Variants in STXBP3 are Associated with Very Early Onset Inflammatory Bowel Disease, Bilateral Sensorineural Hearing Loss and Immune Dysregulation.

BACKGROUND AND AIMS: Very early onset inflammatory bowel disease [VEOIBD] is characterized by intestinal inflammation affecting infants and children less than 6 years of age. To date, over 60 monogenic aetiologies of VEOIBD have been identified, many characterized by highly penetrant recessive or dominant variants in underlying immune and/or epithelial pathways. We sought to identify the genetic cause of VEOIBD in a subset of patients with a unique clinical presentation. METHODS: Whole exome sequencing was performed on five families with ten patients who presented with a similar constellation of symptoms including medically refractory infantile-onset IBD, bilateral sensorineural hearing loss and, in the majority, recurrent infections. Genetic aetiologies of VEOIBD were assessed and Sanger sequencing was performed to confirm novel genetic findings. Western analysis on peripheral blood mononuclear cells and functional studies with epithelial cell lines were employed. RESULTS: In each of the ten patients, we identified damaging heterozygous or biallelic variants in the Syntaxin-Binding Protein 3 gene [STXBP3], a protein known to regulate intracellular vesicular trafficking in the syntaxin-binding protein family of molecules, but not associated to date with either VEOIBD or sensorineural hearing loss. These mutations interfere with either intron splicing or protein stability and lead to reduced STXBP3 protein expression. Knock-down of STXBP3 in CaCo2 cells resulted in defects in cell polarity. CONCLUSION: Overall, we describe a novel genetic syndrome and identify a critical role for STXBP3 in VEOIBD, sensorineural hearing loss and immune dysregulation.

Defining the layers of a sensory cilium with STORM and cryoelectron nanoscopy.

Primary cilia carry out numerous signaling and sensory functions, and defects in them, "ciliopathies," cause a range of symptoms, including blindness. Understanding of their nanometer-scale ciliary substructures and their disruptions in ciliopathies has been hindered by limitations of conventional microscopic techniques. We have combined cryoelectron tomography, enhanced by subtomogram averaging, with superresolution stochastic optical reconstruction microscopy (STORM) to define subdomains within the light-sensing rod sensory cilium of mouse retinas and reveal previously unknown substructures formed by resident proteins. Domains are demarcated by structural features such as the axoneme and its connections to the ciliary membrane, and are correlated with molecular markers of subcompartments, including the lumen and walls of the axoneme, the membrane glycocalyx, and the intervening cytoplasm. Within this framework, we report spatial distributions of key proteins in wild-type (WT) mice and the effects on them of genetic deficiencies in 3 models of Bardet-Biedl syndrome.

Syntaxin 8 and the Endoplasmic Reticulum Processing of ΔF508-CFTR.

BACKGROUND/AIMS: Cystic fibrosis (CF) is a lethal recessive disorder caused by mutations in the CF transmembrane conductance regulator (CFTR). ΔF508, the most common mutation, is a misfolded protein that is retained in the endoplasmic reticulum and degraded, precluding delivery to the cell surface [1]. METHODS: Here we use a combination of western blotting, immunoprecipitation, and short circuit current techniques combined with confocal microscopy to address whether the SNARE attachment protein, STX8 plays a role in ΔF508's processing and movement out of the ER. RESULTS: Although the SNARE protein STX8 is thought to be functionally related and primarily localized to early endosomes, we show that silencing of STX8, particularly in the presence of the Vertex corrector molecule C18, rescues ΔF508-CFTR, allowing it to reach the cell surface and increasing CFTR-dependent chloride currents by approximately 2.5-fold over control values. STX8 silencing reduced the binding of quality control protein, Hsp 27, a protein that targets ΔF508-CFTR for sumoylation and subsequent degradation, to ΔF508-CFTR. STX8 silencing increased the levels of Hsp 60 a protein involving in early events in protein folding. CONCLUSION: STX8 knockdown creates an environment favorable for mature ΔF508 to reach the cell surface. The data also suggest that when present at normal levels, STX8 functions as part of the cell's quality control mechanism.

ATG conjugation-dependent degradation of the inner autophagosomal membrane is a key step for autophagosome maturation.

