin (GAP-43) is a membrane protein that is transported to neuronal growth cones. Zuber and co-workers have proposed that the N-terminal 10 amino acid sequence of neuromodulin is sufficient to target proteins to growth cones. We demonstrate that a neuromodulin(β-galactosidase fusion protein is transported to growth cones of cultured rat neurons, whereas a fusion protein containing the N-terminal 10 amino acids of neuromodulin and (β-galactosidase is not. A mutant neuromodulin lacking cysteines 3 and 4, the palmitylation sites required for membrane attachment, does not target β-galactosi-dase to growth cones. We conclude that membrane attachment is required for growth cone accumulation and that structural elements, in addition to the first 10 amino acids of neuromodulin, may be required for growth cone targeting.

Publisher Summary This chapter focuses on biochemical properties of neuromodulin that may be important for its function in neurons. Two interesting biochemical properties that may be important for the physiological function of neuromodulin are its affinity for CaM and the effect of protein kinase C phosphorylation on CaM interactions. The concentration of neuromodulin in the brain and the affinity of the protein for CaM in the absence of free calcium are sufficient to complex the majority of CaM present. Consequently, interactions between neuromodulin and CaM as well as the regulation of CaM binding by protein kinase C phosphorylation are of considerable interest. The protein kinase C phosphorylation site of neuromodulin is serine-41. This phosphorylation significantly reduces the affinity of neuromodulin for CaM, suggesting that the introduction of negative charge may abolish CaM binding. The chapter addresses this issue by substituting serine-41 with an aspartate or an asparagines residue. The aspartate-41 mutant neuromodulin did not bind to CaM-Sepharose. In contrast, the asparagine-41 mutant bound to CaM-Sepharose in a manner indistinguishable from the wild type protein.

Styryl dyes (also referred to as FM dyes) become highly fluorescent upon binding to membranes and are often used to study synaptic vesicle recycling in neurons. To date, however, no direct comparisons of the fluorescent properties, or time-resolved (millisecond) measurements of dye-membrane binding and unbinding reactions, for all members of this family of probes have been reported. Here, we compare the fluorescence intensities of each member of the FM dye family when bound to membranes. This analysis included SGC5, a new lipophilic fluorescent dye with a unique structure. Fluorescence intensities depended on the length of the lipophilic tail of each dye, with a rank order as follows: SGC5 > FM1-84 > FM1-43 > SynaptoGreen C3 > FM2-10/FM4-64/FM5-95. Stopped-flow measurements revealed that dye hydrophobicity determined the affinity and departitioning rates for dye-membrane interactions. All of the dyes dissociated from membranes on the millisecond timescale, which is orders of magnitude faster than the overall destaining rate (timescale of seconds) of these dyes from presynaptic boutons. Departitioning kinetics were faster at higher temperatures, but were unaffected by pH or cholesterol. The data reported here aid interpretation of dye-release kinetics from single synaptic vesicles, and indicate that these probes dissociate from membranes on more rapid timescales than previously appreciated.

Synaptotagmin I (syt), a transmembrane protein localized to secretory vesicles, functions as a Ca2+ sensor that facilitates SNARE-mediated membrane fusion. The cytoplasmic domain of syt harbors two C2-domains designated C2A and C2B. Upon binding Ca2+, C2A and C2B partially penetrate into membranes that contain anionic phospholipids. However, it is unknown whether these tandem C2-domains engage membranes at the same time, in a sequential manner, or in a mutually exclusive manner. We have used site-directed fluorescent probes to monitor the penetration of syt’s C2-domains into phosphatidylserine-harboring lipid bilayers. We report that, in response to Ca2+, C2A and C2B copenetrate into these bilayers with diffusion-limited kinetics. Membrane penetration was more efficient when synthetic rather than natural phospholipids were used to prepare bilayers. The membrane penetration activity of the intact cytoplasmic domain of syt (C2A-C2B) exhibits significant resistance to changes in ionic strength. In contrast, the ability of isolated C2B to bind membranes in response to Ca2+ can be disrupted by subtle changes in ionic strength. Tethering C2B to a mutant version of C2A that does not bind Ca2+ or membranes significantly increases the stability of Ca2+·C2B·membrane complexes, confirming that C2A affects the membrane-binding properties of the adjacent C2B domain.

Synaptic transmission involves a fast synchronous phase and a slower asynchronous phase of neurotransmitter release that are regulated by distinct Ca(2+) sensors. Though the Ca(2+) sensor for rapid exocytosis, synaptotagmin I, has been studied in depth, the sensor for asynchronous release remains unknown. In a screen for neuronal Ca(2+) sensors that respond to changes in [Ca(2+)] with markedly slower kinetics than synaptotagmin I, we observed that Doc2--another Ca(2+), SNARE, and lipid-binding protein--operates on timescales consistent with asynchronous release. Moreover, up- and downregulation of Doc2 expression levels in hippocampal neurons increased or decreased, respectively, the slow phase of synaptic transmission. Synchronous release, when triggered by single action potentials, was unaffected by manipulation of Doc2 but was enhanced during repetitive stimulation in Doc2 knockdown neurons, potentially due to greater vesicle availability. In summary, we propose that Doc2 is a Ca(2+) sensor that is kinetically tuned to regulate asynchronous neurotransmitter release.

