Expression of the green fluorescent protein in Paramecium tetraurelia

Hauser, Karin; Haynes, W. John; Kung, Ching; Plattner, Helmut; Kissmehl, Roland

doi:10.1078/s0171-9335(04)70016-3

Cited by 44 publications

(45 citation statements)

References 38 publications

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“…The plasmids were designated pPD-N1N2 (IP 3 R N specific) or pPD-C1C2 (CRC-IV-1 specific). As a negative control (pPD-GFP), the coding sequence of green fluorescent protein (GFP) derived from the pPXV-GFP vector (35) was cloned by using KpnI.…”

Section: Methodsmentioning

confidence: 99%

“…6A). As a negative control, the ORF of GFP derived from the pPXV-GFP vector (35,35) was cloned (pPD-GFP), whereas the pPD-ND7 construct was used as a positive control (88), which contains the ORF of the "non-discharge" gene nd7, resulting in impaired exocytosis of trichocysts. In order to show that the feeding constructs pPD-N1N2 and pPD-C1C2 lead to a specific knockdown, silencing of the channels was monitored by analyzing mRNA levels via real-time PCR.…”

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confidence: 99%

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Novel Types of Ca²⁺ Release Channels Participate in the Secretory Cycle of Paramecium Cells

Ladenburger¹,

Sehring²,

Korn³

et al. 2009

Molecular and Cellular Biology

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121

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A database search of the Paramecium genome reveals 34 genes related to Ca 2؉ -release channels of the inositol-1,4,5-trisphosphate (IP 3 ) or ryanodine receptor type (IP 3 R, RyR). Phylogenetic analyses show that these Ca 2؉ release channels (CRCs) can be subdivided into six groups (Paramecium tetraurelia CRC-I to CRC-VI), each one with features in part reminiscent of IP 3 Rs and RyRs. We characterize here the P. tetraurelia CRC-IV-1 gene family, whose relationship to IP 3 Rs and RyRs is restricted to their C-terminal channel domain. CRC-IV-1 channels localize to cortical Ca 2؉ stores (alveolar sacs) and also to the endoplasmic reticulum. This is in contrast to a recently described true IP 3 channel, a group II member (P. tetraurelia IP 3 R N -1), found associated with the contractile vacuole system. Silencing of either one of these CRCs results in reduced exocytosis of dense core vesicles (trichocysts), although for different reasons. Knockdown of P. tetraurelia IP 3 R N affects trichocyst biogenesis, while CRC-IV-1 channels are involved in signal transduction since silenced cells show an impaired release of Ca 2؉ from cortical stores in response to exocytotic stimuli. Our discovery of a range of CRCs in Paramecium indicates that protozoans already have evolved multiple ways for the use of Ca 2؉ as signaling molecule.Ca 2ϩ is an important component of cell activity in all organisms, from protozoa to mammals. Thereby Ca 2ϩ may originate from the outside medium and/or from internal stores (7, 18). Ca 2ϩ release from internal stores is mediated by various Ca 2ϩ release channels (CRCs), of which the inositol-1,4,5-trisphosphate receptor (IP 3 R) and ryanodine receptor (RyR) families have been studied most extensively (8,9,29,63 (14). IP 3 generates and maintains a Ca 2ϩ gradient in the hyphal tip of Neurospora crassa and the IP 3 -sensitive channels have been reconstituted and characterized with the planar bilayer method (87). In summary, these publications suggest that IP 3 -dependent signaling pathways are conserved among unicellular organisms, including protozoa.Despite these data, the molecular characterization of IP 3 or ryanodine receptors in low eukaryotes is currently a challenge since the identification of orthologues has not been possible thus far, probably because of evolutionary sequence divergence (66). Traynor et al. (96) identified an IP 3 receptor-like protein, IplA, in Dictyostelium discoideum, which possesses regions related to IP 3 R sequences, but thus far no evidence for IP 3 interaction exists. We have recently described an IP 3 R in the ciliated protozoa Paramecium tetraurelia (referred to here as P. tetraurelia IP 3 R N ) (53), with features characteristic of mammalian IP 3 Rs in terms of topology and ability for IP 3 binding. The expression level of P. tetraurelia IP 3 R N is modulated by extracellular Ca 2ϩ concentrations ([Ca 2ϩ ] o ) and immunofluorescence studies reveal an unexpected localization to the contractile vacuole complex (CVC), the major organelle involved in osmoregulation...

