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PubMedDate not supplied

Vesicle targeting in hair cells.

The mammalian hair cell has two distinct plasma membrane domains separated by tight junctions, the apical domain which contains the stereocilia and the basolateral domain which contains the presynaptic region. Little is known concerning the mechanisms that regulate vesicle trafficking to these two domains. Using SNAP 25 and syntaxin as baits, we carried out a yeast two-hybrid screen of the organ of Corti. We identified a novel syntaxin interacting protein, ocsyn, that is enriched in inner hair cells and concentrated at the apical pole. Our results are consistent with ocsyn playing a role in vesicle trafficking to the apical membrane of the hair cell.

Animals↗
PubMedDate not supplied

Roles of SNARE proteins and synaptotagmin I in synaptic transmission: studies at the Drosophila neuromuscular synapse.

The roles of SNARE proteins, i.e. neuronal Synaptobrevin (n-Syb), SNAP-25 and Syntaxin 1A (Syx 1A), and Synaptotagmin I (Syt I) in synaptic transmission have been studied in situ using mutant embryos or larvae that lack these molecules or have alterations in them. Because of the ease of genetic manipulation, the Drosophila neuromuscular synapse is widely used for these studies. The functional properties of synaptic transmission have been studied in mutant embryos using the patch-clamp technique, and in larvae by recording with microelectrodes. A major vesicular membrane protein, n-Syb, is indispensable for nerve-evoked synaptic transmission. Spontaneous synaptic currents (minis), however, are present even in embryos totally lacking n-Syb (N-SYB). Furthermore, Ca(2+)-independent enhancement of mini frequency induced by hypertonic sucrose solutions (hypertonicity response) is totally absent in N-SYB. Embryos that have defects in SNAP-25 (SNAP-25) have similar but milder phenotypes than N-SYB. The phenotype in synaptic transmission was most severe in the synapse lacking Syx 1A. Neither nerve-evoked synaptic currents nor minis occur in embryos lacking Syx 1A (SYX 1A). No hypertonicity response was observed in them. Syt I binds Ca(2+) in vitro and probably serves as a Ca(2+) sensor for nerve-evoked synaptic transmission, since nerve-evoked synaptic currents were greatly reduced in embryos lacking Syt I (SYT I). Also, Syt I has a role in vesicle recycling. Interestingly, the Ca(2+)-independent hypertonicity response is also greatly reduced in SYT I. Minis persist in mutant embryos lacking any of these proteins (n-Syb, SNAP-25 and Syt I), except Syx, suggesting that minis have a distinct fusion mechanism from that for fast and synchronized release. It appears that these SNARE proteins and Syt I are coordinated for fast vesicle fusion. Minis, on the other hand, do not require SNARE complex nor Syt I, but Syx is absolutely required for vesicle fusion. The SNARE complex and Syt I are indispensable for the hypertonicity response. None of these molecules seem to serve for selective docking of synaptic vesicles to the release site. For further studies on synaptic transmission, the Drosophila neuromuscular synapse will continue to be a useful model.

Animals↗
PubMed2002

Epimorphin expression in intestinal myofibroblasts induces epithelial morphogenesis.

The formation of the crypt-villus axis during gut ontogeny requires continued reciprocal interactions between the endoderm and mesenchyme. Epimorphin/syntaxin 2 (epimorphin) is a mesenchymal protein expressed in the fetal gastrointestinal tract during villus morphogenesis. To elucidate its role in gut ontogeny, the epimorphin cDNA was transfected, in sense and antisense orientations, into a rat intestinal myofibroblast cell line, MIC 216. To determine the effects of epimorphin on the epithelium, myofibroblasts were cocultured with the Caco2 cell line. Caco2 cells spread in a simple monolayer over antisense-transfected cells lacking epimorphin. In contrast, sense-transfected myofibroblasts induced Caco2 cells to form compact, round clusters with small lumens. These morphologic differences were preserved in Transwell cocultures in which cell-cell contact was prevented, suggesting that epimorphin's effects were mediated by secreted factor(s). To determine the effects of epimorphin on crypt-villus axis formation in an in vivo model, rat gut endoderm was combined with epimorphin-transfected myofibroblasts and implanted into the chick intracoelomic cavity. The grafts in which epimorphin was overexpressed revealed multiple well-formed villi with crypt-like units, whereas those in which epimorphin expression was inhibited developed into round cystic structures without crypts or villi. Of several potential secreted morphogens, only the expression of bone morphogenetic protein 4 (Bmp4) was increased in the epimorphin-transfected cells. Incubation with noggin partially blocked the transfected myofibroblasts' effects on Caco2 colony morphology. These results indicate that mesenchymal epimorphin has profound effects on crypt-villus morphogenesis, mediated in part by secreted factor(s) including the Bmp's.