Although the autophagy-related (ATG) conjugation systems are thought to be important for a late step of autophagosome formation, their precise function has been poorly understood because they are also required for localization of the most important autophagosomal marker LC3. In our recent study we found that, using the autophagosomal SNARE STX17 (syntaxin 17) as an alternative marker, autophagosome-like structures were generated in ATG conjugation system-deficient cells. Those structures could fuse with lysosomes but the degradation of the inner autophagosomal membrane was significantly delayed. We suggest that the ATG conjugation-dependent closure of autophagosomes causes the inner autophagosomal membrane to become sensitive to lysosomal degradation.

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.

Syntaxin-4 is essential for IgE secretion by plasma cells.

The humoral immune system provides a crucial first defense against the invasion of microbial pathogens via the secretion of antigen specific immunoglobulins (Ig). The secretion of Ig is carried out by terminally differentiated B-lymphocytes called plasma cells. Despite the key role of plasma cells in the immune response, the mechanisms by which they constitutively traffic large volumes of Ig out of the cell is poorly understood. The involvement of Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins in the regulation of protein trafficking from cells has been well documented. Syntaxin-4, a member of the Qa SNARE syntaxin family has been implicated in fusion events at the plasma membrane in a number of cells in the immune system. In this work we show that knock-down of syntaxin-4 in the multiple myeloma U266 human plasma cell line results in a loss of IgE secretion and accumulation of IgE within the cells. Furthermore, we show that IgE co-localises with syntaxin-4 in U266 plasma cells suggesting direct involvement in secretion at the plasma membrane. This study demonstrates that syntaxin-4 plays a critical role in the secretion of IgE from plasma cells and sheds some light on the mechanisms by which these cells constitutively traffic vesicles to the surface for secretion. An understanding of this machinery may be beneficial in identifying potential therapeutic targets in multiple myeloma and autoimmune disease where over-production of Ig leads to severe pathology in patients.

Isolation and proteomic analysis of the SYP61 compartment reveal its role in exocytic trafficking in Arabidopsis.

The endomembrane system is a complex and dynamic intracellular trafficking network. It is very challenging to track individual vesicles and their cargos in real time; however, affinity purification allows vesicles to be isolated in their natural state so that their constituent proteins can be identified. Pioneering this approach in plants, we isolated the SYP61 trans-Golgi network compartment and carried out a comprehensive proteomic analysis of its contents with only minimal interference from other organelles. The proteome of SYP61 revealed the association of proteins of unknown function that have previously not been ascribed to this compartment. We identified a complete SYP61 SNARE complex, including regulatory proteins and validated the proteome data by showing that several of these proteins associated with SYP61 in planta. We further identified the SYP121-complex and cellulose synthases, suggesting that SYP61 plays a role in the exocytic trafficking and the transport of cell wall components to the plasma membrane. The presence of proteins of unknown function in the SYP61 proteome including ECHIDNA offers the opportunity to identify novel trafficking components and cargos. The affinity purification of plant vesicles in their natural state provides a basis for further analysis and dissection of complex endomembrane networks. The approach is widely applicable and can afford the study of several vesicle populations in plants, which can be compared with the SYP61 vesicle proteome.

The COG complex interacts directly with Syntaxin 6 and positively regulates endosome-to-TGN retrograde transport.

The conserved oligomeric Golgi (COG) complex has been implicated in the regulation of endosome to trans-Golgi network (TGN) retrograde trafficking in both yeast and mammals. However, the exact mechanisms by which it regulates this transport route remain largely unknown. In this paper, we show that COG interacts directly with the target membrane SNARE (t-SNARE) Syntaxin 6 via the Cog6 subunit. In Cog6-depleted cells, the steady-state level of Syntaxin 6 was markedly reduced, and concomitantly, endosome-to-TGN retrograde traffic was significantly attenuated. Cog6 knockdown also affected the steady-state levels and/or subcellular distributions of Syntaxin 16, Vti1a, and VAMP4 and impaired the assembly of the Syntaxin 6-Syntaxin16-Vti1a-VAMP4 SNARE complex. Remarkably, overexpression of VAMP4, but not of Syntaxin 6, bypassed the requirement for COG and restored endosome-to-TGN trafficking in Cog6-depleted cells. These results suggest that COG directly interacts with specific t-SNAREs and positively regulates SNARE complex assembly, thereby affecting their associated trafficking steps.

