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Helmut Plattner - One of the best experts on this subject based on the ideXlab platform.
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2016Co-Authors: Ivonne Margarete Sehring, Christoph Reiner, Helmut Plattner, Cell DivisionAbstract:phagocytosis Paramecium tetraurelia possesses more actin isoforms than most other cells. With monospecific antibodies against actin subfamily 4 members, we could label cleavage furrow, nascent food vacuoles, oral apparatus, cilia, cell surface and macronucleus. Expression as green fluorescent protein- (GFP-) fusion protein now allowed us to localize more stringently actin4, e.g., in the macronucleus, particularly when enhanced with anti-GFP antibodies. Posttranscriptional gene silencing of actin4 resulted in disturbances at sites where actin4 has been localized. Cell division was impaired already early on, occasionally resulting in deformed cells. Both micro- and macronuclear development during vegetative cell fission were disturbed. Over longer periods, actin4 silencing entailed reduced phagocytotic activity, paralleled by accumulation of "acidosomes " (late endosomes) near the cytopharynx where they normally fuse with nascent phagosomes. In addition, near the cell surface, extensively misshapen "terminal cisternae " (early endosomes) occurred. In deformed cells, both constitutive endocytosis and stimulated Trichocyst exocytosis were impaired. Thus, actin4 exerts pleiotropic effects at widely different sites of the Paramecium cell and disturbances generally coincide with sites where actin4 is normally enriched. Evidently the loss of actin4 cannot easily be compensated for by any other of the large number of actin isoforms occurring in a Paramecium cell
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the actin subfamily ptact4 out of many subfamilies is differentially localized for specific local functions in paramecium tetraurelia cells
European Journal of Cell Biology, 2010Co-Authors: Ivonne Margarete Sehring, Christoph Reiner, Helmut PlattnerAbstract:Abstract Paramecium tetraurelia possesses more actin isoforms than most other cells. With monospecific antibodies against actin subfamily 4 members, we could label cleavage furrow, nascent food vacuoles, oral apparatus, cilia, cell surface and macronucleus. Expression as green fluorescent protein- (GFP-) fusion protein now allowed us to localize more stringently actin4, e.g., in the macronucleus, particularly when enhanced with anti-GFP antibodies. Posttranscriptional gene silencing of actin4 resulted in disturbances at sites where actin4 has been localized. Cell division was impaired already early on, occasionally resulting in deformed cells. Both micro- and macronuclear development during vegetative cell fission were disturbed. Over longer periods, actin4 silencing entailed reduced phagocytotic activity, paralleled by accumulation of “acidosomes” (late endosomes) near the cytopharynx where they normally fuse with nascent phagosomes. In addition, near the cell surface, extensively misshapen “terminal cisternae” (early endosomes) occurred. In deformed cells, both constitutive endocytosis and stimulated Trichocyst exocytosis were impaired. Thus, actin4 exerts pleiotropic effects at widely different sites of the Paramecium cell and disturbances generally coincide with sites where actin4 is normally enriched. Evidently the loss of actin4 cannot easily be compensated for by any other of the large number of actin isoforms occurring in a Paramecium cell.
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Distinct Subcellular Localization of a Group of Synaptobrevin-Like SNAREs in Paramecium tetraurelia and Effects of Silencing SNARE-Specific Chaperone NSF
Eukaryotic cell, 2009Co-Authors: Christina Schilde, Barbara Schönemann, Ivonne Margarete Sehring, Helmut PlattnerAbstract: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.
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Seventeen a-subunit isoforms of paramecium V-ATPase provide high specialization in localization and function.
Molecular Biology of the Cell, 2006Co-Authors: Thomas Wassmer, Roland Kissmehl, Jean Cohen, Helmut PlattnerAbstract: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.
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Seventeen a-subunit isoforms of Paramecium V-ATPase provide high specialization in localization and function
2006Co-Authors: Thomas Wassmer, Jean Cohen, Helmut Plattner, Howard RiezmanAbstract: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. This article was published online ahead of print in MBC in Pres
Roland Kissmehl - One of the best experts on this subject based on the ideXlab platform.
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Seventeen a-subunit isoforms of paramecium V-ATPase provide high specialization in localization and function.