Actins↗
PubMed2000

Syntaxin 1A is expressed in airway epithelial cells, where it modulates CFTR Cl(-) currents.

The CFTR Cl(-) channel controls salt and water transport across epithelial tissues. Previously, we showed that CFTR-mediated Cl(-) currents in the Xenopus oocyte expression system are inhibited by syntaxin 1A, a component of the membrane trafficking machinery. This negative modulation of CFTR function can be reversed by soluble syntaxin 1A peptides and by the syntaxin 1A binding protein, Munc-18. In the present study, we determined whether syntaxin 1A is expressed in native epithelial tissues that normally express CFTR and whether it modulates CFTR currents in these tissues. Using immunoblotting and immunofluorescence, we observed syntaxin 1A in native gut and airway epithelial tissues and showed that epithelial cells from these tissues express syntaxin 1A at >10-fold molar excess over CFTR. Syntaxin 1A is seen near the apical cell surfaces of human bronchial airway epithelium. Reagents that disrupt the CFTR-syntaxin 1A interaction, including soluble syntaxin 1A cytosolic domain and recombinant Munc-18, augmented cAMP-dependent CFTR Cl(-) currents by more than 2- to 4-fold in mouse tracheal epithelial cells and cells derived from human nasal polyps, but these reagents did not affect CaMK II-activated Cl(-) currents in these cells.

Animals↗
PubMed2003

Regulation of growth cone extension by SNARE proteins.

Recent studies have suggested that the soluble N-ethylmaleimide-sensitive factor attached protein (SNAP) receptor (SNARE)-mediated membrane fusion system is involved in vesicle fusion with the surface plasma membrane, which leads to neurite elongation. There have been several reports analyzing the effects of neurite outgrowth by inhibition of SNAREs. We studied this mechanism by overexpressing GFP-fusion SNAREs including VAMP-2, SNAP-25A, and syntaxin1A in PC12 cells to investigate the role of SNAREs in neurite outgrowth. When overexpressed in PC12 cells, VAMP-2 promoted neurite elongation, whereas SNAP-25A stimulated neurite sprouting. On the other hand, overexpression of syntaxin1A neither promoted nor inhibited neurite outgrowth. Thus, VAMP-2 and SNAP-25A play different roles in neurite elongation and sprouting.

Animals↗
PubMed2002

Subcellular distribution of 3 functional platelet SNARE proteins: human cellubrevin, SNAP-23, and syntaxin 2.

Morphologic studies have demonstrated a process by which alpha-granule contents are released from platelets. Studies aimed at defining the molecular mechanisms of this release have demonstrated that SNARE proteins are required for alpha-granule secretion. These observations raise the possibility that morphologic features of alpha-granule secretion may be influenced by the subcellular distribution of SNARE proteins in the platelet. To evaluate this possibility, we analyzed the subcellular distribution of 3 functional platelet SNARE proteins-human cellubrevin, SNAP-23, and syntaxin 2. Exposure of streptolysin O-permeabilized platelets to antihuman cellubrevin antibody inhibited Ca(++)-induced alpha-granule secretion by approximately 50%. Inhibition of alpha-granule secretion by antihuman cellubrevin was reversed by a blocking peptide. Syntaxin 2 and SNAP-23 have previously been demonstrated to mediate platelet granule secretion. The subcellular localization of the 3 SNARE proteins was determined by ultrastructural studies, using a pre-embedding immunonanogold method, and by immunoblot analysis of subcellular fractions. Immunonanogold localization demonstrated that approximately 80% of human cellubrevin in resting platelets was localized to platelet granule membranes. In contrast, SNAP-23 localized predominantly to plasma membrane, whereas syntaxin 2 was more evenly distributed among membranes of alpha-granules, the open canalicular system, and plasma membrane. Thus, each of these SNARE proteins has a distinct subcellular distribution in platelets, and each of these membrane compartments demonstrates a unique SNARE protein composition. This distribution provides a basis for several characteristics of alpha-granule secretion that include homotypic alpha-granule fusion and the fusion of alpha-granules with the open canalicular system and plasma membrane.