Syntaxin 3 is necessary for cAMP- and cGMP-regulated exocytosis of CFTR: implications for enterotoxigenic diarrhea.

Enterotoxins elaborated by Vibrio cholerae and Escherichia coli cannot elicit fluid secretion in the absence of functional cystic fibrosis transmembrane conductance regulator (CFTR) chloride channels. After enterotoxin exposure, CFTR channels are rapidly recruited from endosomes and undergo exocytic insertion into the apical plasma membrane of enterocytes to increase the number of channels on the cell surface by at least fourfold. However, the molecular machinery that orchestrates exocytic insertion of CFTR into the plasma membrane is largely unknown. The present study used immunofluorescence, immunoblotting, surface biotinylation, glutathione S-transferase (GST) pulldown assays, and immunoprecipitation to identify components of the exocytic soluble N-ethylmaleimide (NEM)-sensitive factor attachment receptor (SNARE) vesicle fusion machinery in cyclic nucleotide-activated exocytosis of CFTR in rat jejunum and polarized intestinal Caco-2(BB)e cells. Syntaxin 3, an intestine-specific SNARE, colocalized with CFTR on the apical domain of enterocytes in rat jejunum and polarized Caco-2(BB)e cells. Coimmunoprecipitation and GST binding studies confirmed that syntaxin 3 interacts with CFTR in vivo. Moreover, heat-stable enterotoxin (STa) activated exocytosis of both CFTR and syntaxin 3 to the surface of rat jejunum. Silencing of syntaxin 3 by short hairpin RNA (shRNA) interference abrogated cyclic nucleotide-stimulated exocytosis of CFTR in cells. These observations reveal a new and important role for syntaxin 3 in the pathophysiology of enterotoxin-elicited diarrhea.

Spo5 phosphorylation is essential for its own timely degradation and for successful meiosis in Schizosaccharomyces pombe.

Protein phosphorylation is pivotal for meiotic progression, but little is known about its regulatory mechanisms. We show that before meiosis I, the meiosis-specific Schizosaccharomyces pombe protein Spo5 is phosphorylated in vivo on T29, T55, S59 and/or T63. In a mutant strain expressing Spo5 fused to green fluorescent protein with alanine substitutions of these amino acid sites (GFP; Spo5-4A-GFP), the timely degradation of Spo5 at meiosis II was not observed. Additionally, Spo5-4A-GFP signals were retained after metaphase II and were localized to the nucleus. This was accompanied by the nuclear mislocalization of Psy1, a marker of the forespore membrane (FSM), and the generation of empty cells, in which cytoplasm had leaked from the ruptured membrane, as well as by the appearance of asci harboring deformed spores. Indeed, thin-section electron microscopy (TEM) revealed fragile-looking spo5-4A-GFP ascospores with ruffled spore walls. In contrast, a mutant strain expressing a constitutively-phosphorylated form of Spo5 (Spo5-4D-GFP) was phenotypically indistinguishable from a strain expressing wild-type (WT) protein (Spo5-WT-GFP). Taken together, these results indicate that Spo5 phosphorylation ensures the timely degradation of Spo5 during meiosis and the proper localization of Psy1, leading to the production of viable spores with robust FSMs and strong walls.

Syntaxin-11 is expressed in primary human monocytes/macrophages and acts as a negative regulator of macrophage engulfment of apoptotic cells and IgG-opsonized target cells.

Syntaxin-11 is a member of a family of membrane-trafficking proteins referred to as soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs). Recent studies have shown that syntaxin-11 is expressed in natural killer cells and cytotoxic T cells and is likely to play a role in the granule exocytosis pathway. However, the biological role of syntaxin-11 in other immune cells has remained elusive. This study found that stimulation with interferon-gamma upregulated syntaxin-11 expression in primary monocytes. Experiments using monocytes from patients with familial haemophagocytic lymphohistiocytosis harbouring mutations in the gene encoding syntaxin-11 (STX11), or monocytes from healthy individuals in which syntaxin-11 was downregulated using specific short-interfering RNA, demonstrated that syntaxin-11 was not required for antibody-dependent cellular cytotoxicity. On the other hand, silencing of syntaxin-11 expression in primary macrophages enhanced the phagocytosis of apoptotic target cells with a concomitant increase in macrophage secretion of tumour necrosis factor-alpha. Moreover, Fcgamma-receptor-mediated uptake of target cells was also enhanced following silencing of syntaxin-11 expression in macrophages. In addition, syntaxin-11 localized to the plasma membrane in macrophages ingesting apoptotic cell corpses. Syntaxin-11 thus appears to act as a negative regulator of human macrophage engulfment of apoptotic cells and IgG-opsonized red blood cells.

