scholarly journals Molecular Identification of a SNAP-25-Like SNARE Protein in Paramecium

2008 ◽  
Vol 7 (8) ◽  
pp. 1387-1402 ◽  
Author(s):  
Christina Schilde ◽  
Kaya Lutter ◽  
Roland Kissmehl ◽  
Helmut Plattner
Keyword(s):  
Fluorescent Protein ◽  
Heptad Repeat ◽  
Paramecium Tetraurelia ◽  
Snare Protein ◽  
Membrane Structures ◽  
Fusion Construct ◽  
Genome Duplications ◽  
Botulinum Neurotoxins ◽  
Food Vacuoles ◽  
Green Fluorescent

ABSTRACT Using database searches of the completed Paramecium tetraurelia macronuclear genome with the metazoan SNAP-25 homologues, we identified a single 21-kDa Qb/c-SNARE in this ciliated protozoan, named P. tetraurelia SNAP (PtSNAP), containing the characteristic dual heptad repeat SNARE motifs of SNAP-25. The presence of only a single Qb/c class SNARE in P. tetraurelia is surprising in view of the multiple genome duplications and the high number of SNAREs found in other classes of this organism. As inferred from the subcellular localization of a green fluorescent protein (GFP) fusion construct, the protein is localized on a variety of intracellular membranes, and there is a large soluble pool of PtSNAP. Similarly, the PtSNAP that is detected with a specific antibody in fixed cells is associated with a number of intracellular membrane structures, including food vacuoles, the contractile vacuole system, and the sites of constitutive endo- and exocytosis. Surprisingly, using gene silencing, we could not assign a role to PtSNAP in the stimulated exocytosis of dense core vesicles (trichocysts), but we found an increased number of food vacuoles in PtSNAP-silenced cells. In conclusion, we identify PtSNAP as a Paramecium homologue of metazoan SNAP-25 that shows several divergent features, like resistance to cleavage by botulinum neurotoxins.

scholarly journals Double-Labeled Rabies Virus: Live Tracking of Enveloped Virus Transport

2007 ◽  
Vol 82 (1) ◽  
pp. 237-245 ◽  
Author(s):  
Yvonne Klingen ◽  
Karl-Klaus Conzelmann ◽  
Stefan Finke
Keyword(s):  
Cell Surface ◽  
Rabies Virus ◽  
Fluorescent Protein ◽  
Signal Sequence ◽  
Surface Membrane ◽  
Fusion Construct ◽  
Virus Particles ◽  
Enveloped Virus ◽  
Green Fluorescent

ABSTRACT Here we describe a strategy to fluorescently label the envelope of rabies virus (RV), of the Rhabdoviridae family, in order to track the transport of single enveloped viruses in living cells. Red fluorescent proteins (tm-RFP) were engineered to comprise the N-terminal signal sequence and C-terminal transmembrane spanning and cytoplasmic domain sequences of the RV glycoprotein (G). Two variants of tm-RFP were transported to and anchored in the cell surface membrane, independent of glycosylation. As shown by confocal microscopy, tm-RFP colocalized at the cell surface with the RV matrix and G protein and was incorporated into G gene-deficient virus particles. Recombinant RV expressing the membrane-anchored tm-RFP in addition to G yielded infectious viruses with mosaic envelopes containing both tm-RFP and G. Viable double-labeled virus particles comprising a red fluorescent envelope and a green fluorescent ribonucleoprotein were generated by expressing in addition an enhanced green fluorescent protein-phosphoprotein fusion construct (S. Finke, K. Brzozka, and K. K. Conzelmann, J. Virol. 78:12333-12343, 2004). Individual enveloped virus particles were observed under live cell conditions as extracellular particles and inside endosomal vesicles. Importantly, double-labeled RVs were transported in the retrograde direction over long distances in neurites of in vitro-differentiated NS20Y neuroblastoma cells. This indicates that the typical retrograde axonal transport of RV to the central nervous system involves neuronal transport vesicles in which complete enveloped RV particles are carried as a cargo.