Antibodies↗
PubMed2005

Epimorphin expression in interstitial pneumonia.

Epimorphin modulates epithelial morphogenesis in embryonic mouse organs. We previously suggested that epimorphin contributes to repair of bleomycin-induced pulmonary fibrosis in mice via epithelium-mesenchyme interactions. To clarify the role of epimorphin in human lungs, we evaluated epimorphin expression and localization in normal lungs, lungs with nonspecific interstitial pneumonia (NSIP), and lungs with usual interstitial pneumonia (UIP); we also studied the effect of recombinant epimorphin on cultured human alveolar epithelial cells in vitro. Northern and Western blotting analyses revealed that epimorphin expression in NSIP samples were significantly higher than those in control lungs and lungs with UIP. Immunohistochemistry showed strong epimorphin expression in mesenchymal cells of early fibrotic lesions and localization of epimorphin protein on mesenchymal cells and extracellular matrix of early fibrotic lesions in the nonspecific interstitial pneumonia group. Double-labeled fluorescent images revealed expression of matrix metalloproteinase 2 in re-epithelialized cells overlying epimorphin-positive early fibrotic lesions. Immunohistochemistry and metalloproteinase activity assay demonstrated augmented expression of metalloproteinase induced by recombinant epimorphin in human alveolar epithelial cells. These findings suggest that epimorphin contributes to repair of pulmonary fibrosis in nonspecific interstitial pneumonia, perhaps partly by inducing expression of matrix metalloproteinase 2, which is an important proteolytic factor in lung remodeling.

Cells, Cultured↗
PubMed2005

Localization of plasma membrane t-SNAREs syntaxin 2 and 3 in intracellular compartments.

BACKGROUND: Membrane fusion requires the formation of a complex between a vesicle protein (v-SNARE) and the target membrane proteins (t-SNAREs). Syntaxin 2 and 3 are t-SNAREs that, according to previous over-expression studies, are predominantly localized at the plasma membrane. In the present study we investigated localization of the endogenous syntaxin 2 and 3. RESULTS: Endogenous syntaxin 2 and 3 were found in NRK cells in intracellular vesicular structures in addition to regions of the plasma membrane. Treatment of these cells with N-ethylmaleimide (NEM), which is known to inactivate membrane fusion, caused syntaxin 3 to accumulate in the trans-Golgi network and syntaxin 2 in perinuclear membrane vesicles. Kinetic analysis in the presence of NEM indicated that this redistribution of syntaxin 2 and 3 takes place via actin containing structures. CONCLUSION: Our data suggest that syntaxin 2 cycles between the plasma membrane and the perinuclear compartment whereas syntaxin 3 cycles between the plasma membrane and the trans-Golgi network. It is possible that this cycling has an important role in the regulation of t-SNARE function.

Actins↗
PubMed2004

Crystal structure of the Habc domain of neuronal syntaxin from the squid Loligo pealei reveals conformational plasticity at its C-terminus.

