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PubMed2001

Protein localization and mRNA expression of epimorphin in mouse and human kidneys.

Epimorphin is a mesenchymal cell surface protein which induces epithelial branching morphogenesis. However, the role of epimorphin in the kidney has not been addressed. In the present study, the localization of epimorphin protein and the expression of its mRNA were investigated in the developing mouse and adult human kidneys using immunohistochemistry and semiquantitative RT-PCR. The in vitro expression of epimorphin protein and its mRNA was also explored in cultured mouse and human mesangial cells. Epimorphin protein was expressed in the renal interstitium and the circumference of the comma-shaped body at day 16 of gestation. The intensity and distribution of epimorphin were gradually increased during kidney differentiation and maturation. Epimorphin was first observed in glomeruli at 1 week of age. The localization of epimorphin in glomerular mesangial cells and interstitial fibroblasts was confirmed by immunoelectron microscopy of 2-week-old mouse kidneys. The highest mRNA expression of epimorphin was observed at day 16 of gestation, thereafter it diminished with the maturation of the kidney. A similar localization of epimorphin was observed in a normal adult human kidney. Cultured human mesangial cells expressed epimorphin mRNA 150-kD protein. These results suggest that epimorphin may play a role in the development of the kidney and in the differentiation of fibroblast and mesangial cells.

Animals↗

PubMed2004

SNAP-23 functions in docking/fusion of granules at low Ca2+.

Ca(2+)-triggered exocytosis of secretory granules mediates the release of hormones from endocrine cells and neurons. The plasma membrane protein synaptosome-associated protein of 25 kDa (SNAP-25) is thought to be a key component of the membrane fusion apparatus that mediates exocytosis in neurons. Recently, homologues of SNAP-25 have been identified, including SNAP-23, which is expressed in many tissues, albeit at different levels. At present, little is known concerning functional differences among members of this family of proteins. Using an in vitro assay, we show here that SNAP-25 and SNAP-23 mediate the docking of secretory granules with the plasma membrane at high (1 microM) and low (100 nM) Ca(2+) levels, respectively, by interacting with different members of the synaptotagmin family. In intact endocrine cells, expression of exogenous SNAP-23 leads to high levels of hormone secretion under basal conditions. Thus, the relative expression levels of SNAP-25 and SNAP-23 might control the mode (regulated vs. basal) of granule release by forming docking complexes at different Ca(2+) thresholds.

Animals↗

PubMed2004

Heterogeneous expression of neuroendocrine marker proteins in human undifferentiated carcinoma of the colon and rectum.

The expression of neuroendocrine marker proteins in undifferentiated colorectal cancers has not yet been studied in great detail. Therefore, the survival of 20 patients with small cell undifferentiated colorectal cancers treated at our institution between 1982 and 1997 (0.8% of all operated colorectal carcinomas) was correlated with the extent of neuroendocrine differentiation. Chromogranin A, synaptophysin, syntaxin1, VAMP2, SNAP25, and alpha/beta-SNAP were used as neuroendocrine markers. Based on the degree of immunoreactivity for these marker proteins, tumors were divided into group 0 (<2% cells stained positive for neuroendocrine markers) and group 1 (>2% cells stained positive). Patients were followed up for at least 5 years or until death. Nine of twenty (45%) undifferentiated colorectal tumors expressed neuroendocrine markers (group 1). Only one patient of this group survived 2 years (11%), whereas the 2-year survival rate was 45.4% in group 0. Nine of eleven patients of group 0 were diagnosed in UICC stage I-III, whereas eight of nine tumors with expression of neuroendocrine markers were diagnosed in UICC stage IV (P = 0.002). Our results show that neuroendocrine differentiation is often seen in small cell undifferentiated colorectal cancer. It correlates with a more aggressive course of the disease.

Adult↗

PubMed2000

VAMP-2 promotes neurite elongation and SNAP-25A increases neurite sprouting in PC12 cells.