Components of intestinal epithelial hypoxia activate the virulence circuitry of Pseudomonas

2005 ◽  
Vol 288 (5) ◽  
pp. G1048-G1054 ◽  
Author(s):  
Jonathan E. Kohler ◽  
Olga Zaborina ◽  
Licheng Wu ◽  
Yingmin Wang ◽  
Cindy Bethel ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Surgical Stress ◽  
Recovery Period ◽  
Intestinal Lumen ◽  
Bacterial Cells ◽  
Intestinal Epithelial ◽  
Promoter Activation ◽  
Fusion Construct ◽  
Green Fluorescent ◽  
Ambient Hypoxia

We have previously shown that a lethal virulence trait in Pseudomonas aeruginosa, the PA-I lectin, is expressed by bacteria within the intestinal lumen of surgically stressed mice. The aim of this study was to determine whether intestinal epithelial hypoxia, a common response to surgical stress, could activate PA-I expression. A fusion construct was generated to express green fluorescent protein downstream of the PA-I gene, serving as a stable reporter strain for PA-I expression in P. aeruginosa. Polarized Caco-2 monolayers were exposed to ambient hypoxia (0.1–0.3% O2) for 1 h, with or without a recovery period of normoxia (21% O2) for 2 h, and then inoculated with P. aeruginosa containing the PA-I reporter construct. Hypoxic Caco-2 monolayers caused a significant increase in PA-I promoter activity relative to normoxic monolayers (165% at 1 h; P < 0.001). Similar activation of PA-I was also induced by cell-free apical, but not basal, media from hypoxic Caco-2 monolayers. PA-I promoter activation was preferentially enhanced in bacterial cells that physically interacted with hypoxic epithelia. We conclude that the virulence circuitry of P. aeruginosa is activated by both soluble and contact-mediated elements of the intestinal epithelium during hypoxia and normoxic recovery.

scholarly journals Visualization of Phosphoinositides That Bind Pleckstrin Homology Domains: Calcium- and Agonist-induced Dynamic Changes and Relationship to Myo-[3H]inositol-labeled Phosphoinositide Pools

1998 ◽  
Vol 143 (2) ◽  
pp. 501-510 ◽  
Author(s):  
Péter Várnai ◽  
Tamás Balla
Keyword(s):  
Fluorescent Probe ◽  
Fluorescent Protein ◽  
Dynamic Imaging ◽  
Ph Domain ◽  
Pleckstrin Homology ◽  
Protein Fusion ◽  
Fusion Construct ◽  
Cellular Expression ◽  
Ph Domains ◽  
Green Fluorescent

Phosphatidylinositol 4,5-bisphosphate (PtdIns[4,5]P2) pools that bind pleckstrin homology (PH) domains were visualized by cellular expression of a phospholipase C (PLC)δ PH domain–green fluorescent protein fusion construct and analysis of confocal images in living cells. Plasma membrane localization of the fluorescent probe required the presence of three basic residues within the PLCδ PH domain known to form critical contacts with PtdIns(4,5)P2. Activation of endogenous PLCs by ionophores or by receptor stimulation produced rapid redistribution of the fluorescent signal from the membrane to cytosol, which was reversed after Ca2+ chelation. In both ionomycin- and agonist-stimulated cells, fluorescent probe distribution closely correlated with changes in absolute mass of PtdIns(4,5)P2. Inhibition of PtdIns(4,5)P2 synthesis by quercetin or phenylarsine oxide prevented the relocalization of the fluorescent probe to the membranes after Ca2+ chelation in ionomycin-treated cells or during agonist stimulation. In contrast, the synthesis of the PtdIns(4,5)P2 imaged by the PH domain was not sensitive to concentrations of wortmannin that had been found inhibitory of the synthesis of myo-[3H]inositol– labeled PtdIns(4,5)P2. Identification and dynamic imaging of phosphoinositides that interact with PH domains will further our understanding of the regulation of such proteins by inositol phospholipids.

scholarly journals Construction and use of aCupriavidus necatorH16 soluble hydrogenase promoter (PSH) fusion togfp(green fluorescent protein)

