Inhibition of trichocyst exocytosis and calcium influx in Paramecium by amiloride and divalent cations

Summary— Regulated exocytosis of defensive secretory organelles, the trichocysts, as well as a transient Ca²⁺-influx can be induced in Paramecium by aminoethyldextran (Kerb?uf and Cohen, J Cell Biol (1990) 111, 2527). Knoll et al (Febs Lett (1992) 304, 265) reported that veratridine was also a secretagogue for Paramecium. Here we show that, like aminoethyldextran, veratridine induces a transient Ca²⁺-influx. Both aminoethyldextran-and veratridine-induced exocytosis and associated Ca²⁺-influx were: i) blocked in the nd12 thermosensitive mutant at the non-permissive temperature; and ii) inhibited by amiloride and four divalent cations, Ba²⁺, Mg²⁺, Sr²⁺ and Co²⁺. This suggests that, although of different chemical nature, aminoethyldextran and veratridine act through the same physiological pathway. In addition, the inhibitory doses are comparable to the ones found to inhibit a hyperpolarization-sensitive Ca²⁺-current described in Paramecium (Preston et al (1992) J Gen Physiol 100, 233). The possibility that the activation of this Ca²⁺-current by the secretagogue represents an early step in the regulation of trichocyst exocytosis is discussed.     

Rediscovery of Paramecium nephridiatum Gelei, 1925 and its Characteristics

Paramecium nephridiatum Gelei. 1925, was rediscovered. It is a euryhaline brackish-water species that morphologically resembles Paramecium woodruffi . but with multiple contractile vacuole pores. The general morphology, morphometry. and random amplified polymorphic DNA (RAPD) fingerprint patterns are presented for a number of the stocks collected around the world.     

The Interface Between Air and Water: A Perturbation Source Eliciting Adaptive Behaviour in Ciliates

ABSTRACT Interference with the water-air interface, both direct (by contact with a flat, rigid surface) and indirect (by inducing a meniscus) caused the ciliated protozoa we investigated to actively collect in the water column or on the substrate directly under the area of altered surface tension. A crowding effect is observed in this "rest area" reaching plateau values within one hour after onset of the experiment. The simple experimental procedures described here induced analogous behaviour in both Paramecium caudatum (a swimmer) and Oxytricha bifaria (a crawler). The ciliates seem in this reaction to be seeking a refuge from vibrations transmitted by the free interface. Our discovery is discussed in its implications for the adaptive biology and ecology of these micro-organisms.     

SK channels and calmodulin

Calcium ions are Nature's most widely used signaling mechanism, mediating communication between pathways at virtually every physiological level. Ion channels are no exception, as the activities of a wide range of ion channels are intricately shaped by fluctuations in intracellular Ca²⁺ levels. Mirroring the importance and the breadth of Ca²⁺ signaling, free Ca²⁺ levels are tightly controlled, and a myriad of Ca²⁺ binding proteins transduce Ca²⁺ signals, each with its own nuance, comprising a constantly changing symphony of metabolic activity. The founding member of Ca²⁺ binding proteins is calmodulin (CaM), a small, acidic, modular protein endowed with gymnastic-like flexibility and E-F hand motifs that chelate Ca²⁺ ions. In this review, I will trace the history that led to the realization that CaM serves as the Ca²⁺-gating cue for SK channels, the experiments that revealed that CaM is an intrinsic subunit of SK channels, and itself a target of regulation.     

Effect of Adenine on Paramecium multimicronucleatum*

SYNOPSIS. The addition of adenine to the culture medium of Paramecium multimicronucleatum caused an inhibition of conjugation and fission and also an increase in the number of food vacuoles. The inhibition of conjugation was observed at the stage of adhesion of cell membranes. With adenine, the duration of fission was prolonged and L-shaped cells were formed. Adenine apparently repressed the excretion of food vacuoles and thus caused an accumulation of food vacuoles within the cells. All these effects were observed at similar concentrations of adenine higher than 2 mM. The addition of uridine to the adenine-containing medium reversed the inhibition of fission; conjugation and vacuole accumulation remained unaffected.     