STED microscopy with continuous wave beams.

We report stimulated emission depletion (STED) fluorescence microscopy with continuous wave (CW) laser beams. Lateral fluorescence confinement from the scanning focal spot delivered a resolution of 29-60 nm in the focal plane, corresponding to a 5-8-fold improvement over the diffraction barrier. Axial spot confinement increased the axial resolution by 3.5-fold. We observed three-dimensional (3D) subdiffraction resolution in 3D image stacks. Viable for fluorophores with low triplet yield, the use of CW light sources greatly simplifies the implementation of this concept of far-field fluorescence nanoscopy.

The EGFR inhibitor gefitinib suppresses ligand-stimulated endocytosis of EGFR via the early/late endocytic pathway in non-small cell lung cancer cell lines.

The drug gefitinib (Iressa), which is a specific inhibitor of EGFR tyrosine kinase, has been shown to suppress the activation of EGFR signaling for survival and proliferation in non-small cell lung cancer (NSCLC) cell lines. A recent study demonstrated rapid down-regulation of ligand-induced EGFR in a gefitinib-sensitive cell line and inefficient down-regulation of EGFR in a gefitinib-resistant cell line in the exponential phase of growth; this implies that each cell type employs a different unknown down-regulation mechanism occurs. However, the mechanism of drug sensitivity to gefitinib remains unclear. In this study, to further substantiate the effect of gefitinib on the EGFR down-regulation pathway and to understand the detailed internalization mechanism of gefitinib-sensitive PC9 and gefitinib-resistant QG56 cell lines, we examined the internalization of Texas red-EGF in the absence or presence of gefitinib in both cell lines. The distribution of internalized Texas red-EGF, early endosomes, and late endosomes/lysosomes was then assessed by confocal immunofluorescence microscopy. Here, we provide novel evidence that efficient endocytosis of EGF-EGFR occurs via the endocytic pathway in the PC9 cells, because the internalized Texas red-EGF-positive small punctate vesicles were transported to the late endosomes/lysosomes and then degraded within the lysosomes after 60 min of internalization. Additionally, gefitinib exerted a strong inhibitory effect on the endocytosis of EGFR in PC9 cells, and the internalization rate of EGFR from the plasma membrane via the early endosomes to the late endosomes/lysosomes was considerably delayed. This indicates that gefitinib efficiently suppresses ligand-stimulated endocytosis of EGFR via the early/late endocytic pathway in PC9 cells. In contrast, the internalization rate of ligand-induced EGFR was not significantly changed by gefitinib in QG56 cells because even in the absence of gefitinib, internalized EGFR accumulation was noted in the early and late endosomes after 60 min of internalization instead of its delivery to the lysosomes in QG56 cells. This suggests that the endocytic machinery of EGFR might be basically impaired at the level of the early/late endosomes. Taken together, this is the first report demonstrating that the suppressive effect of gefitinib on the endocytosis of EGFR is much stronger with PC9 cells than QG56 cells. Thus, impairment in some steps of the EGF-EGFR traffic out of early endosomes toward the late endosomes/lysosomes might confer gefitinib-resistance in NSCLC cell lines.

Four-dimensional imaging of filter-grown polarized epithelial cells.