PeerJ ◽  
2016 ◽  
Vol 4 ◽  
pp. e2269 ◽  
Author(s):  
Bat-Erdene Jugder ◽  
Jeffrey Welch ◽  
Nady Braidy ◽  
Christopher P. Marquis
Keyword(s):  
Green Fluorescent Protein ◽  
Promoter Activity ◽  
Fluorescent Protein ◽  
Small Scale ◽  
Growth Media ◽  
Reporter System ◽  
Screening Assay ◽  
Fluorescent Reporter ◽  
Fusion Construct ◽  
Green Fluorescent

Hydrogenases are metalloenzymes that reversibly catalyse the oxidation or production of molecular hydrogen (H2). Amongst a number of promising candidates for application in the oxidation of H2is a soluble [Ni–Fe] uptake hydrogenase (SH) produced byCupriavidus necatorH16. In the present study, molecular characterisation of the SH operon, responsible for functional SH synthesis, was investigated by developing a green fluorescent protein (GFP) reporter system to characterise PSHpromoter activity using several gene cloning approaches. A PSHpromoter-gfp fusion was successfully constructed and inducible GFP expression driven by the PSHpromoter under de-repressing conditions in heterotrophic growth media was demonstrated in the recombinantC. necatorH16 cells. Here we report the first successful fluorescent reporter system to study PSHpromoter activity inC. necatorH16. The fusion construct allowed for the design of a simple screening assay to evaluate PSHactivity. Furthermore, the constructed reporter system can serve as a model to develop a rapid fluorescent based reporter for subsequent small-scale process optimisation experiments for SH expression.

scholarly journals Distinct Subcellular Localization of a Group of Synaptobrevin-Like SNAREs in Paramecium tetraurelia and Effects of Silencing SNARE-Specific Chaperone NSF

2009 ◽  
Vol 9 (2) ◽  
pp. 288-305 ◽  
Author(s):  
Christina Schilde ◽  
Barbara Schönemann ◽  
Ivonne M. Sehring ◽  
Helmut Plattner
Keyword(s):  
Cell Surface ◽  
Membrane Trafficking ◽  
Fluorescent Protein ◽  
Close Association ◽  
Secretory Vesicle ◽  
Food Vacuole ◽  
Paramecium Tetraurelia ◽  
Content Type ◽  
Early Endosomes ◽  
Green Fluorescent

ABSTRACT We have identified new synaptobrevin-like SNAREs and localized the corresponding gene products with green fluorescent protein (GFP)-fusion constructs and specific antibodies at the light and electron microscope (EM) levels. These SNAREs, named Paramecium tetraurelia synaptobrevins 8 to 12 (PtSyb8 to PtSyb12), showed mostly very restricted, specific localization, as they were found predominantly on structures involved in endo- or phagocytosis. In summary, we found PtSyb8 and PtSyb9 associated with the nascent food vacuole, PtSyb10 near the cell surface, at the cytostome, and in close association with ciliary basal bodies, and PtSyb11 on early endosomes and on one side of the cytostome, while PtSyb12 was found in the cytosol. PtSyb4 and PtSyb5 (identified previously) were localized on small vesicles, PtSyb5 probably being engaged in trichocyst (dense core secretory vesicle) processing. PtSyb4 and PtSyb5 are related to each other and are the furthest deviating of all SNAREs identified so far. Because they show no similarity with any other R-SNAREs outside ciliates, they may represent a ciliate-specific adaptation. PtSyb10 forms small domains near ciliary bases, and silencing slows down cell rotation during depolarization-induced ciliary reversal. NSF silencing supports a function of cell surface SNAREs by revealing vesicles along the cell membrane at sites normally devoid of vesicles. The distinct distributions of these SNAREs emphasize the considerable differentiation of membrane trafficking, particularly along the endo-/phagocytic pathway, in this protozoan.

scholarly journals Saccharomyces cerevisiae Aqr1 Is an Internal-Membrane Transporter Involved in Excretion of Amino Acids

2004 ◽  
Vol 3 (6) ◽  
pp. 1492-1503 ◽  
Author(s):  
Isabel Velasco ◽  
Sandra Tenreiro ◽  
Isabel L. Calderon ◽  
Bruno André
Keyword(s):  
Amino Acids ◽  
Reverse Genetics ◽  
Fluorescent Protein ◽  
Negative Control ◽  
Yeast Cells ◽  
Green Fluorescent Protein Fusion ◽  
Protein Fusion ◽  
Membrane Structures ◽  
Internal Membrane ◽  
Green Fluorescent

