Snapin mediates insulin secretory granule docking, but not trans-SNARE complex formation.

Secretory granule exocytosis is a tightly regulated process requiring granule targeting, tethering, priming, and membrane fusion. At the heart of this process is the SNARE complex, which drives fusion through a coiled-coil zippering effect mediated by the granule v-SNARE protein, VAMP2, and the plasma membrane t-SNAREs, SNAP-25 and syntaxin-1A. Here we demonstrate that in pancreatic ß-cells the SNAP-25 accessory protein, snapin, C-terminal H2 domain binds SNAP-25 through its N-terminal Sn-1 domain. Interestingly whilst snapin binds SNAP-25, there is only modest binding of this complex with syntaxin-1A under resting conditions. Instead synataxin-1A appears to be recruited in response to secretory stimulation. These results indicate that snapin plays a role in tethering insulin granules to the plasma membrane through coiled coil interaction of snapin with SNAP-25, with full granule fusion competency only resulting after subsequent syntaxin-1A recruitment triggered by secretory stimulation.

Synaptic vesicles studied by dynamic light scattering.

The size polydispersity distribution of synaptic vesicles (SVs) is characterized under quasi-physiological conditions by dynamic light scattering (DLS). Highly purified fractions of SVs obtained from rat brain still contain a small amount of larger contaminant structures, which can be quantified by DLS and further reduced by asymmetric-flow field-flow (AFFF) fractionation. The intensity autocorrelation functions g (2)(τ) recorded from these samples are analyzed by a constrained regularization method as well as by an alternative direct modeling approach. The results are in quantitative agreement with the polydispersity obtained from cryogenic electron microscopy of vitrified SVs. Next, different vesicle fusion assays based on samples composed of SVs and small unilamellar proteoliposomes with the fusion proteins syntaxin 1 and SNAP-25A are characterized by DLS. The size increase of the proteoliposomes due to SNARE-dependent fusion with SVs is quantified by DLS under quasi-physiological conditions.

The panel of syntaxin 1 and insulinoma-associated protein 1 outperforms classic neuroendocrine markers in pulmonary neuroendocrine neoplasms.

Syntaxin-1 (STX1) is a recently described highly sensitive and specific neuroendocrine marker. We evaluated the applicability of STX1 as an immunohistochemical marker in pulmonary neuroendocrine neoplasms (NENs). We compared STX1 with established neuroendocrine markers, including insulinoma-associated protein 1 (INSM1). Typical carcinoids (n = 33), atypical carcinoids (n = 7), small cell lung carcinomas ([SCLCs] n = 30), and large cell neuroendocrine lung carcinomas (n = 17) were immunostained using tissue microarray for STX1, chromogranin A, synaptophysin, CD56, and INSM1. Eighty-four of eighty-seven (96.5%) NENs showed STX1 positivity. Carcinoids and LCNECs typically presented a combined strong membranous and weak cytoplasmic staining pattern; cytoplasmic expression was predominately observed in SCLCs. The sensitivity of STX1 was 90% in SCLCs and 100% in typical carcinoids, atypical carcinoids, and large cell neuroendocrine lung carcinomas. The overall sensitivity of STX1 in pulmonary NENs was 96.6%, and the sensitivity of the other markers was as follows: chromogranin A (85.2%), synaptophysin (85.2%), CD56 (92.9%), and INSM1 (97.7%). STX1 was found to be an excellent neuroendocrine marker of pulmonary NENs, with sensitivity and specificity surpassing that of classic markers. We propose a panel of STX1 and INSM1 for the routine immunohistochemical workup of pulmonary NENs.

Changes in concentrations of NMDA receptor subunit GluN2B, Arc and syntaxin-1 in dorsal hippocampus Schaffer collateral synapses in a rat learned helplessness model of depression.

Major depressive disorder involves changes in synaptic structure and function, but the molecular underpinnings of these changes are still not established. In an initial pilot experiment, whole-brain synaptosome screening with quantitative western blotting was performed to identify synaptic proteins that may show concentration changes in a congenital rat learned helplessness model of depression. We found that the N-methyl-d-aspartate receptor (NMDAR) subunits GluN2A/GluN2B, activity-regulated cytoskeleton-associated protein (Arc) and syntaxin-1 showed significant concentration differences between congenitally learned helpless (LH) and nonlearned helpless (NLH) rats. Having identified these three proteins, we then performed more elaborate quantitative immunogold electron microscopic analyses of the proteins in a specific synapse type in the dorsal hippocampus: the Schaffer collateral synapse in the CA1 region. We expanded the setup to include also unstressed wild-type (WT) rats. The concentrations of the proteins in the LH and NLH groups were compared to WT animals. In this specific synapse, we found that the concentration of NMDARs was increased in postsynaptic spines in both LH and NLH rats. The concentration of Arc was significantly increased in postsynaptic densities in LH animals as well as in presynaptic cytoplasm of NLH rats. The concentration of syntaxin-1 was significantly increased in both presynaptic terminals and postsynaptic spines in LH animals, while pre- and postsynaptic syntaxin-1 concentrations were significantly decreased in NLH animals. These protein changes suggest pathways by which synaptic plasticity may be increased in dorsal hippocampal Schaffer collateral synapses during depression, corresponding to decreased synaptic stability.