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Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

Novel Types of Ca²⁺ Release Channels Participate in the Secretory Cycle of Paramecium Cells

Ladenburger¹,

Sehring²,

Korn³

et al. 2009

Molecular and Cellular Biology

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121

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“…PtSNAP-specific PCR products obtained with the oligonucleotides SNAP-O and SNAP-A or SNAP-K and SNAP-A (Table 1) were cloned into the enhanced green fluorescent protein (eGFP) expression plasmid pPXV-GFP (27) in front of the eGFP gene, as described by Wassmer et al (77), between the SpeI and XhoI restriction sites of the plasmid, using conventional cloning procedures (58). Thus, because the actual start codon was unknown in the beginning, a short version and a long version of a GFP fusion protein were constructed.…”

Section: Methodsmentioning

confidence: 99%

Molecular Identification of a SNAP-25-Like SNARE Protein inParamecium

et al. 2008

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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.Membrane trafficking in eukaryotic cells involves budding of vesicles from a donor compartment and transport to and fusion with the acceptor compartment. The soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) are of central importance in the mediation of membrane fusions (32). The crystal structure of the synaptic SNARE complex has been resolved (70). The ternary synaptic SNARE complex consists of the SNARE motifs of synaptobrevin-2 (VAMP2) and syntaxin-1A and the two SNARE motifs from the synaptosome-associated protein of 25 kDa (SNAP-25). Structures of different SNARE complexes revealed a highly conserved four-helix structure, with the difference that the positions of the two SNARE motifs from SNAP-25 can be contributed by two different SNARE proteins (7). The highly conserved pattern of SNARE pairing has led to the so-called 3Q-plus-1R rule (21). According to this rule, fusogenic SNARE complexes always contain three SNARE motifs containing a glutamine residue in the center of the SNARE motif (Q-SNARE) and one SNARE displaying an arginine at the same position (R-SNARE). Furthermore, Qa-, Qb-, Qc-, and R-SNAREs can be recognized by specific sequence features (40).Identification of the SNARE components of the synaptic SNARE complex and functional analysis have been greatly facilitated by the availability of specific inhibitors, e.g., by Clostridium botulinum neurotoxins (BoNTs), that specifically cleave certain neuronal SNAREs (46). BoNTs are zinc-dependent proteases which, by cleaving SNARE proteins, inhibit neurotransmitter release. The structural basis for the specificity of SNAP-25 cleavage by BoNT/A and BoNT/E has been solved, and the interacting amino acid...

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“…12j-l). However, it should be noted that a relatively high concentration of GFP is needed to reach the detection limit in Paramecium (Hauser et al, 2000a). This means that a low concentration of the endogenous F-and F-GFP-subunits remains after 16 hours, because the cell biological defects caused by V 1 silencing just start to become visible after 16 hours.…”

Section: Silencing Of V-atpase Subunits Disturbs Maturation Of Dense mentioning

confidence: 99%

“…The eGFP-gene presented in Hauser et al (Hauser et al, 2000a) was cloned into the Acc65I site of the Paramecium overexpression vector described in Haynes et al (Haynes et al, 1995) referred to as pPXV-GFP. All genes were cloned between SpeI and XhoI sites in-frame with the gene encoding GFP.…”

Section: Gfp Constructsmentioning

confidence: 99%

The vacuolar proton-ATPase plays a major role in several membrane-bounded organelles inParamecium