Understanding how epithelial cells generate and maintain polarity and function requires live cell imaging. In order for cells to become fully polarized, it is necessary to grow them on a permeable membrane filter; however, the translucent filter obstructs the microscope light path required for quantitative live cell imaging. Alternatively, the membrane filter may be excised but this eliminates selective access to apical and basolateral surfaces. Conversely, epithelial cells cultured directly on glass exhibit different phenotypes and functions from filter grown cells. Here, we describe a new method for culturing polarized epithelial cells on a Transwell filter insert that allows superior live cell imaging with spatial and temporal image resolution previously unachievable using conventional methods. Cells were cultured on the underside of a filter support. Epithelial cells grown in this inverted configuration exhibit a fully polarized architecture, including the presence of functional tight junctions. This new culturing system permits four-dimensional (three spatial dimension over time) imaging of endosome and Golgi apparatus dynamics, and permits selective manipulation of the apical and basolateral surfaces. This new technique has wide applicability for visualization and manipulation of polarized epithelial cells.

Heavy chain of cytoplasmic dynein is a major component of the postsynaptic density fraction.

A protein with an apparent molecular size of 490 kDa was found in the postsynaptic density (PSD) fraction isolated from porcine cerebral cortices and rat forebrains, and this 490 kDa protein accounted for approximately 3% of the total protein of these samples. Matrix-assisted laser desorption ionization-time of flight mass spectrometric and Western blotting analyses consistently indicated that this 490 kDa protein consisted primarily of the heavy chain of cytoplasmic dynein (cDHC). Immunocytochemical analyses showed that cDHC was found in 92% and 89% of the phalloidin-positive protrusions that were themselves associated with discrete clusters of synaptophysin, a presynaptic terminal marker, and PSD-95, a postsynaptic marker, on neuronal processes, respectively. Quantitative Western blotting analyses of various subcellular fractions isolated from porcine cerebral cortices and rat forebrains further showed that not only the heavy but also the intermediate chains of dynein are enriched in the PSD fraction. Cytoplasmic dynein is a microtubule-associated motor protein complex that drives the movement of various cargos toward the minus ends of microtubules and plays many other diverse functions in the cell. Our results that cDHC is a major component of the PSD fraction, that both dynein heavy and intermediate chains are enriched in the PSD fraction and that cDHC is present in dendritic spines raise the possibilities that cytoplasmic dynein may play structural and functional roles in the postsynaptic terminal.

High-throughput identification of IMCD proteins using LC-MS/MS.

The inner medullary collecting duct (IMCD) is an important site of vasopressin-regulated water and urea transport. Here we have used protein mass spectrometry to investigate the proteome of the IMCD cell and how it is altered in response to long-term vasopressin administration in rats. IMCDs were isolated from inner medullas of rats, and IMCD proteins were identified by liquid chromatography/tandem mass spectrometry (LC-MS/MS). We present a WWW-based "IMCD Proteome Database" containing all IMCD proteins identified in this study (n = 704) and prior MS-based identification studies (n = 301). We used the isotope-coded affinity tag (ICAT) technique to identify IMCD proteins that change in abundance in response to vasopressin. Vasopressin analog (dDAVP) or vehicle was infused subcutaneously in Brattleboro rats for 3 days, and IMCDs were isolated for proteomic analysis. dDAVP and control samples were labeled with different cleavable ICAT reagents (mass difference 9 amu) and mixed. This was followed by one-dimensional SDS-PAGE separation, in-gel trypsin digestion, biotin-avidin affinity purification, and LC-MS/MS identification and quantification. Responses to vasopressin for a total of 165 proteins were quantified. Quantification, based on semiquantitative immunoblotting of 16 proteins for which antibodies were available, showed a high degree of correlation with ICAT results. In addition to aquaporin-2 and gamma-epithelial Na channel (gamma-ENaC), five of the immunoblotted proteins were substantially altered in abundance in response to dDAVP, viz., syntaxin-7, Rap1, GAPDH, heat shock protein (HSP)70, and cathepsin D. A 28-protein vasopressin signaling network was constructed using literature-based network analysis software focusing on the newly identified proteins, providing several new hypotheses for future studies.

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.

Quantitative membrane proteomics reveals new cellular targets of viral immune modulators.