Need for speed: Super-resolving the dynamic nanoclustering of syntaxin-1 at exocytic fusion sites.

Communication between cells relies on regulated exocytosis, a multi-step process that involves the docking, priming and fusion of vesicles with the plasma membrane, culminating in the release of neurotransmitters and hormones. Key proteins and lipids involved in exocytosis are subjected to Brownian movement and constantly switch between distinct motion states which are governed by short-lived molecular interactions. Critical biochemical reactions between exocytic proteins that occur in the confinement of nanodomains underpin the precise sequence of priming steps which leads to the fusion of vesicles. The advent of super-resolution microscopy techniques has provided the means to visualize individual molecules on the plasma membrane with high spatiotemporal resolution in live cells. These techniques are revealing a highly dynamic nature of the nanoscale organization of the exocytic machinery. In this review, we focus on soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) syntaxin-1, which mediates vesicular fusion. Syntaxin-1 is highly mobile at the plasma membrane, and its inherent speed allows fast assembly and disassembly of syntaxin-1 nanoclusters which are associated with exocytosis. We reflect on recent studies which have revealed the mechanisms regulating syntaxin-1 nanoclustering on the plasma membrane and draw inferences on the effect of synaptic activity, phosphoinositides, N-ethylmaleimide-sensitive factor (NSF), α-soluble NSF attachment protein (α-SNAP) and SNARE complex assembly on the dynamic nanoscale organization of syntaxin-1. This article is part of the special issue entitled 'Mobility and trafficking of neuronal membrane proteins'.

Syntaxins 3 and 4 are concentrated in separate clusters on the plasma membrane before the establishment of cell polarity.

Syntaxins 3 and 4 localize to the apical and basolateral plasma membrane, respectively, of epithelial cells where they mediate vesicle fusion. Here, we report that before establishment of cell polarity, syntaxins 3 and 4 are confined to mutually exclusive, submicron-sized clusters. Syntaxin clusters are remarkably uniform in size, independent of expression levels, and are distinct from caveolae and clathrin-coated pits. SNAP-23 partially colocalizes with both syntaxin 3 and 4 clusters. Deletion of the apical targeting signal of syntaxin 3 does not prevent sorting into clusters away from syntaxin 4. Syntaxin 3 and 4 cluster formation depends on different mechanisms because the integrity of syntaxin 3 clusters depends on intact microtubules, whereas syntaxin 4 clusters depend on intact actin filaments. Cholesterol depletion causes dispersion of syntaxin 3 but not syntaxin 4 clusters. In migrating cells, syntaxin clusters polarize to the leading edge, suggesting a role in polarized exocytosis. These results suggest that exocytosis occurs at small fusion sites exhibiting high local concentrations of SNARE proteins that may be required for efficient membrane fusion. The establishment of separate clusters for each syntaxin suggests that the plasma membrane is inherently polarized on an ultrastructural level even before the establishment of true cell polarity.

Nanobodies reveal an extra-synaptic population of SNAP-25 and Syntaxin 1A in hippocampal neurons.

Synaptic vesicle fusion (exocytosis) is a precisely regulated process that entails the formation of SNARE complexes between the vesicle protein synaptobrevin 2 (VAMP2) and the plasma membrane proteins Syntaxin 1 and SNAP-25. The sub-cellular localization of the latter two molecules remains unclear, although they have been the subject of many recent investigations. To address this, we generated two novel camelid single domain antibodies (nanobodies) specifically binding to SNAP-25 and Syntaxin 1A. These probes penetrated more easily into samples and detected their targets more efficiently than conventional antibodies in crowded regions. When investigated by super-resolution imaging, the nanobodies revealed substantial extra-synaptic populations for both SNAP-25 and Syntaxin 1A, which were poorly detected by antibodies. Moreover, extra-synaptic Syntaxin 1A molecules were recruited to synapses during stimulation, suggesting that these are physiologically-active molecules. We conclude that nanobodies are able to reveal qualitatively and quantitatively different organization patterns, when compared to conventional antibodies.

Preparation of polysomal fractions from mouse brain synaptoneurosomes and analysis of polysomal-bound mRNAs.