Recent studies suggest that the soluble N-ethylmaleimide-sensitive factor attached protein (SNAP) receptor (SNARE)-mediated membrane fusion system is involved in vesicle fusion in the plasma membrane that allows expansion for neurite elongation. There have been several reports analyzing the effects of neurite outgrowth by inhibition of SNAREs. In this study, we took the opposite approach by overexpressing green fluorescent protein (GFP)-fusion SNAREs, including VAMP-2, SNAP-25A, and syntaxin1A, in PC12 cells to investigate the role of SNAREs in the neurite outgrowth of PC12 cells. Neurite outgrowth analysis demonstrated that: (1) GFP-VAMP-2 increased the length of individual neurites, without changing the number of neurites per cell; (2) GFP-SNAP-25A increased the number of neurites per cell, with no change in the length of the individual neurites. In both cases, the total length of neurites per cell was increased; (3) GFP-syntaxin1A resulted in no significant change, either in neurite length, or in the number of neurites per cell. These findings suggest that when overexpressed in PC12 cells, VAMP-2 can promote neurite elongation, while SNAP-25A can stimulate neurite sprouting. On the other hand, overexpression of syntaxin1A neither promotes nor inhibits neurite outgrowth. Thus VAMP-2 and SNAP-25A play different roles in neurite elongation and sprouting.

Animals↗

PubMed2000

Incorporation and differentiation of hippocampus-derived neural stem cells transplanted in injured adult rat retina.

PURPOSE: In a previous study it has been shown that adult rat hippocampus-derived neural stem cells can be successfully transplanted into neonatal retinas, where they differentiate into neurons and glia, but they cannot be transplanted into adult retinas. In the current study, the effect of mechanical injury to the adult retina on the survival and differentiation of the grafted hippocampal stem cells was determined. METHODS: Mechanical injury was induced in the adult rat retina by a hooked needle. A cell suspension (containing 90,000 neural stem cells) was slowly injected into the vitreous space. The specimens were processed for immunohistochemical studies at 1, 2, and 4 weeks after the transplantation. RESULTS: In the best case, incorporation of grafted stem cells was seen in 50% of the injured retinas. Most of these cells located from the ganglion cell layer through the inner nuclear layer close to the injury site. Immunohistochemically, at 1 week, more than half of the grafted cells expressed nestin. At 4 weeks, some grafted cells showed immunoreactivity for microtubule-associated protein (MAP) 2ab, MAP5, and glial fibrillary acidic protein (GFAP), suggesting progress in differentiation into cells of neuronal and astroglial lineages. However, they showed no immunoreactivity for HPC-1, calbindin, and rhodopsin, which suggests that they did not differentiate into mature retinal neurons. Immunoelectron microscopy revealed the formation of synapse-like structures between graft and host cells. CONCLUSIONS: By the manipulation of mechanical injury, the incorporation and subsequent differentiation of the grafted stem cells into neuronal and glial lineage, including the formation of synapse-like structures, can be achieved, even in the adult rat retina.

Animals↗

PubMed2002

Role of secretory carrier membrane protein SCAMP2 in granule exocytosis.

In secretory carrier membrane proteins (SCAMPs), the most conserved structural segment is between transmembrane spans 2 and 3, facing the cytosol. A synthetic peptide, CWYRPIYKAFR (E peptide), from this segment of SCAMP2 potently inhibits exocytosis in permeabilized neuroendocrine (PC12) cells. E peptide blocked discharge of (35)S-labeled secretogranin with the same structural selectivity and potency as observed for hexosaminidase secretion in mast cells. SCAMPs 1 and 2 are concentrated primarily on intracellular membranes in PC12 cells. Both, however, are found on plasma membranes, but neither is present on large dense-core vesicles. Yet, large dense-core vesicles marked by secretogranin attach to plasma membranes at foci containing SCAMP2 along with syntaxin1 and complexin at putative cell-surface docking/fusion sites. Regulated overexpression of SCAMP2 with point mutations in its E peptide but not of normal SCAMP2 caused dose-dependent inhibition of depolarization-induced secretion. The SCAMP2 mutants also inhibited secretion stimulated by elevated calcium. Inhibition was largely overcome by adding lysophosphatidylcholine to the medium at concentrations that do not otherwise affect secretion. Although overexpression of normal or mutant SCAMP2 slightly inhibits endocytosis, this effect does not appear to be related to the specific effect of the mutant SCAMP on stimulated exocytosis. Thus, SCAMP2 not only colocalizes with fusion sites but also appears to have an essential function in granule exocytosis through actions mediated by its E peptide-containing domain.