Summary Decades ago it was proposed that exocytosis involves invagination of the target membrane, resulting in a highly localized site of contact between the bilayers destined to fuse. The vesicle protein synaptotagmin-I (syt) bends membranes in response to Ca 2+ , but whether this drives localized invagination of the target membrane to accelerate fusion has not been determined. Previous studies relied on reconstituted vesicles that were already highly curved and used mutations in syt that were not selective for membrane-bending activity. Here, we directly address this question by utilizing vesicles with different degrees of curvature. A tubulation-defective syt mutant was able to promote fusion between highly curved SNARE-bearing liposomes but exhibited a marked loss of activity when the membranes were relatively flat. Moreover, bending of flat membranes by adding an N-BAR domain rescued the function of the tubulation-deficient syt mutant. Hence, syt-mediated membrane bending is a critical step in membrane fusion.

A single-vesicle, fluorescence-based, SNARE-driven fusion assay enables simultaneous measurement of lipid mixing and content release with 5 ms/frame, or even 1 ms/frame, time resolution. The v-SNARE vesicles, labeled with lipid and content markers of different color, dock and fuse with a planar t-SNARE bilayer supported on glass. A narrow (

Murine cDNA that encodes neuromodulin, a neurospecific calmodulin binding protein, was inserted into the plasmid pKK223-3 for expression in Escherichia coli. After being transformed into E. coli strain SG20252 (lon-), the expression vector directed the synthesis of a protein that was recognized by polyclonal antibodies raised against bovine neuromodulin. The recombinant protein expressed in E. coli was found to be tightly associated with insoluble cell material and was extractable only with guanidine hydrochloride or sodium dodecyl sulfate. Following solubilization with guanidine hydrochloride, the protein was purified to apparent homogeneity by a single CaM-Sepharose affinity column step with a yield of 0.2 mg of protein/L of E. coli culture. The availability of the purified recombinant neuromodulin made it possible to answer several specific questions concerning the structure and function of the protein. Despite the fact that murine neuromodulin is 12 amino acid residues shorter than the bovine protein and the recombinant protein expressed in E. coli may lack any posttranslational modifications, the two proteins displayed similar biochemical properties in almost all respects examined. They both had higher affinity for CaM-Sepharose in the absence of Ca2+ than in its presence; they were both phosphorylated in vitro by protein kinase C in a Ca2+- and phospholipid-dependent manner; neither form of the proteins was autophosphorylated, and the phosphorylated form of the proteins did not bind calmodulin. The recombinant neuromodulin and neuromodulin purified from bovine brain had similar, but not identical, affinities of calmodulin, indicating that the palmitylation of the protein that occurs in animal cells is not crucial for calmodulin interactions.(ABSTRACT TRUNCATED AT 250 WORDS)

The synaptic vesicle protein synaptobrevin (VAMP) has recently been implicated as one of the key proteins involved in exocytotic membrane fusion. It interacts with the synaptic membrane proteins syntaxin I and synaptosome-associated protein (SNAP)-25 to form a complex which precedes exocytosis [Sollner et al. (1993b) Cell, 75, 409-418]. Here we demonstrate that the majority of synaptobrevin is bound to the vesicle protein synaptophysin in detergent extracts. No syntaxin I was found in this complex when synaptophysin-specific antibodies were used for immunoprecipitation. Conversely, no synaptophysin was associated with the synaptobrevin-syntaxin I complex when syntaxin-specific antibodies were used for immunoprecipitation. Thus, the synaptobrevin pool bound to synaptophysin is not available for binding to syntaxin I and SNAP-25, and vice versa. Synaptobrevin-synaptophysin binding was also demonstrated by chemical cross-linking in isolated nerve terminals. Furthermore, recombinant synaptobrevin II efficiently bound synaptophysin and its isoform synaptoporin, but not the more distantly related synaptic vesicle protein p29. Recombinant synaptobrevin I bound with similar efficiency, whereas the non-neuronal isoform cellubrevin displayed a lower affinity towards synaptophysin. Treatment with high NaCl concentrations resulted in a dissociation of the synaptobrevin-synaptophysin complex. In addition, the interaction of synaptobrevin with synaptophysin was irreversibly abolished by low amounts of SDS, while the interaction with syntaxin I was enhanced. We conclude that synaptophysin selectively interacts with synaptobrevin in a complex which excludes the t-SNAP receptors syntaxin I and SNAP-25, suggesting a role for synaptophysin in the control of exocytosis.

The membrane proteins SNAP-25, syntaxin, and synaptobrevin (vesicle-associated membrane protein) have recently been implicated as central elements of an exocytotic membrane fusion complex in neurons. Here we report that SNAP-25 binds directly to both syntaxin and synaptobrevin. The SNAP-25-binding domain of syntaxin lies between residues 199 and 243, within the region previously shown to mediate synaptobrevin binding (Calakos, N., Bennett, M. K., Peterson, K. E., and Scheller, R. H. (1994) Science 263, 1146-1149). The syntaxin-binding domain of SNAP-25 encompasses most of the amino-terminal half of SNAP-25, including its putative palmitoylation sites. Truncation of the carboxyl-terminal 9 residues of SNAP-25, which yields a fragment corresponding to that generated by botulinum neurotoxin A, diminishes the interaction of SNAP-25 with synaptobrevin, but not with syntaxin. Sequence analysis revealed that the regions that mediate the interaction between SNAP-25 and syntaxin contain heptad repeats characteristic of certain classes of alpha-helices. Similar repeats are also present at the carboxyl terminus of SNAP-25 and in synaptobrevin. These domains have a moderate to high probability of forming coiled coils. We conclude that SNAP-25 can interact with both syntaxin and synaptobrevin and that binding may be mediated by alpha-helical domains that form intermolecular coiled-coil structures.