BACKGROUND: Here we describe a detailed, reliable protocol for isolation of polysomal fractions from mouse brain synaptoneurosomes. This method is an important tool to study local protein synthesis in neurons. NEW METHOD: We combined rapid preparation of synaptoneurosomes by filtration with polysome profiling. We provide a detailed protocol highlighting difficulties and critical steps of: i) preparation of synaptoneurosomes; ii) polyribosome fractionation from synaptoneurosomes; iii) extraction of proteins and RNA from sucrose gradient fractions. RESULTS: and Comparison with Existing Methods We fractionated polyribosomes from synaptoneurosomes and detected the association of Mmp9, Camk2a and Stx1B mRNA with polysomes in the unstimulated conditions. Synaptic stimulation led to increased levels of Mmp9 and Camk2a mRNA in the heavy polysomal fractions. We compared our protocol with existing methods CONCLUSIONS: We have developed a reliable, effective method to prepare polyribosomal fractions from synaptoneurosomes to study polyribosomal binding of mRNAs as an aspect of synaptic translation in vitro.

Tomosyn is expressed in beta-cells and negatively regulates insulin exocytosis.

Tomosyn, a syntaxin-binding protein, is capable of dissociating mammalian homolog of the Caenorhabditis elegans unc-18 gene from syntaxin and is involved in the regulation of exocytosis. We have investigated the expression, cellular localization, and functional role of tomosyn in pancreatic beta-cells. Western blotting revealed a 130-kDa protein corresponding to tomosyn in insulin-secreting beta-cell lines. RT-PCR amplification showed that b-, m-, and s-tomosyn isoform mRNAs are expressed in beta-cell lines and rat pancreatic islets. Immunohistochemistry revealed punctate tomosyn immunoreactivity in the cytoplasm of insulin-, glucagon-, pancreatic polypeptide-, and somatostatin-containing islet cells. Syntaxin 1 coimmunoprecipitated with tomosyn in extracts of insulin-secreting cells. Overexpression of m-tomosyn in mouse beta-cells significantly decreased exocytosis, whereas inhibition of tomosyn expression by small interfering RNA increased exocytosis. Hence, in the pancreatic beta-cell, tomosyn negatively regulates insulin exocytosis.

Syntaxin 4 is concentrated on plasma membrane of astrocytes.

Syntaxins are a family of transmembrane proteins that participate in SNARE complexes to mediate membrane fusion events including exocytosis. Different syntaxins are thought to participate in exocytosis in different compartments of the nervous system such as the axon, the soma/dendrites or astrocytes. It is well known that exocytosis of synaptic vesicles at axonal presynaptic terminals involves syntaxin 1 but distributions of syntaxins on neuronal somal and dendritic, postsynaptic or astroglial plasma membranes are less well characterized. Here, we use pre-embedding immunogold labeling to compare the distribution of two plasma membrane-enriched syntaxins (1 and 4) in dissociated rat hippocampal cultures as well as in perfusion-fixed mouse brains. Comparison of Western blots of neuronal cultures, consisting of a mixture of hippocampal neurons and glia, with glial cultures, consisting of mostly astrocytes, shows that syntaxin 1 is enriched in neuronal cultures, whereas syntaxin 4 is enriched in glial cultures. Electron microscopy (EM)-immunogold labeling shows that syntaxin 1 is most abundant at the plasma membranes of axons and terminals, while syntaxin 4 is most abundant at astroglial plasma membranes. This differential distribution was evident even at close appositions of membranes at synapses, where syntaxin 1 was localized to the plasma membrane of the presynaptic terminal, including that at the active zone, while syntaxin 4 was localized to nearby peri-synaptic astroglial processes. These results show that syntaxin 4 is available to support exocytosis in astroglia.

Phenotypic characterization of retinoblastoma for the presence of putative cancer stem-like cell markers by flow cytometry.

PURPOSE: Retinoblastoma (Rb) is an intraocular tumor that grows rapidly and poses a threat to sight and life. Similar to other tumors, there is increasing speculation that the Rb tumor also contains cancer stem-like cells that could influence the prognosis and response to therapy. This study was undertaken in an attempt to identify putative stem-like cells by characterizing different subpopulations of cells in retinoblastoma. METHODS: Freshly isolated tumor cells obtained from unfixed eye specimens (n=7) were analyzed for the presence of CD44, ABCG2, CXCR4, CD133, and CD90 using flow cytometry. RT-PCR was performed to analyze the expression of human Syntaxin1A, PROX1, CD133, and NSE in the sorted subpopulation of tumor cells. RESULTS: Two different subpopulations of cells were observed in seven samples. The small cells, assigned FSC(lo)/SSC(lo) (forward scatter low/side scatter low, ranging from 1.7% to 17.7%) were characterized as positive for CD44 and negative for CD133, CXCR4, and CD90. The large cells were designated as FSC(hi)/SSC(lo) (ranging from 2.7% to 35.1%) and characterized as positive for all markers. RT-PCR analysis revealed that sorted cells of FSC(lo)/SSC(lo) subpopulation expressed the retinal progenitor cell markers PROX1 and Syntaxin1A. CONCLUSIONS: Retinoblastoma, on flow cytometric analysis, revealed two distinct subpopulations with variable expression of stem cell and retinal progenitor markers. In these populations, the FSC(lo)/SSC(lo) subpopulation appeared to be more primitive, since they expressed stem cell (CD44) and retinal progenitor markers (PROX1 and Syntaxin 1A) combined with a relatively lower percentage of differentiated markers. Moreover, the FSC(hi)/SSC(lo) subpopulation showed a higher percentage of differentiated markers (CD90 and CD133).