Molecular Biology of the Cell, 2006Co-Authors: Thomas Wassmer, Roland Kissmehl, Jean Cohen, Helmut PlattnerAbstract: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.
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the vacuolar proton atpase plays a major role in several membrane bounded organelles in paramecium
Journal of Cell Science, 2005Co-Authors: Thomas Wassmer, Marine Froissard, Roland Kissmehl, Helmut Plattner, Jean CohenAbstract:The vacuolar proton-ATPase (V-ATPase) is a multisubunit enzyme complex that is able to transfer protons over membranes against an electrochemical potential under ATP hydrolysis. The enzyme consists of two subcomplexes: V0, which is membrane embedded; and V1, which is cytosolic. V0 was also reported to be involved in fusion of vacuoles in yeast. We identified six genes encoding c-subunits (proteolipids) of V0 and two genes encoding F-subunits of V1 and studied the role of the V-ATPase in trafficking in Paramecium. Green fluorescent protein (GFP) fusion proteins allowed a clear subcellular localization of c- and F-subunits in the contractile vacuole complex of the osmoregulatory system and in food vacuoles. Several other organelles were also detected, in particular dense core secretory granules (Trichocysts). The functional significance of the V-ATPase in Paramecium was investigated by RNA interference (RNAi), using a recently developed feeding method. A novel strategy was used to block the expression of all six c- or both F-subunits simultaneously. The V-ATPase was found to be crucial for osmoregulation, the phagocytotic pathway and the biogenesis of dense core secretory granules. No evidence was found supporting participation of V0 in membrane fusion.
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Identification of isoforms of the exocytosis-sensitive phosphoprotein PP63/parafusin in Paramecium tetraurelia and demonstration of phosphoglucomutase activity
Biochemical Journal, 1997Co-Authors: Karin Hauser, Roland Kissmehl, Jürgen U. Linder, Joachim E. Schultz, Friedrich Lottspeich, Helmut PlattnerAbstract:PP63 (parafusin) is a 63 kDa phosphoprotein which is very rapidly (within 80 ms) dephosphorylated (to P63) during triggered Trichocyst exocytosis; this occurs selectively in exocytosis-competent Paramecium tetraurelia strains. In the present work, two cDNAs coding for PP63/parafusin have been isolated, one of which is a new isoform. These isoforms are 99.6% identical and are derived from two different genes. Similarity searches revealed 43-51% identity of the deduced amino acid sequences with known phosphoglucomutases from yeast and mammals. The sequences of two proteolytic peptides obtained from PP63/parafusin isolated from Paramecium are identical to parts of the amino acid sequence deduced from the major cDNA. The major cDNA was mutated from the macronuclear ciliate genetic code into the universal genetic code and expressed in Escherichia coli. The recombinant protein shows the same biochemical and immunological characteristics as the (P)P63/parafusin originally isolated from Paramecium. It has the same specific phosphoglucomutase activity as phosphoglucomutase from chicken muscle. We also show that recombinant P63-1 parafusin 1 is a substrate of an endogenous casein kinase from Paramecium, as is the originally isolated P63/parafusin. Polyclonal antibodies against recombinant P63-1/parafusin 1 were raised which recognized phosphoglucomutases from different sources. Thus we show that PP63/parafusin and phosphoglucomutase in Paramecium are identical.
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identification of isoforms of the exocytosis sensitive phosphoprotein pp63 parafusin in paramecium tetraurelia and demonstration of phosphoglucomutase activity
Biochemical Journal, 1997Co-Authors: Karin Hauser, Roland Kissmehl, Jürgen U. Linder, Joachim E. Schultz, Friedrich Lottspeich, Helmut PlattnerAbstract:PP63 (parafusin) is a 63 kDa phosphoprotein which is very rapidly (within 80 ms) dephosphorylated (to P63) during triggered Trichocyst exocytosis; this occurs selectively in exocytosis-competent Paramecium tetraurelia strains. In the present work, two cDNAs coding for PP63/parafusin have been isolated, one of which is a new isoform. These isoforms are 99.6% identical and are derived from two different genes. Similarity searches revealed 43-51% identity of the deduced amino acid sequences with known phosphoglucomutases from yeast and mammals. The sequences of two proteolytic peptides obtained from PP63/parafusin isolated from Paramecium are identical to parts of the amino acid sequence deduced from the major cDNA. The major cDNA was mutated from the macronuclear ciliate genetic code into the universal genetic code and expressed in Escherichia coli. The recombinant protein shows the same biochemical and immunological characteristics as the (P)P63/parafusin originally isolated from Paramecium. It has the same specific phosphoglucomutase activity as phosphoglucomutase from chicken muscle. We also show that recombinant P63-1 parafusin 1 is a substrate of an endogenous casein kinase from Paramecium, as is the originally isolated P63/parafusin. Polyclonal antibodies against recombinant P63-1/parafusin 1 were raised which recognized phosphoglucomutases from different sources. Thus we show that PP63/parafusin and phosphoglucomutase in Paramecium are identical.