ABSTRACT Excretion of amino acids by yeast cells was reported long ago but has not been characterized in molecular terms. It is typically favored by overproduction of the amino acid and/or impairment of its uptake. Here we describe the construction of a yeast strain excreting threonine and homoserine. Using this excretor strain, we then applied a reverse-genetics approach and found that the transporter encoded by the YNL065w/AQR1 gene, a protein thought to mediate H+ antiport, is involved in homoserine and threonine excretion. Furthermore, overexpression of AQR1 led to increased excretion of several amino acids (alanine, aspartate, and glutamate) known to be relatively abundant in the cytosol. Transcription of the AQR1 gene is induced severalfold by a number of amino acids and appears to be under the negative control of Gcn4. An Aqr1-green fluorescent protein fusion protein is located in multiple internal membrane structures and appears to cycle continuously between these compartments and the plasma membrane. The Aqr1 sequence is significantly similar to the vesicular amine transporters of secretory vesicles of neuronal cells. We propose that Aqr1 catalyzes transport of excess amino acids into vesicles, which then release them in the extracellular space by exocytosis.

PKC Modulation of Transmitter Release by SNAP-25 at Sensory-to-Motor Synapses in Aplysia

2007 ◽  
Vol 97 (1) ◽  
pp. 134-143 ◽  
Author(s):  
Gry Houeland ◽  
Arash Nakhost ◽  
Wayne S. Sossin ◽  
Vincent F. Castellucci
Keyword(s):  
Motor Neuron ◽  
Transmitter Release ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Phosphorylation Site ◽  
Snare Protein ◽  
Vesicle Docking ◽  
Putative Target ◽  
Sensory Motor ◽  
Green Fluorescent

Activation of phosphokinase C (PKC) can increase transmitter release at sensory–motor neuron synapses in Aplysia, but the target of PKC phosphorylation has not been determined. One putative target of PKC at synapses is the synaptosomal-associated protein of 25 kDa (SNAP-25), a member of the SNARE protein complex implicated in synaptic vesicle docking and fusion. To determine whether PKC regulated transmitter release through phosphorylation of SNAP-25, we cloned Aplysia SNAP-25 and expressed enhanced green fluorescent protein (EGFP)–coupled SNAP-25 constructs mutated at the PKC phosphorylation site Ser198 in Aplysia sensory neurons. We found several distinct effects of expression of EGFP–SNAP-25 constructs. First, the rates of synaptic depression were slowed when cells contained SNAP-25 with phosphomimetic residues Glu or Asp. Second, PDBu-mediated increases in transmitter release at naïve synapses were blocked in cells expressing nonphosphorylated-state SNAP-25. Finally, expression of EGFP-coupled SNAP-25 but not uncoupled SNAP-25 inhibited 5-HT–mediated reversal of depression and the ability of EGFP-coupled SNAP-25 to inhibit the reversal of depression was affected by changes at Ser198. These results suggest SNAP-25 and phosphorylation of SNAP-25 by PKC can regulate transmitter release at Aplysia sensory–motor neuron synapses by a number of distinct processes.

scholarly journals A Yeast Platform for the Production of Single-Chain Antibody-Green Fluorescent Protein Fusions

2006 ◽  
Vol 72 (12) ◽  
pp. 7748-7759 ◽  
Author(s):  
Dagang Huang ◽  
Eric V. Shusta
Keyword(s):  
Green Fluorescent Protein ◽  
Fusion Protein ◽  
Protein Secretion ◽  
Fusion Proteins ◽  
Fluorescent Protein ◽  
Expression System ◽  
Single Chain ◽  
Single Chain Antibody ◽  
Fusion Construct ◽  
Green Fluorescent

