NSF regulates membrane traffic along multiple pathways in<i>Paramecium</i>

Kissmehl, Roland; Froissard, Marine; Plattner, Helmut; Momayezi, Massoud; Cohen, Jean

doi:10.1242/jcs.00079

Cited by 36 publications

(42 citation statements)

References 63 publications

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“…The presence of the SNARE-forming proteins syntaxin, VAMP and SNAP, involved in synaptic vesicle exocytosis, and of NSF, the SNARE-specific chaperone, has been recently demonstrated in Paramecium Kissmehl et al, 2002;Kissmehl et al, 2007;Schilde et al, 2006;Schilde et al, 2008). Seven subfamilies of synaptobrevins encoded by 12 genes, as well as at least 26 syntaxins grouped into 15 subfamilies, were identified (Kissmehl et al, 2007;Schilde et al, 2006).…”

Section: +mentioning

confidence: 99%

γ-Amino butyric acid (GABA) release in the ciliated protozoonParameciumoccurs by neuronal-like exocytosis

Ramoino

Milanese

Candiani

et al. 2010

Journal of Experimental Biology

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SUMMARYParamecium primaurelia expresses a significant amount of -amino butyric acid (GABA). Paramecia possess both glutamate decarboxylase (GAD)-like and vesicular GABA transporter (vGAT)-like proteins, indicating the ability to synthesize GABA from glutamate and to transport GABA into vesicles. Using antibodies raised against mammalian GAD and vGAT, bands with an apparent molecular weight of about 67kDa and 57kDa were detected. The presence of these bands indicated a similarity between the proteins in Paramecium and in mammals. VAMP, syntaxin and SNAP, putative proteins of the release machinery that form the so-called SNARE complex, are present in Paramecium. Most VAMP, syntaxin and SNAP fluorescence is localized in spots that vary in size and density and are primarily distributed near the plasma membrane. Antibodies raised against mammal VAMP-3, sintaxin-1 or SNAP-25 revealed protein immunoblot bands having molecular weights consistent with those observed in mammals. Moreover, P. primaurelia spontaneously releases GABA into the environment, and this neurotransmitter release significantly increases after membrane depolarization. The depolarization-induced GABA release was strongly reduced not only in the absence of extracellular Ca 2+ but also by pre-incubation with bafilomycin A1 or with botulinum toxin C1 serotype. It can be concluded that GABA occurs in Paramecium, where it is probably stored in vesicles capable of fusion with the cell membrane; accordingly, GABA can be released from Paramecium by stimulus-induced, neuronal-like exocytotic mechanisms.

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Section: +mentioning

confidence: 99%

γ-Amino butyric acid (GABA) release in the ciliated protozoonParameciumoccurs by neuronal-like exocytosis

Ramoino

Milanese

Candiani

et al. 2010

Journal of Experimental Biology

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“…SNARE-mediated fusion is a common feature of all eukaryotic cells, and all of the above-mentioned components of the SNARE fusion machinery have also been identified in the ciliated protozoan Paramecium tetraurelia (22,36,37,61). Paramecium, which must perform all of the autonomous functions of an entire organism, possesses highly diversified membrane trafficking pathways (53).…”

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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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“…It is hoped that electroporation or "bioballistic" methods (bombardment with DNA-coated gold particles) will allow us to achieve stable transfectants (Boileau et al, 1999). Homology-dependent gene silencing by injection of a large number of ORFs, without flanking regions (Ruiz et al, 1998;Bastin et al, 2001;Galvani and Sperling, 2001), has already proved useful in analyzing some aspects of membrane trafficking in Paramecium Kissmehl et al, 2002). Its effect is not fully 207 -.…”

Section: Discussionmentioning

confidence: 99%

“…It allows us to design appropriate primers and to clone the respective genes. Cloning of the Paramecium NSF gene, in collaboration with the Cohen group, was a pivotal step Kissmehl et al, 2002). Because NSF is a chaperone for SNAREs and because these interact with additional components, this now opens the door to many other components of the docking/fusion machinery.…”

Section: B Recent Molecular Approaches To the Study Of Membrane Trafmentioning

confidence: 99%

“…NSF gene-silencing experiments have resulted in the inhibition of phagocytotic vacuole formation, decreasing cell size, and finally in cell death Kissmehl et al, 2002). Immunofluorescence labeling by anti-NSF ABs was visible most clearly at the beginning and at the end of the digestive cycle, i.e., at the cytopharynx and the cytoproct, respectively.…”

Section: Phagocytosis and Lysosomal Systemmentioning

confidence: 99%

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Molecular Aspects of Membrane Trafficking in Paramecium

Plattner

Kissmehl

2003

International Review of Cytology

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Results achieved in tile molecular biology of Paramecium have shed new light on its elaborate membrane trafficking system. Paramecium disposes not only of the standard routes (endoplasmic reticulum ---+ Golgi ---+ Iysosomes or secretory vesicles; endo-and phagosomes ---+ Iysosomes/digesting vacuoles), but also of some unique features, e.g. and elaborate phagocytic route with the cytoproct and membrane recycling to the cytopharynx, as well as the osmoregulatory system with multiple membrane fusion sites. Exocytosis sites for trichocysts (dense-core secretory vesicles), parasomal sacs (coated pits), and terminal cisternae (early endosomes) display additional regularly arranged predetermined fusion/fission sites, which now can be discussed on a molecular basis. Considering the regular, repetitive arrangements of membrane components, availability of mutants for complementation studies, sensitivity to gene silencing, and so on, Paramecium continues to be a valuable model system for analyzing membrane interactions. This review intends to set a new baseline for ongoing work along these lines.

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NSF regulates membrane traffic along multiple pathways inParamecium

Cited by 36 publications

References 63 publications

γ-Amino butyric acid (GABA) release in the ciliated protozoonParameciumoccurs by neuronal-like exocytosis

γ-Amino butyric acid (GABA) release in the ciliated protozoonParameciumoccurs by neuronal-like exocytosis

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

Molecular Aspects of Membrane Trafficking in Paramecium

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