Wassmer

Froissard

Plattner

et al. 2005

Journal of Cell Science

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IntroductionThe vacuolar-proton-ATPase (V-ATPase) translocates protons across membranes against their electrochemical potential through ATP hydrolysis. The V-ATPase is responsible for the acidification of organelles like phagosomes, lysosomes, early endosomes, the trans-Golgi-network, dense core secretory granules and vacuoles of plants (Sun-Wada et al., 2003a;Nelson, 2003;Kluge et al., 2003). In some specialized cells of higher organisms, the enzyme is also localized in the plasma membrane and serves to secrete protons to the extracellular space. For example, the acidification of the extracellular matrix by proton secretion of osteoclasts is important for bone resorption (Toyomura et al., 2003). An important aspect of the proton translocation by the V-ATPase is the creation of an electrochemical potential that can be used for secondary active transport of ions. This mechanism is used in neuronal cells, where the neurotransmitters glutamate or γ-amino-butyric-acid are concentrated in the lumen of synaptic vesicles (Roseth et al., 1995;Fonnum et al., 1998) and in chromaffin granules (Apps, 1997).The V-ATPase is composed of two subcomplexes: the cytosolic V 1 -sector, where ATP binding and hydrolysis takes place; and a transmembranous V 0 -sector, through which protons are tunneled. The holoenzyme in Saccharomyces cerevisiae consists of 14 different subunits (Kawasaki-Nishi et al., 2004;Sambade and Kane, 2004). V 1 contains subunits A-H in the stoichiometry A 3 B 3 CDEFG 2 H 1-2 . V 0 is formed by the subunits a, c, c′, c′′, d and e in a stoichiometry of ade x c 4 c′c′′. The V-ATPase was shown to act by a rotationary mechanism similar to that of the structurally related F-ATPase (Imamura et al., 2003).The enzyme activity can be regulated by reversible dissociation of V 1 from V 0 . It was found in yeast that the ratio of assembled/disassembled enzyme is strongly influenced by the culture conditions. Most of the V 1 -domains dissociate from their V 0 counterparts at vacuoles in yeast when glucose is depleted in the medium (Kane and Smardon, 2003). Whether other physiological conditions exist that lead to a dissociation of the enzyme is unclear.Apart from its enzymatic action as a proton pump, the V 0 -subcomplex of the V-ATPase was found to be localized in the plasma membrane and to be involved in the release of neurotransmitter in mammalian cells (Falk-Vairant et al., 1996;Morel, 2003). A role of the V 0 -sector was also shown in homotypic membrane fusion of vacuoles in yeast (Peters et al., 2001). V 0 -subcomplexes were reported to build so-called 'trans-complexes' in opposing membranes and were postulated to facilitate membrane fusion. A knockout mutant of the vacuolar a-subunit of V 0 (vph1) was shown to cause fragmentation of vacuoles, also indicating an involvement of the V-ATPase in membrane dynamics (Bayer et al., 2003).Paramecium is a unicellular model organism, in which

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Expression of the green fluorescent protein in Paramecium tetraurelia

Cited by 44 publications

References 38 publications

Novel Types of Ca²⁺ Release Channels Participate in the Secretory Cycle of Paramecium Cells

Novel Types of Ca²⁺ Release Channels Participate in the Secretory Cycle of Paramecium Cells

Molecular Identification of a SNAP-25-Like SNARE Protein inParamecium

The vacuolar proton-ATPase plays a major role in several membrane-bounded organelles inParamecium

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Expression of the green fluorescent protein in Paramecium tetraurelia

Cited by 44 publications

References 38 publications

Novel Types of Ca2+ Release Channels Participate in the Secretory Cycle of Paramecium Cells

Novel Types of Ca2+ Release Channels Participate in the Secretory Cycle of Paramecium Cells

Molecular Identification of a SNAP-25-Like SNARE Protein inParamecium

The vacuolar proton-ATPase plays a major role in several membrane-bounded organelles inParamecium

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Novel Types of Ca²⁺ Release Channels Participate in the Secretory Cycle of Paramecium Cells

Novel Types of Ca²⁺ Release Channels Participate in the Secretory Cycle of Paramecium Cells