Adaptor Proteins, Vesicular Transport↗

PubMed2004

Fluorescence resonance energy transfer reports properties of syntaxin1a interaction with Munc18-1 in vivo.

Syntaxin1A, a neural-specific N-ethylmaleimide-sensitive factor attachment protein receptor protein essential to neurotransmitter release, in isolation forms a closed conformation with an N-terminal alpha-helix bundle folded upon the SNARE motif (H3 domain), thereby limiting interaction of the H3 domain with cognate SNAREs. Munc18-1, a neural-specific member of the Sec1/Munc18 protein family, binds to syntaxin1A, stabilizing this closed conformation. We used fluorescence resonance energy transfer (FRET) to characterize the Munc18-1/syntaxin1A interaction in intact cells. Enhanced cyan fluorescent protein-Munc18-1 and a citrine variant of enhanced yellow fluorescent protein-syntaxin1A, or mutants of these proteins, were expressed as donor and acceptor pairs in human embryonic kidney HEK293-S3 and adrenal chromaffin cells. Apparent FRET efficiency was measured using two independent approaches with complementary results that unambiguously verified FRET and provided a spatial map of FRET efficiency. In addition, enhanced cyan fluorescent protein-Munc18-1 and a citrine variant of enhanced yellow fluorescent protein-syntaxin1A colocalized with a Golgi marker and exhibited FRET at early expression times, whereas a strong plasma membrane colocalization, with similar FRET values, was apparent at later times. Trafficking of syntaxin1A to the plasma membrane was dependent on the presence of Munc18-1. Both syntaxin1A(L165A/E166A), a constitutively open conformation mutant, and syntaxin1A(I233A), an H3 domain point mutant, demonstrated apparent FRET efficiency that was reduced approximately 70% from control. In contrast, the H3 domain mutant syntaxin1A(I209A) had no effect. By using phosphomimetic mutants of Munc18-1, we also established that Ser-313, a Munc18-1 protein kinase C phosphorylation site, and Thr-574, a cyclin-dependent kinase 5 phosphorylation site, regulate Munc18-1/syntaxin1A interaction in HEK293-S3 and chromaffin cells. We conclude that FRET imaging in living cells may allow correlated regulation of Munc18-1/syntaxin1A interactions to Ca(2+)-regulated secretory events.

Animals↗

PubMed1998

Differential turnover of syntaxin and SNAP-25 during synaptogenesis in cultured cerebellar granule neurons.

In order to investigate the molecular mechanism underlying synaptogenesis, we examined the dynamics and stability of syntaxin 1A and SNAP-25 in cultured cerebellar granule cells. In neurons cultured for less than 5 days in vitro (DIV), syntaxin was highly expressed with a half-life of >48 hours. SNAP-25 was also expressed at 5 DIV, but at a lower level and with a much shorter half-life of 16 hours. As the neurons matured and established synpatic connections, the expression of both proteins increased steadily, with the more rapid increase between 5 DIV and 8 DIV associated with SNAP-25. The half-life of syntaxin was slightly increased in the mature neurons. SNAP-25, however, showed an increased half-life of about 35 hours. These results suggested that the dynamics and stability of the t-SNAREs are differentially modulated during synaptogenesis, which may be important in establishing and maintaining synaptic connections.

Animals↗

PubMed1997

Non-functional role of syntaxin 2 in insulin exocytosis by pancreatic beta cells.

This study was designed in order to examine the expression and functional role of syntaxin 2/epimorphin in pancreatic beta cells. Northern blot analysis revealed that syntaxin 2 mRNA was able to be detected in mouse beta TC3 cells, but not in isolated mouse islets. In agreement with this result, immunoblot analysis detected an appreciable amount of syntaxin 2 protein in beta TC3 cells, but not in mouse islets. Immunohistochemistry of the mouse pancreas demonstrated that syntaxin 2 was little evident in islet cells of Langerhans, and somewhat predominant in exocrine tissues. In order to examine whether syntaxin 2 is anchored to cell surfaces in beta TC3 cells, living cells were incubated with a monoclonal antibody against syntaxin 2 (MC-1). The antibody bound to their surfaces, indicating that syntaxin 2 was localized on cell surfaces. The addition of MC-1 to the culture medium of beta TC3 cells did not affect insulin release under the presence or absence of 11 mM glucose, indicating that syntaxin 2 is not associated with insulin exocytosis. Thus, the expression of syntaxin 2 in islets of Langerhans is very low and the function of this protein is probably unrelated to the insulin exocytosis pathway.