BACKGROUND: Intracellular membrane fusion processes are mediated by the spatial and temporal control of SNARE complex assembly that results in the formation of a four-helical bundle, composed of one vesicle SNARE and three target membrane SNARE polypeptide chains. Syntaxins are essential t-SNAREs and are characterized by an N-terminal Habc domain, a flexible linker region, a coiled-coil or SNARE motif and a membrane anchor. The N-terminal Habc domain fulfills important regulatory functions while the coiled-coil motif, present in all SNAREs, is sufficient for SNARE complex formation, which is thought to drive membrane fusion. RESULTS: Here we report the crystal structure of the Habc domain of neuronal syntaxin from the squid Loligo pealei, s-syntaxin. Squid Habc crystallizes as a dimer and the monomer structure consists of a three-helical bundle. One molecule is strikingly similar to mammalian syntaxin 1A while the second one shows a structural deviation from the common fold in that the C-terminal part of helix C unwinds and adopts an extended conformation. CONCLUSION: Conservation of surface residues indicates that the cytosolic part of s-syntaxin can adopt an auto-inhibitory closed conformation that may bind squid neuronal Sec1, s-Sec1, in the same manner as observed in structure of the rat nSec1/syntaxin 1A complex. Furthermore, despite the overall structural similarity, the observed changes at the C-terminus of one molecule indicate structural plasticity in neuronal syntaxin. Implications of the structural conservation and the changes are discussed with respect to potential Habc domain binding partners such as Munc13, which facilitates the transition from the closed to the open conformation.

Amino Acid Sequence↗
PubMed2003

42nd Annual Meeting of the American Society for Cell Biology, San Francisco, California, USA, 14-18 December 2002.

The Annual Meeting of the American Society for Cell Biology (ASCB) is a diverse conference covering all topics in cell biology. While all of the basic biology presented at this meeting may potentially contribute to breast cancer research, there were a significant number of presentations and posters directly pertinent to this field. Here we have summarized the research that is of greatest immediate relevance to breast cancer, with particular emphasis on mammary gland development and tumorigenesis in vivo, three-dimensional in vitro models of mammary morphogenesis, alterations of signal transduction pathways in breast cancer, and global studies in expression profiling and drug screening.

Animals↗
PubMed2003

Effect of inherited abnormalities of calcium regulation on human neuromuscular transmission.

Synaptotagmins are abundant synaptic proteins that represent the best candidate for the calcium sensor at the nerve terminal. The pore-forming, voltage-sensing transmembrane alpha-1 subunit of the P/Q voltage-gated calcium channel (or Ca(v)2.1) encoded by the CACNA1A gene is another major component of the process of action potential-evoked exocytosis at the adult mammalian neuromuscular junction. Defects of these proteins, in nonhuman species, result in severe disruption of rapid synaptic transmission. This paper investigates the molecular bases of inherited presynaptic deficits of neuromuscular transmission in humans. Patients with congenital presynaptic failure, including two patients with episodic ataxia type 2 (EA-2) due to CACNA1A mutations, were studied with muscle biopsy, microelectrode studies, electron microscopy, DNA amplification, and sequencing. All patients, including EA-2 patients, showed selective failure of the action potential-dependent release without reduction of the spontaneous release of neurotransmitter. In addition, patients with EA-2 showed partial blockade of neuromuscular transmission with the N-type blocker omega-conotoxin not seen in controls. The EM showed a varied degree of increased complexity of postsynaptic folds. Mutational analysis in candidate genes, including human synaptotagmin II, syntaxin 1A, synaptobrevin I, SNAP 25, CACNA1A, CACNB2, and Rab3A, was unrevealing. Although no mutations in candidate genes were found in patients with inborn presynaptic failure, functional and structural similarities between this group and patients with EA-2 due to CACNA1A mutations suggest a common pathogenic mechanism.

Action Potentials↗
PubMed1997

Characterization of two distinct intracellular GLUT4 membrane populations in muscle fiber. Differential protein composition and sensitivity to insulin.