Experimental Microbial Populations Thirty-five Years Later: The Influence of Food on the Symbiosis of Paramecium aurelia Syngen 4, 51.7

Changes in the nutrition of Paramecium aurelia affect its ability to serve as host for the bacteroid parasite, kappa, and the presence or absence of kappa affects its ability to grow in axenic culture. Loss of kappa, tested by the presence or absence of killer reaction, occurred in cultures of P. aurelia growing at a reduced division rate on autoclaved Enterobacter aerogenes in suspensions of lettuce and yeast autolysate 14–17 days after they had been rendered bacteria-free by washing. Killer Paramecium sterilized of bacteria by treatment with an antibiotic mixture of penicillin-G and streptomycin in combination with a nonbacterial nonliving culture medium, lost the ability to kill after from 6 to 48 hours in the sterilizing medium. The ciliates from which kappa had been lost during exposure to antibiotics could be transferred immediately and maintained in axenic culture, but those washed free of bacteria could not be maintained axenically until kappa had been lost during cultivation in a medium containing killed bacteria. It is suggested that a knowledge of the nutritional requirements of symbiotic microorganisms is essential for understanding the ecological aspects of eutrophication of aquatic environments.     

Three-Dimensional Structure of Macronuclei from Paramecium Determined by Scanning Electron Microscopy*

SYNOPSIS. Macronuclei of Paramecium primaurelia were isolated and examined by scanning electron microscopy. These nuclei consisted of a closely packed array of chromatin bodies measuring ～ 0.2 μm in diameter. We estimated there were ～ 30,000 such bodies/macronucleus, 20 times more than the number of unit genome equivalents. This suggests that a unit genome is physically shared by several chromatin bodies.     

Quaternary assembly and crystal structure of GDP-D-mannose 4,6 dehydratase from Paramecium bursaria Chlorella virus

GDP-D-mannose 4,6 dehydratase is the first enzyme in the de novo biosynthetic pathway of GDP-L-fucose, the activated form of L-fucose, a monosaccharide found in organisms ranging from bacteria to mammals. We determined the three-dimensional structure of GDP-D-mannose 4,6 dehydratase from the Paramecium bursaria Chlorella virus at 3.8A resolution. Unlike other viruses that use the host protein machinery to glycosylate their proteins, P. bursaria Chlorella virus modifies its structural proteins using many glycosyltransferases, being the first virus known to encode enzymes involved in sugar metabolism. P. bursaria Chlorella virus GDP-D-mannose 4,6 dehydratase belongs to the short-chain dehydrogenase/reductase protein superfamily. Accordingly, the family fold and the specific Thr, Tyr, and Lys catalytic triad are well conserved in the viral enzyme.     

A multigene family encoding R-SNAREs in the ciliate Paramecium tetraurelia

SNARE proteins (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) mediate membrane interactions and are conventionally divided into Q-SNAREs and R-SNAREs according to the possession of a glutamine or arginine residue at the core of their SNARE domain. Here, we describe a set of R-SNAREs from the ciliate Paramecium tetraurelia consisting of seven families encoded by 12 genes that are expressed simultaneously. The complexity of the endomembrane system in Paramecium can explain this high number of genes. All P. tetraurelia synaptobrevins (PtSybs) possess a SNARE domain and show homology to the Longin family of R-SNAREs such as Ykt6, Sec22 and tetanus toxin-insensitive VAMP (TI-VAMP). We localized four exemplary PtSyb subfamilies with GFP constructs and antibodies on the light and electron microscopic level. PtSyb1-1, PtSyb1-2 and PtSyb3-1 were found in the endoplasmic reticulum, whereas PtSyb2 is localized exclusively in the contractile vacuole complex. PtSyb6 was found cytosolic but also resides in regularly arranged structures at the cell cortex (parasomal sacs), the cytoproct and oral apparatus, probably representing endocytotic compartments. With gene silencing, we showed that the R-SNARE of the contractile vacuole complex, PtSyb2, functions to maintain structural integrity as well as functionality of the osmoregulatory system but also affects cell division.