Animals↗

PubMed2000

Synaptic proteins in rat taste bud cells: appearance in the Golgi apparatus and relationship to alpha-gustducin and the Lewis(b) and A antigens.

Taste receptor cells are continuously replaced during the life of the animal, but many of their sensory axons respond primarily to stimuli belonging to a single taste quality. This suggests that a newly arising taste cell must form a synapse with an appropriate sensory axon, requiring cell recognition that is likely to be mediated by surface markers. As an approach to studying this process, we attempted to locate synapses by immunolabeling taste buds of rats for proteins involved in neurotransmitter release. In taste bud cells of vallate papillae and nasoincisor ducts, double-labeling experiments showed that syntaxin-1, SNAP-25, synaptobrevin, and synaptophysin colocalized with the Golgi marker beta COP in elongated cytoplasmic compartments that extended from the perinuclear region into apical and basal processes of the cells. Labeled cells were spindle-shaped, identifying them as light cells. Syntaxin-1 appeared only in taste cells, but SNAP-25, synaptobrevin, and synaptophysin were also seen in nerve fibers. The synaptic vesicle glycoprotein SV2 appeared only in nerve fibers. Taste cells of fungiform papillae did not show immunoreactivity for presynaptic proteins or Golgi markers, but axonal labeling was similar to that in other regions. Taste cells with alpha-gustducin could express either presynaptic proteins or the carbohydrate blood group antigen Lewis(b), but not both. Therefore, Lewis(b) and presynaptic proteins are not expressed during the same period in the life of a taste bud cell. Most taste cells expressing syntaxin-1 (82%) also expressed the A blood group antigen, whether or not they expressed alpha-gustducin.

Animals↗

PubMed2000

Fusion of membranes during the acrosome reaction: a tale of two SNAREs.

During spermiogenesis, hydrolytic enzymes are sorted from the Golgi apparatus to the acrosome, a supranuclear megavesicle. At fertilization, the enzymatic content of the acrosome is released by exocytosis when a portion of the plasma membrane enveloping the sperm head fuses with the outer membrane of the acrosome. Membrane fusion involves the interaction of a specific pair of proteins, called SNAREs (for soluble N-ethylmaleimide sensitive factor attachment protein receptor). v-SNARE is presumably associated with the membrane of the acrosomal vesicle. Target t-SNARE is associated with the plasma membrane. The interaction of v-SNARE and t-SNARE requires two additional proteins: Rab proteins, members of a family of small GTPases related to the Ras proteins, and a complex of two proteins, NSF-SNAP, recruited by the interacting v-SNARE-tSNARE pair. Syntaxin 2, a v-SNARE member, and Rab3A, a member of the Rab GTPases, have been localized in the acrosome of rodent sperm.

Acrosome Reaction↗

PubMed2002

Refinement of the genomic structure of STX1A and mutation analysis in nondeletion Williams syndrome patients.

Williams syndrome (WS) is a contiguous gene deletion disorder in which the commonly deleted region contains at least 17 genes. One of these genes, Syntaxin 1A (STX1A), codes for a protein that is highly expressed in the nervous system and is essential for the docking of synaptic vesicles with the presynaptic plasma membrane. In this study, we refine the complete genomic structure of the human STX1A gene by direct sequencing and primer walking of bacterial artificial chromosome (BAC) clones and show that STX1A contains at least 10 exons and 9 introns. The length of exons range from 27 bp to 138 bp and all splice sites conform to the GT-AG rule. Investigation of the STX1A gene sequence in five WS patients without detectable deletions did not identify any point mutations. Although the regulatory elements that control STX1A transcription were not examined, these results do not support a role for STX1A in the WS phenotype.