Karin Hauser - One of the best experts on this subject based on the ideXlab platform.
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Identification of isoforms of the exocytosis-sensitive phosphoprotein PP63/parafusin in Paramecium tetraurelia and demonstration of phosphoglucomutase activity
Biochemical Journal, 1997Co-Authors: Karin Hauser, Roland Kissmehl, Jürgen U. Linder, Joachim E. Schultz, Friedrich Lottspeich, Helmut PlattnerAbstract:PP63 (parafusin) is a 63 kDa phosphoprotein which is very rapidly (within 80 ms) dephosphorylated (to P63) during triggered Trichocyst exocytosis; this occurs selectively in exocytosis-competent Paramecium tetraurelia strains. In the present work, two cDNAs coding for PP63/parafusin have been isolated, one of which is a new isoform. These isoforms are 99.6% identical and are derived from two different genes. Similarity searches revealed 43-51% identity of the deduced amino acid sequences with known phosphoglucomutases from yeast and mammals. The sequences of two proteolytic peptides obtained from PP63/parafusin isolated from Paramecium are identical to parts of the amino acid sequence deduced from the major cDNA. The major cDNA was mutated from the macronuclear ciliate genetic code into the universal genetic code and expressed in Escherichia coli. The recombinant protein shows the same biochemical and immunological characteristics as the (P)P63/parafusin originally isolated from Paramecium. It has the same specific phosphoglucomutase activity as phosphoglucomutase from chicken muscle. We also show that recombinant P63-1 parafusin 1 is a substrate of an endogenous casein kinase from Paramecium, as is the originally isolated P63/parafusin. Polyclonal antibodies against recombinant P63-1/parafusin 1 were raised which recognized phosphoglucomutases from different sources. Thus we show that PP63/parafusin and phosphoglucomutase in Paramecium are identical.
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identification of isoforms of the exocytosis sensitive phosphoprotein pp63 parafusin in paramecium tetraurelia and demonstration of phosphoglucomutase activity
Biochemical Journal, 1997Co-Authors: Karin Hauser, Roland Kissmehl, Jürgen U. Linder, Joachim E. Schultz, Friedrich Lottspeich, Helmut PlattnerAbstract:PP63 (parafusin) is a 63 kDa phosphoprotein which is very rapidly (within 80 ms) dephosphorylated (to P63) during triggered Trichocyst exocytosis; this occurs selectively in exocytosis-competent Paramecium tetraurelia strains. In the present work, two cDNAs coding for PP63/parafusin have been isolated, one of which is a new isoform. These isoforms are 99.6% identical and are derived from two different genes. Similarity searches revealed 43-51% identity of the deduced amino acid sequences with known phosphoglucomutases from yeast and mammals. The sequences of two proteolytic peptides obtained from PP63/parafusin isolated from Paramecium are identical to parts of the amino acid sequence deduced from the major cDNA. The major cDNA was mutated from the macronuclear ciliate genetic code into the universal genetic code and expressed in Escherichia coli. The recombinant protein shows the same biochemical and immunological characteristics as the (P)P63/parafusin originally isolated from Paramecium. It has the same specific phosphoglucomutase activity as phosphoglucomutase from chicken muscle. We also show that recombinant P63-1 parafusin 1 is a substrate of an endogenous casein kinase from Paramecium, as is the originally isolated P63/parafusin. Polyclonal antibodies against recombinant P63-1/parafusin 1 were raised which recognized phosphoglucomutases from different sources. Thus we show that PP63/parafusin and phosphoglucomutase in Paramecium are identical.