Three-gene prognostic classifier for early-stage non small-cell lung cancer.

PURPOSE: Several microarray studies have reported gene expression signatures that classify non-small-cell lung carcinoma (NSCLC) patients into different prognostic groups. However, the prognostic gene lists reported to date overlap poorly across studies, and few have been validated independently using more quantitative assay methods. PATIENTS AND METHODS: The expression of 158 putative prognostic genes identified in previous microarray studies was analyzed by reverse transcription quantitative polymerase chain reaction in the tumors of 147 NSCLC patients. Concordance indices and risk scores were used to identify a stage-independent set of genes that could classify patients with significantly different prognoses. RESULTS: We have identified a three-gene classifier (STX1A, HIF1A, and CCR7) for overall survival (hazard ratio = 3.8; 95% CI, 1.7 to 8.2; P < .001). The classifier was also able to stratify stage I and II patients and further improved the predictive ability of clinical factors such as histology and tumor stage. The predictive value of this three-gene classifier was validated in two large independent microarray data sets from Harvard and Duke Universities. CONCLUSION: We have identified a new three-gene classifier that is independent of and improves on stage to stratify early-stage NSCLC patients with significantly different prognoses. This classifier may be tested further for its potential value to improve the selection of resected NSCLC patients in adjuvant therapy.

Loss of SNAP-25 and rabphilin 3a in sensory-motor cortex in Huntington's disease.

Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder caused by a CAG-expansion in the gene encoding the protein huntingtin. The disease is characterized by progressive motor disturbances, cognitive defects, dementia, and weight loss. Using western blotting and immunohistochemistry we have assessed the expression levels and patterns of a number of proteins involved in neurotransmitter release in post-mortem frontal cortex samples from 10 HD cases with different disease grades. We report a loss of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein, synaptosome-associated protein 25 (SNAP 25) in HD brains of grades I-IV. Moreover, in brains of grade III and IV we found a reduction in rabphilin 3a, a protein involved in vesicle docking and recycling. These losses appear to be specific and not due to a general loss of synapses in the HD cortex. Thus, levels of synaptobrevin II, syntaxin 1, rab3a or synaptophysin are unaltered in the same patient samples. SNAP 25 and rabphilin 3a are crucial for neurotransmitter release. Therefore, we suggest that a deficient pre-synaptic transmitter release may underlie some of the symptoms of HD.

Synaptic proteins associate with a sub-set of lipid rafts when isolated from nerve endings at physiological temperature.

Although the high presence of cholesterol in nerve terminals is well documented, specific roles of this lipid in transmitter release have remained elusive. Since cholesterol is a highly enriched component in the membrane microdomains known as lipid rafts, it is probable that these domains are very important in synaptic function. The extraction of lipid rafts using Brij 98 at 37 degrees C avoids the formation of nonspecific membrane aggregates at low temperature, allowing the isolation of more physiologically relevant lipid rafts. In the present work, we examine, by means of buoyancy analysis in sucrose gradients after solubilization of the membranes with Brij 98 or with Lubrol WX, the presence of proteins involved in exocytosis in detergent-resistant membranes (DRM) using rat brain synaptosomes as a neurological model. Significant proportions of the proteins tested in the present work, which are involved in neurotransmitter release, are found in Brij 98 raft fractions, demonstrating that significant pools of synaptic proteins are segregated in specific parts of the membrane at physiological temperature. On the other hand, Lubrol WX is unable to solubilize the major fraction of the proteins tested. Treatment of synaptosomes with methyl-beta-cyclodextrin (mbetaCD) causes alteration in the buoyancy properties of proteins initially present in Brij- as well as in Lubrol-resistant membranes, indicating the cholesterol-dependency of both kinds of microdomains. Finally, we detect the depolarization-induced enhancement of the cholesterol-dependent association of syntaxin 1 with Brij 98-rafts, under the same conditions in which prolonged neurotransmitter release is stimulated.