Adolescent↗

PubMed2005

Cellular distribution and subcellular localization of molecular components of vesicular transmitter release in horizontal cells of rabbit retina.

The mechanism underlying transmitter release from retinal horizontal cells is poorly understood. We investigated the possibility of vesicular transmitter release from mammalian horizontal cells by examining the expression of synaptic proteins that participate in vesicular transmitter release at chemical synapses. Using immunocytochemistry, we evaluated the cellular and subcellular distribution of complexin I/II, syntaxin-1, and synapsin I in rabbit retina. Strong labeling for complexin I/II, proteins that regulate a late step in vesicular transmitter release, was found in both synaptic layers of the retina, and in somata of A- and B-type horizontal cells, of gamma-aminobutyric acid (GABA)- and glycinergic amacrine cells, and of ganglion cells. Immunoelectron microscopy demonstrated the presence of complexin I/II in horizontal cell processes postsynaptic to rod and cone ribbon synapses. Syntaxin-1, a core protein of the soluble N-ethylmaleimide-sensitive-factor attachment protein receptor (SNARE) complex known to bind to complexin, and synapsin I, a synaptic vesicle-associated protein involved in the Ca(2+)-dependent recruitment of synaptic vesicles for transmitter release, were also present in the horizontal cells and their processes at photoreceptor synapses. Photoreceptors and bipolar cells did not express any of these proteins at their axon terminals. The presence of complexin I/II, syntaxin-1, and synapsin I in rabbit horizontal cell processes and tips suggests that a vesicular mechanism may underlie transmitter release from mammalian horizontal cells.

Adaptor Proteins, Vesicular Transport↗

PubMed1993

Yeast syntaxins Sso1p and Sso2p belong to a family of related membrane proteins that function in vesicular transport.

The yeast SEC1 gene encodes a hydrophilic protein that functions at the terminal stage in secretion. We have cloned two yeast genes, SSO1 and SSO2, which in high copy number can suppress sec1 mutations and also mutations in several other late acting SEC genes, such as SEC3, SEC5, SEC9 and SEC15. SSO1 and SSO2 encode small proteins with N-terminal hydrophilic domains and C-terminal hydrophobic tails. The two proteins are 72% identical in sequence and together perform an essential function late in secretion. Sso1p and Sso2p show significant sequence similarity to six other proteins. Two of these, Sed5p and Pep12p, are yeast proteins that function in transport from ER to Golgi and from Golgi to the vacuole, respectively. Also related to Sso1p and Sso2p are three mammalian proteins: epimorphin, syntaxin A/HPC-1 and syntaxin B. A nematode cDNA product also belongs to the new protein family. The new protein family is thus present in a wide variety of eukaryotic cells, where its members function at different stages in vesicular transport.

Amino Acid Sequence↗

PubMed1993

Botulinum neurotoxin C1 blocks neurotransmitter release by means of cleaving HPC-1/syntaxin.

The anaerobic bacterium Clostridium botulinum produces several related neurotoxins that block exocytosis of synaptic vesicles in nerve terminals and that are responsible for the clinical manifestations of botulism. Recently, it was reported that botulinum neurotoxin type B as well as tetanus toxin act as zinc-dependent proteases that specifically cleave synaptobrevin, a membrane protein of synaptic vesicles (Link et al., Biochem. Biophys. Res. Commun., 189, 1017-1023; Schiavo et al., Nature, 359, 832-835). Here we report that inhibition of neurotransmitter release by botulinum neurotoxin type C1 was associated with the proteolysis of HPC-1 (= syntaxin), a membrane protein present in axonal and synaptic membranes. Breakdown of HPC-1/syntaxin was selective since no other protein degradation was detectable. In vitro studies showed that the breakdown was due to a direct interaction between HPC-1/syntaxin and the toxin light chain which acts as a metallo-endoprotease. Toxin-induced cleavage resulted in the generation of a soluble fragment of HPC-1/syntaxin that is 2-4 kDa smaller than the native protein. When HPC-1/syntaxin was translated in vitro, cleavage occurred only when translation was performed in the presence of microsomes, although a full-length product was obtained in the absence of membranes. However, susceptibility to toxin cleavage was restored when the product of membrane-free translation was subsequently incorporated into artificial proteoliposomes. In addition, a translated form of HPC-1/syntaxin, which lacked the putative transmembrane domain at the C-terminus, was soluble and resistant to toxin action. We conclude that HPC-1/syntaxin is involved in exocytotic membrane fusion.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed2002

Malignant rhabdoid tumor shows incomplete neural characteristics as revealed by expression of SNARE complex.

