RNA interference by feeding in Paramecium

Galvani, Angélique; Sperling, Linda

doi:10.1016/s0168-9525(01)02548-3

Cited by 170 publications

(192 citation statements)

References 10 publications

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“…As a negative control, the plasmid pL4440 was used. Positive control was the plasmid pL4440/NSF (Galvani and Sperling, 2002). Part of the dsRNA is evidently capable of leaving the food vacuole during feeding and arrives in the cytoplasm, where it is thought to be diced into siRNA.…”

Section: Gene Silencing 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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Section: Gene Silencing 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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“…The recent advances in RNAi methodology in ciliated protozoa (Galvani and Sperling 2002;Mollenbeck et al 2003) may make this approach more feasible, and using this approach, evidence has been presented that p43 appears to anchor telomerase in the Euplotes macronucleus (Mollenbeck et al 2003). In addition, given that Tetrahymena is much more genetically tractable than Euplotes, the finding of a p43 ortholog in Tetrahymena, if confirmed, would allow further elucidation of the function of ciliate La-motif proteins in telomerase biogenesis and catalysis.…”

Section: Telomerase Stimulation By P43mentioning

confidence: 99%

The Euplotes telomerase subunit p43 stimulates enzymatic activity and processivity in vitro

Aigner

Cech²

2004

RNA

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Telomerase is a reverse transcriptase that synthesizes telomeric DNA repeats at the ends of eukaryotic chromosomes. Although it is minimally composed of a conserved catalytic protein subunit (TERT) and an RNA component, additional accessory factors present in the holoenzyme play crucial roles in the biogenesis and function of the enzyme complex. Telomerase from the ciliate Tetrahymena can be reconstituted in active form in vitro. Using this system, we show that p43, a telomerase-specific La-motif protein from the ciliate Euplotes, stimulates activity and increases repeat addition processivity of telomerase. Activity enhancement by p43 requires its incorporation into a TERT•RNA•p43 ternary complex but is independent of other dissociable protein factors functioning in telomerase complex assembly. Stimulation is enhanced at elevated temperatures, supporting a role for p43 in structural stabilization of a critical region of the RNA subunit. To our knowledge, this represents the first demonstration that an authentic telomerase accessory protein can directly affect the enzymatic activity of the core enzyme in vitro.

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“…Knockdown in Paramecium can be achieved by feeding transformed bacteria which produce (30). To achieve a knockdown phenotype of whole receptor families, tandem constructs representing both isoforms of IP 3 R N and CRC-IV-1 gene families were cloned into the pPD gene silencing vector (see Materials and Methods).…”

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

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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RNA interference by feeding in Paramecium

Cited by 170 publications

References 10 publications

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

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

The Euplotes telomerase subunit p43 stimulates enzymatic activity and processivity in vitro

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

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RNA interference by feeding in Paramecium

Cited by 170 publications

References 10 publications

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

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

The Euplotes telomerase subunit p43 stimulates enzymatic activity and processivity in vitro

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

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