A major objective for the understanding of muscle glucose disposal is the elucidation of the intracellular trafficking pathway of GLUT4 glucose carriers in the muscle fiber. In this report, we provide functional and biochemical characterization of two distinct intracellular GLUT4 vesicle pools obtained from rat skeletal muscle. The two pools showed a differential response to insulin; thus, one showed a marked decrease in GLUT4 levels but the other did not. They also showed a markedly different protein composition as detected by quantitative vesicle immunoisolation analysis. The GLUT4 pool showing no response to insulin contained SCAMP proteins and the vSNARE proteins VAMP2 and cellubrevin, whereas only VAMP2 was found in the insulin-recruitable GLUT4 pool. SDS-PAGE and further silver staining of the immunoprecipitates revealed discrete polypeptide bands associated to the insulin-sensitive pool, and all these polypeptide bands were found in the insulin-insensitive population. Furthermore, some polypeptide bands were exclusive to the insulin-insensitive population. The presence of cellubrevin and SCAMP proteins, endosomal markers, suggest that the insulin-insensitive GLUT4 membrane population belongs to an endosomal compartment. In addition, we favor the view that the insulin-sensitive GLUT4 membrane pool is segregated from the endosomal GLUT4 population and is undergoes exocytosis to the cell surface in response to insulin. Intracellular GLUT4 membranes obtained from skeletal muscle contain cellubrevin, and VAMP2 and GLUT4-vesicles from cardiomyocytes also contain cellubrevin. This suggests that vSNARE proteins are key constituents of GLUT4 vesicles. The presence of the tSNARE protein SNAP25 in skeletal muscle membranes and SNAP25 and syntaxin 1A and syntaxin 1B in cardiomyocyte plasma membranes further suggest a role of the SNAREs in GLUT4 trafficking in muscle.

Animals↗
PubMed1999

Regulation of neuronal and glial proteins by leptin: implications for brain development.

The complete absence of leptin causes severe obesity in mice and humans, but its physiological roles are incompletely defined. Earlier studies reported decreased brain weight and impaired myelination in ob/ob and db/db mice. Here we have examined the effects of leptin deficiency and postnatal leptin treatment on brain weight, the expression of a broad array of neuronal and glial markers, and locomotor activity. ob/ob and db/db mice have reduced brain weight and an immature pattern of expression of synaptic and glial proteins, with growth-associated protein being elevated in the neocortex and hippocampus, and syntaxin-1, synaptosomal-associated protein-25, and synaptobrevin being decreased. The expression of myelin basic protein, proteolipid protein, and glial fibrillary acidic protein was also decreased in the neocortex, hippocampus, and striatum of ob/ob and db/db mice. Six weeks of leptin treatment initiated at week 4 increased brain weight and protein content, increased locomotor activity, and normalized levels of growth-associated protein, syntaxin-1, and synaptosomal-associated protein-25 in ob/ob mice without affecting synaptobrevin and glial proteins. In contrast with ob/ob and db/db mice, obese agouti (AY/a) mice had normal brain weight and expression of synaptic and glial proteins. These findings suggest that leptin, a peripheral signal of energy stores in adult animals, is required for normal neuronal and glial maturation in the mouse nervous system.

Animals↗
PubMed2004

Genetic characterization of the Drosophila homologue of coronin.

We report cloning and characterization of coro, which codes for the Drosophila homologue of the F-actin binding protein coronin. Viable alleles of coro produce a variety of phenotypes in leg, wing and eye development, which are similar to the phenotypes observed as a result of mutations in genes associated with the actin cytoskeleton and/or membrane trafficking. Homozygous lethal mutations in coro results in the disruption of the actin cytoskeleton in wing imaginal discs. Formation of both basolateral septate junctions and apical adherens junctions are also adversely affected in epithelial cells. Both viable and lethal alleles of coro show genetic interactions with syntaxin1A, a gene required for membrane trafficking. They also show enhanced response to over-expression of Decapentaplegic (Dpp) and its receptor Thick vein. Tracing of Dpp morphogen using a Dpp::GFP fusion construct suggested defects in the endocytic pathway, which resulted in uniform distribution of Dpp along the AP axis rather than a gradient from the AP boundary. Our results provide a genetic link between endocytosis/exocytosis events involving F actin-coated vesicles and the establishment of morphogen gradient.