Plattner Helmut - One of the best experts on this subject based on the ideXlab platform.
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Trichocysts - Paramecium's Projectile-like Secretory Organelles : Reappraisal of their Biogenesis, Composition, Intracellular Transport, and Possible Functions
'Wiley', 2017Co-Authors: Plattner HelmutAbstract:This review summarizes biogenesis, composition, intracellular transport, and possible functions of Trichocysts. Trichocyst release by Paramecium is the fastest dense core-secretory vesicle exocytosis known. This is enabled by the crystalline nature of the Trichocyst "body" whose matrix proteins (tmp), upon contact with extracellular Ca2+, undergo explosive recrystallization that propagates cooperatively throughout the organelle. Membrane fusion during stimulated Trichocyst exocytosis involves Ca2+ mobilization from alveolar sacs and tightly coupled store-operated Ca2+-influx, initiated by activation of ryanodine receptor-like Ca2+ -release channels. Particularly, aminoethyldextran perfectly mimics a physiological function of Trichocysts, i.e. defense against predators, by vigorous, local Trichocyst discharge. The tmp's contained in the main "body" of a Trichocyst are arranged in a defined pattern, resulting in crossstriation, whose period expands upon expulsion. The second part of a Trichocyst, the "tip", contains secretory lectins which diffuse upon discharge. Repulsion from predators may not be the only function of Trichocysts. We consider ciliary reversal accompanying stimulated Trichocyst exocytosis (also in mutants devoid of depolarization-activated Ca2+ channels) a second, automatically superimposed defense mechanism. A third defensive mechanism may be effectuated by the secretory lectins of the Trichocyst tip; they may inhibit toxicyst exocytosis in Dileptus by crosslinking surface proteins (an effect mimicked in Paramecium by antibodies against cell surface components). Some of the proteins, body and tip, are glycosylated as visualized by binding of exogenous lectins. This reflects the biogenetic pathway, from the endoplasmic reticulum via the Golgi apparatus, which is also supported by details from molecular biology. There are fragile links connecting the matrix of a Trichocyst with its membrane; these may signal the filling state, full or empty, before and after tmp release upon exocytosis, respectively. This is supported by experimentally produced "frustrated exocytosis", i.e. membrane fusion without contents release, followed by membrane resealing and entry in a new cycle of reattachment for stimulated exocytosis. There are some more puzzles to be solved: Considering the absence of any detectable Ca2+ and of acidity in the organelle, what causes the striking effects of silencing the genes of some specific Ca2+ -release channels and of subunits of the H+ -ATPase? What determines the inherent polarity of a Trichocyst? What precisely causes the inability of Trichocyst mutants to dock at the cell membrane? Many details now call for further experimental work to unravel more secrets about these fascinating organelles.publishe
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Molecular aspects of calcium signalling at the crossroads of unikont and bikont eukaryote evolution : The ciliated protozoan Paramecium in focus
'Elsevier BV', 2015Co-Authors: Plattner HelmutAbstract:The ciliated protozoan, Paramecium tetraurelia has a high basic Ca2+ leakage rate which is counteracted mainly by export through a contractile vacuole complex, based on its V-type H+-ATPase activity. In addition Paramecium cells dispose of P-type Ca2+-ATPases, i.e. a plasmamembrane and a sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (PMCA, SERCA). Antiporter systems are to be expected, as inferred from indirect evidence. Among the best known cytosolic Ca2+-binding proteins, calmodulin activates Ca2+ influx channels in the somatic cell membrane, but inactivates Ca2+ influx channels in cilia, where it, thus, ends ciliary reversal induced by depolarization via channels in the somatic cell membrane. Centrin inactivates Ca2+ signals after stimulation by its high capacity/low affinity binding sites, whereas its high affinity sites regulate some other functions. Cortical Ca2+ stores (alveolar sacs) are activated during stimulated Trichocyst exocytosis and thereby mediate store-operated