To elucidate the biological differences in neural phenotype between malignant rhabdoid tumor (MRT) and neuroblastoma cell lines, we examined the expression of solube N-ethylmaleimide-sensitive fusion protein attachment protein receptor (SNARE) complex proteins in MRT cell lines under differentiation induction with 12-O-tetradecanoylphorbol-13-acetate (TPA). Six MRT cell lines (TM87-16, STM91-01, TTC642, TTC549, YAM-RTK1, and TTC1240) and six neuroblastoma cell lines (IMR-32, NH12, SCCH26, TGW, NB-1, and NB-NR) were used in this study. Expression of SNAREs: the vesicle SNARE (synaptotagmin, synaptophysin, and synaptobrevin-2) and the target SNARE (syntaxin 1A, SNAP-25A/B) was examined. Our results showed that in MRT cells, only two cell lines (TM87-16, TTC642) expressed the vesicle SNARE and the target SNARE with the exception of SNAP-25B, while all neuroblastoma cells expressed the entire SNARE complex. During differentiation, synaptotagmin was upregulated in these two MRT cell lines. Interestingly, synaptophysin was downregulated in these MRT cell lines in contrast with the neuroblastoma cell lines. SNAP-25B was not expressed in MRT cells after differentiation with TPA. MRT cells having a neural phenotype morphologically looked like neuroblastoma cells after treatment with TPA. However, the expression of SNARE complex was incomplete in MRT cells. Our results suggest that the biological characteristics of MRT cells with neural phenotype are distinct from those of neuroblastoma cells.

Blotting, Western↗

PubMed2003

Minimal residues in linker domain of syntaxin 1A required for binding affinity to Ca2+/calmodulin-dependent protein kinase II.

The linker domain is important for the conformational change syntaxin 1A, which enables it to act as a SNARE for exocytosis. We found that when applied exogenously, the linker domain is a potent inhibitor of exocytosis through inhibiting interaction between autophosphorylated CaMKII and endogenous syntaxin 1A (Ohyama et al. [2002] J. Neurosci. 22:3342-3351). To identify the simplest and the most potent inhibitor for exocytosis, we further characterized the linker domain and determined the minimal number of residues required for CaMKII binding. The minimal length of the CaMKII-binding site was 145-172 residues and a loss of G172 considerably weakened affinity for CaMKII. The basic amino acid clusters, R151 and K146, were indispensable for binding, whereas R148 was not. A comparison of the CaMKII-binding in several syntaxin isoforms revealed that the substitution of S162 attenuated CaMKII-binding activity. These results suggest that S162 is an important residue as well as the basic amino acid cluster within region 145-172 of the linker domain.

Amino Acid Motifs↗

PubMed2005

Expression and transport function of the glutamate transporter EAAC1 in Xenopus oocytes is regulated by syntaxin 1A.

The function of several membrane proteins is regulated by interaction with the SNARE protein syntaxin 1A; this includes regulation of GAT1, the transporter for the dominating inhibitory neurotransmitter gamma-aminobutyric acid (GABA). Here we demonstrate that also EAAC1, the transporter for the dominating excitatory neurotransmitter, is down-regulated by interaction with syntaxin 1A. This is shown by coexpression of EAAC1 and syntaxin 1A in Xenopus oocytes. Total EAAC1 expression is not significantly affected by the coexpression of syntaxin 1A, but more proteins become targeted to the membrane as demonstrated by biotinylation. Colocalization by coimmunoprecipitation suggests direct interaction between the two proteins. In contrast to the number of transporters, the glutamate transport activity becomes reduced, and even stronger inhibition is observed for the EAAC1-mediated conductance uncoupled from glutamate translocation. We conclude that the interaction of syntaxin 1A with EAAC1 particularly disrupts the structure of the conductance pathway of EAAC1.

Amino Acid Transport System X-AG↗