ABSTRACT Fusion proteins comprised of a binding domain and green fluorescent protein (GFP) have the potential to act as one-step binding reagents. In this study, eight single-chain antibodies (scFv) and one single-chain T-cell receptor (scTCR) were secreted as fusions to GFP using a Saccharomyces cerevisiae expression system. Fusion protein secretion levels ranged over 3 orders of magnitude, from 4 μg/liter to 4 mg/liter, and correlated well with the secretion levels of the unfused scFv/scTCR. Three fusion types with various linker lengths and fusion orientations were tested for each scFv/scTCR. Although the fusion protein secretion levels were not significantly affected by the nature of the fusion construct, the properties of the fusion protein were clearly influenced. The fluorescence yield per fusion molecule was increased by separating the scFv/scTCR and GFP with an extended (GGGGS)3 linker, and fusions with scFv/scTCR at the carboxy-terminus were more resistant to degradation. By evaluating leader sequence processing and using GFP fluorescence to track intracellular processing, it was determined that the majority of fusion protein synthesized by the yeast was not secreted and in most cases was accumulating in an immature, although active, endoplasmic-reticulum (ER)-processed form. This contrasted with unfused scFv, which accumulated in both immature ER-processed and mature post-Golgi forms. The results indicated that yeast can be used as an effective host for the secretion of scFv/scTCR-GFP fusion proteins and that as a result of intracellular secretory bottlenecks, there is considerable yeast secretory capacity remaining to be exploited.

scholarly journals Seventeen a-Subunit Isoforms of Paramecium V-ATPase Provide High Specialization in Localization and Function

2006 ◽  
Vol 17 (2) ◽  
pp. 917-930 ◽  
Author(s):  
Thomas Wassmer ◽  
Roland Kissmehl ◽  
Jean Cohen ◽  
Helmut Plattner
Keyword(s):  
Secretory Pathway ◽  
Fluorescent Protein ◽  
Single Gene ◽  
Amino Acid Identity ◽  
Specific Form ◽  
Contractile Vacuole ◽  
Paramecium Tetraurelia ◽  
A Subunit ◽  
Targeting Signal ◽  
Green Fluorescent

In the Paramecium tetraurelia genome, 17 genes encoding the 100-kDa-subunit (a-subunit) of the vacuolar-proton-ATPase were identified, representing by far the largest number of a-subunit genes encountered in any organism investigated so far. They group into nine clusters, eight pairs with >82% amino acid identity and one single gene. Green fluorescent protein-tagging of representatives of the nine clusters revealed highly specific targeting to at least seven different compartments, among them dense core secretory vesicles (trichocysts), the contractile vacuole complex, and phagosomes. RNA interference for two pairs confirmed their functional specialization in their target compartments: silencing of the trichocyst-specific form affected this secretory pathway, whereas silencing of the contractile vacuole complex-specific form altered organelle structure and functioning. The construction of chimeras between selected a-subunits surprisingly revealed the targeting signal to be located in the C terminus of the protein, in contrast with the N-terminal targeting signal of the a-subunit in yeast. Interestingly, some chimeras provoked deleterious effects, locally in their target compartment, or remotely, in the compartment whose specific a-subunit N terminus was used in the chimera.

scholarly journals Green Fluorescent Protein as a Marker inRickettsia typhi Transformation

1999 ◽  
Vol 67 (7) ◽  
pp. 3308-3311 ◽  
Author(s):  
Jill Michelle Troyer ◽  
Suzana Radulovic ◽  
Abdu F. Azad
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Fragment Length Polymorphism ◽  
Vero Cells ◽  
Intracellular Pathogen ◽  
Protein Variant ◽  
Cell Sorter ◽  
Fusion Construct ◽  
Green Fluorescent ◽  
Green Fluorescent Protein Variant

ABSTRACT Transformation of rickettsiae is a recent accomplishment, but utility of this technique is limited due to the paucity of selectable markers suitable for use in this intracellular pathogen. We chose a green fluorescent protein variant optimized for fluorescence under UV lights (GFPUV) as a fluorometric marker and transformed Rickettsia typhi with anrpoB-GFPUV fusion construct. The rickettsiae were subsequently grown in Vero cells, and cultures were screened by PCR and restriction fragment length polymorphism (RFLP) to confirm incorporation of the rpoB-GFPUV construct. Cultures were then analyzed by flow cytometry for detection of GFPUV expression, and transformed R. typhi were isolated in a fluorescence-activated cell sorter. This is the first report of transformation of rickettsiae with a nonrickettsial (GFPUV) gene.

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