Ca2+ entry (SOCE). Ca2+ release channels (CRCs) localised to alveoli and underlying SOCE are considered as Ryanodine receptor-like proteins (RyR-LPs) which are members of a CRC family with 6 subfamilies. These also encompass genuine inositol 1,4,5-trisphosphate receptors (IP3Rs) and intermediates between the two channel types. All IP3R/RyR-type CRCs possess six carboxyterminal transmembrane domains (TMD), with a pore domain between TMD 5 and 6, endowed with a characteristic selectivity filter. There are reasons to assume a common ancestor molecule for such channels and diversification further on in evolution. The distinct distribution of specific CRCs in the different vesicles undergoing intracellular trafficking suggests constitutive formation of very locally restricted Ca2+ signals during vesicle–vesicle interaction. In summary, essential steps of Ca2+ signalling already occur at this level of evolution, including an unexpected multitude of CRCs. For dis-/similarities with other bikonts see “Conclusions”
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Novel types of Ca2+ release channels participate in the secretory cycle of Paramecium cells
'American Society for Microbiology', 2009Co-Authors: Ladenburger Eva-maria, Sehring Ivonne, Korn Iris, Plattner HelmutAbstract:A database search of the Paramecium genome reveals 34 genes related to Ca2+-release channels of the inositol-1,4,5-trisphosphate (IP3) or ryanodine receptor type (IP3R, RyR). Phylogenetic analyses show that these Ca2+ release channels (CRCs) can be subdivided into six groups (Paramecium tetraurelia CRC-I to CRC-VI), each one with features in part reminiscent of IP3Rs and RyRs. We characterize here the P. tetraurelia CRC-IV-1 gene family, whose relationship to IP3Rs and RyRs is restricted to their C-terminal channel domain. CRC-IV-1 channels localize to cortical Ca2+ stores (alveolar sacs) and also to the endoplasmic reticulum. This is in contrast to a recently described true IP3 channel, a group II member (P. tetraurelia IP3RN-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 IP3RN affects Trichocyst biogenesis, while CRC-IV-1 channels are involved in signal transduction since silenced cells show an impaired release of Ca2+ 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 Ca2+ as signaling molecule
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Seventeen a-subunit isoforms of Paramecium V-ATPaseprovide high specialization in localization and function
'American Society for Cell Biology (ASCB)', 2006Co-Authors: Wassmer Thomas, Kissmehl Roland, Cohen Jean, Plattner HelmutAbstract: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 o 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
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Immunolocalization of actin in Paramecium cells
'SAGE Publications', 2004Co-Authors: Kissmehl Roland, Sehring, Ivonne Margarete, Wagner Erika, Plattner HelmutAbstract:We have selected a conserved immunogenic region from several actin genes of Paramecium, recently cloned in our laboratory, to prepare antibodies for Western blots and immunolocalization. According to cell fractionation analysis, most actin is structurebound. Immunofluorescence shows signal enriched in the cell cortex, notably around ciliary basal bodies (identified by anti-centrin antibodies), as well as around the oral cavity, at the cytoproct and in association with vacuoles (phagosomes) up to several μm in size. Subtle strands run throughout the cell body. Postembedding immunogold labeling/EM analysis shows that actin in the cell cortex emanates, together with the infraciliary lattice, from basal bodies to around Trichocyst tips. Label was also enriched around vacuoles and vesicles of different size including discoidal vesicles that serve the formation of new phagosomes. By all methods used, we show actin in cilia. Although none of the structurally welldefined filament systems in Paramecium are exclusively formed by actin, actin does display some ordered, though not very conspicuous, arrays throughout the cell. F-actin may somehow serve vesicle trafficking and as a cytoplasmic scaffold. This is particularly supported by the postembedding/EM labeling analysis we used, which would hardly allow for any largescale redistribution during preparation
Jean Cohen - One of the best experts on this subject based on the ideXlab platform.