5' Untranslated Regions↗
PubMed2004

Syntaxin 8 impairs trafficking of cystic fibrosis transmembrane conductance regulator (CFTR) and inhibits its channel activity.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cyclic AMP-dependent chloride channel that mediates electrolyte transport across the luminal surface of epithelial cells. In this paper, we describe the CFTR regulation by syntaxin 8, a t-SNARE protein (target soluble N-ethylmaleimide-sensitive factor attachment protein receptor) involved in the SNARE endosomal complex. Syntaxin family members are key molecules implicated in diverse vesicle docking and membrane fusion events. We found that syntaxin 8 physically interacts with CFTR: recombinant syntaxin 8 binds CFTR in vitro and both proteins co-immunoprecipitate in HT29 cells. Syntaxin 8 regulates CFTR-mediated currents in chinese hamster ovary (CHO) cells stably expressing CFTR and syntaxin 8. Iodide efflux and whole-cell patch-clamp experiments on these cells indicate a strong inhibition of CFTR chloride current by syntaxin 8 overexpression. At the cellular level, we observed that syntaxin 8 overexpression disturbs CFTR trafficking. Confocal microscopy shows a dramatic decrease in green fluorescent protein-tagged CFTR plasma membrane staining, when syntaxin 8 is coexpressed in COS-7 cells. Using antibodies against Lamp-1, TfR or Rab11 we determined by immunofluorescence assays that both proteins are mainly accumulated in recycling endosomes. Our results evidence that syntaxin 8 contributes to the regulation of CFTR trafficking and chloride channel activity by the SNARE machinery.

Animals↗
PubMed2006

Tomosyn-1 is involved in a post-docking event required for pancreatic beta-cell exocytosis.

Although the assembly of a ternary complex between the SNARE proteins syntaxin-1, SNAP25 and VAMP2 is known to be crucial for insulin exocytosis, the mechanisms controlling this key event are poorly understood. We found that pancreatic beta-cells express different isoforms of tomosyn-1, a syntaxin-1-binding protein possessing a SNARE-like motif. Using atomic force microscopy we show that the SNARE-like domain of tomosyn-1 can form a complex with syntaxin-1 and SNAP25 but displays binding forces that are weaker than those observed for VAMP2 (237+/-13 versus 279+/-3 pN). In pancreatic beta-cells tomosyn-1 was found to be concentrated in cellular compartments enriched in insulin-containing secretory granules. Silencing of tomosyn-1 in the rat beta-cell line INS-1E by RNA interference did not affect the number of secretory granules docked at the plasma membrane but led to a reduction in stimulus-induced exocytosis. Replacement of endogenous tomosyn-1 with mouse tomosyn-1, which differs in the nucleotide sequence from its rat homologue and escapes silencing, restored a normal secretory rate. Taken together, our data suggest that tomosyn-1 is involved in a post-docking event that prepares secretory granules for fusion and is necessary to sustain exocytosis of pancreatic beta-cells in response to insulin secretagogues.

Animals↗
PubMed1999

Blockade of membrane transport and disassembly of the Golgi complex by expression of syntaxin 1A in neurosecretion-incompetent cells: prevention by rbSEC1.

The t-SNAREs syntaxin1A and SNAP-25, i.e. the members of the complex involved in regulated exocytosis at synapses and neurosecretory cells, are delivered to their physiological site, the plasma membrane, when transfected into neurosecretion-competent cells, such as PC12 and AtT20. In contrast, when transfection is made into cells incompetent for neurosecretion, such as those of a defective PC12 clone and the NRK fibroblasts, which have no endogenous expression of these t-SNAREs, syntaxin1A (but neither two other syntaxin family members nor SNAP-25) remains stuck in the Golgi-TGN area with profound consequences to the cell: blockade of both membrane (SNAP-25, GAT-1) and secretory (chromogranin B) protein transport to the cell surface; progressive disassembly of the Golgi complex and TGN; ultimate disappearance of the latter structures, with intermixing of their markers (mannosidase II; TGN-38) with those of the endoplasmic reticulum (calreticulin) and with syntaxin1A itself. When, however, syntaxin 1A is transfected together with rbSec1, a protein known to participate in neurosecretory exocytosis via its dynamic interaction with the t-SNARE, neither the blockade nor the alterations of the Golgi complex take place. Our results demonstrate that syntaxin1A, in addition to its role in exocytosis at the cell surface, possesses a specific potential to interfere with intracellular membrane transport and that its interaction with rbSec1 is instrumental to its physiological function not only at the plasma membrane but also within the cell. At the latter site, the rbSec1-induced conversion of syntaxin1A into a form that can be transported and protects the cell from the development of severe structural and membrane traffic alterations.

Animals↗