Immunomodulators of pathogens frequently affect multiple cellular targets, thus preventing recognition by different immune cells. For instance, the K5 modulator of immune recognition (MIR2) from Kaposi sarcoma-associated herpesvirus prevents activation of cytotoxic T cells, natural killer cells, and natural killer T cells by downregulating major histocompatibility complex (MHC) class I molecules, the MHC-like molecule CD1, the cell adhesion molecules ICAM-1 and PECAM, and the co-stimulatory molecule B7.2. K5 belongs to a family of viral- and cellular-membrane-spanning RING ubiquitin ligases. While a limited number of transmembrane proteins have been shown to be targeted for degradation by this family, it is unknown whether additional targets exist. We now describe a quantitative proteomics approach to identify novel targets of this protein family. Using stable isotope labeling by amino acids, we compared the proteome of plasma, Golgi, and endoplasmic reticulum membranes in the presence and absence of K5. Mass spectrometric protein identification revealed four proteins that were consistently underrepresented in the plasma membrane of K5 expression cells: MHC I (as expected), bone marrow stromal antigen 2 (BST-2, CD316), activated leukocyte cell adhesion molecule (ALCAM, CD166) and Syntaxin-4. Downregulation of each of these proteins was independently confirmed by immunoblotting with specific antibodies. We further demonstrate that ALCAM is a bona fide target of both K5 and the myxomavirus homolog M153R. Upon exiting the endoplasmic reticulum, ALCAM is ubiquitinated in the presence of wild-type, but not RING-deficient or acidic motif-deficient, K5, and is targeted for lysosomal degradation via the multivesicular body pathway. Since ALCAM is the ligand for CD6, a member of the immunological synapse of T cells, its removal by viral immune modulators implies a role for CD6 in the recognition of pathogens by T cells. The unbiased global proteome analysis therefore revealed novel immunomodulatory functions of pathogen proteins.

Intracellular interaction between syntaxin and Munc 18-1 revealed by fluorescence resonance energy transfer.

Neurosecretion is catalyzed by assembly of a soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor (SNARE)-complex composed of SNAP-25, synaptobrevin and syntaxin. Munc 18-1 is known to bind to syntaxin in vitro. This interaction prevents assembly of the SNARE-complex, but might also affect intracellular targeting of the proteins. We have fused syntaxin and Munc 18 to the yellow- (YFP) or cyan-fluorescence-protein (CFP) and expressed the constructs in CHO- and MDCK-cells. We have studied their localization with confocal microscopy and a possible protein-protein interaction with fluorescence-resonance energy transfer (FRET). YFP-syntaxin localizes to intracellular membranes. CFP-Munc 18 is present in the cytoplasm as expected for a protein lacking membrane targeting domains. However, Munc 18 is redirected to internal membranes when syntaxin is coexpressed, but only limited transport of the proteins to the plasma membrane was observed. An interaction between Munc 18 and syntaxin could be demonstrated by FRET using two methods, sensitized acceptor fluorescence and acceptor photobleaching. A mutation in syntaxin (L165A, E166A), which is known to inhibit binding to Munc 18 in vitro, prevents colocalization of the proteins and also the FRET signal. Thus, a protein-protein interaction between Munc 18 and syntaxin occurs on intracellular membranes, which is required but not sufficient for quantitative transport of both proteins to the plasma membrane.

Envenoming bites by kraits: the biological basis of treatment-resistant neuromuscular paralysis.

Beta-bungarotoxin, a neurotoxic phospholipase A2 is a major fraction of the venom of kraits. The toxin was inoculated into one hind limb of young adult rats. The inoculated hind limb was paralysed within 3 h, and remained paralysed for 2 days. The paralysis was associated with the loss of synaptic vesicles from motor nerve terminal boutons, a decline in immunoreactivity of synaptophysin, SNAP-25 and syntaxin, a loss of muscle mass and the upregulation of NaV(1.5) mRNA and protein. Between 3 and 6 h after the inoculation of toxin, some nerve terminal boutons exhibited clear signs of degeneration. Others appeared to be in the process of withdrawing from the synaptic cleft and some boutons were fully enwrapped in terminal Schwann cell processes. By 12 h all muscle fibres were denervated. Re-innervation began at 3 days with the appearance of regenerating nerve terminals, a return of neuromuscular function in some muscles and a progressive increase in the immunoreactivity of synaptophysin, SNAP-25 and syntaxin. Full recovery occurred at 7 days. The data were compared with recently published clinical data on envenoming bites by kraits and by extrapolation we suggest that the acute, reversible denervation caused by beta-bungarotoxin is a credible explanation for the clinically important, profound treatment-resistant neuromuscular paralysis seen in human subjects bitten by these animals.