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Copyright Q 1992 by the Genetics Society of America Interactions Between Genes Involved in Exocytotic Membrane Fusion in Paramecium
2013Co-Authors: Hugues Bonnemain, Tadeusz Gulik-krzywicki, Jean CohenAbstract:Crosses between members of two independent collections of Paramecium tetraurelia mutants blocked in the final membrane fusion step of Trichocyst release (nd mutants) allowed us to define 13 complementation groups comprising 23 alleles. The mutant nd9 “ was then used as a target in a mutagenesis experiment designed to screen both revertants and new mutants in order to identify interacting genes. This mutant was chosen because it is the best known of its class to date and seems to be altered in assembly of the material connecting the Trichocyst membrane to the plasma membrane and in assembly of the “rosette, ” a complex array of intramembranous particles in the plasma membrane at the Trichocyst insertion sites. No revertants were obtained but two new mutants deficient for rosette assembly were identified, nd16 ’ and nd18, whose gene products appear to interact with that of nd9. Indeed, the double mutants grown at 18”, a permissive temperature for each of the single mutants, are characterized by a deficiency in exocytosis and in rosette assembly, as are also double mutants combining other allelic forms of the same genes. Moreover, aberrant dominance relationships among alleles of nd9 and of nd16 indicate the existence of interactions between identical subunits, which most likely assemble into multimeric structures. The nd16 gene product was show
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Seventeen a-subunit isoforms of paramecium V-ATPase provide high specialization in localization and function.
Molecular Biology of the Cell, 2006Co-Authors: Thomas Wassmer, Roland Kissmehl, Jean Cohen, Helmut PlattnerAbstract: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.
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Seventeen a-subunit isoforms of Paramecium V-ATPase provide high specialization in localization and function
2006Co-Authors: Thomas Wassmer, Jean Cohen, Helmut Plattner, Howard RiezmanAbstract: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. This article was published online ahead of print in MBC in Pres
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nd6p a novel protein with rcc1 like domains involved in exocytosis in paramecium tetraurelia
Eukaryotic Cell, 2005Co-Authors: Delphine Gogendeau, Annemarie Keller, Akira Yanagi, Jean CohenAbstract:In Paramecium tetraurelia, the regulated secretory pathway of dense core granules called Trichocysts can be altered by mutation and genetically studied. Seventeen nondischarge (ND) genes controlling exocytosis have already been identified by a genetic approach. The site of action of the studied mutations is one of the three compartments, the cytosol, Trichocyst, or plasma membrane. The only ND genes cloned to date correspond to mutants affected in the cytosol or in the Trichocyst compartment. In this work, we investigated a representative of the third compartment, the plasma membrane, by cloning the ND6 gene. This gene encodes a 1,925-amino-acid protein containing two domains homologous to the regulator of chromosome condensation 1 (RCC1). In parallel, 10 new alleles of the ND6 gene were isolated. Nine of the 12 available mutations mapped in the RCC1-like domains, showing their importance for the Nd6 protein (Nd6p) function. The RCC1 protein is well known for its guanine exchange factor activity towards the small GTPase Ran but also for its involvement in membrane fusion during nuclear envelope assembly. Other proteins with RCC1-like domains are also involved in intracellular membrane fusion, but none has been described yet as involved in exocytosis. The case of Nd6p is thus the first report of such a protein with a documented role in exocytosis.
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the vacuolar proton atpase plays a major role in several membrane bounded organelles in paramecium
Journal of Cell Science, 2005Co-Authors: Thomas Wassmer, Marine Froissard, Roland Kissmehl, Helmut Plattner, Jean CohenAbstract:The vacuolar proton-ATPase (V-ATPase) is a multisubunit enzyme complex that is able to transfer protons over membranes against an electrochemical potential under ATP hydrolysis. The enzyme consists of two subcomplexes: V0, which is membrane embedded; and V1, which is cytosolic. V0 was also reported to be involved in fusion of vacuoles in yeast. We identified six genes encoding c-subunits (proteolipids) of V0 and two genes encoding F-subunits of V1 and studied the role of the V-ATPase in trafficking in Paramecium. Green fluorescent protein (GFP) fusion proteins allowed a clear subcellular localization of c- and F-subunits in the contractile vacuole complex of the osmoregulatory system and in food vacuoles. Several other organelles were also detected, in particular dense core secretory granules (Trichocysts). The functional significance of the V-ATPase in Paramecium was investigated by RNA interference (RNAi), using a recently developed feeding method. A novel strategy was used to block the expression of all six c- or both F-subunits simultaneously. The V-ATPase was found to be crucial for osmoregulation, the phagocytotic pathway and the biogenesis of dense core secretory granules. No evidence was found supporting participation of V0 in membrane fusion.
