Identification of possible phosducins in the ciliate Blepharisma japonicum

Examination of ciliate Blepharisma japonicum whole cell lysates with an antibody against phosphoserine and in vivo labeling of cells with radioactive phosphate revealed that the photophobic response in the ciliate is accompanied by a rapid dephosphorylation of a 28 kDa protein and an enhanced phosphorylation of a 46 kDa protein. Analysis with antibodies raised against rat phosducin or human phosducin-like proteins, identified one major protein of a molecular weight of 28 kDa, and two protein bands of 40 kDa and 93 kDa. While the identified ciliate phosducin is phosphorylated in a light-dependent manner, both phosducin-like proteins exhibit no detectable dependence of phosphorylation upon illumination. An immunoprecipitation assay also showed that the ciliate phosducin is indeed phosphorylated on a serine residue and exists in a phosphorylated form in darkness and that its dephosphorylation occurs in light. Immunocytochemical experiments showed that protozoan phosducin and phosducin-like proteins are localized almost uniformly within the cytoplasm of cells adapted to darkness. Cell exposure to light caused a pronounced displacement of the cell phosducin to the vicinity of the plasma membrane; however, no translocation of phosducin-like proteins was observed upon cell illumination. The obtained results are the first demonstration of the presence and morphological localization of a possible phosducin and phosducin-like proteins in ciliate protists. Phosducin and phosducin-like proteins were found to bind and sequester the betagamma-subunits of G-proteins with implications for regulation of G-protein-mediated signaling pathways in various eukaryotic cells. The findings presented in this study suggest that the identified phosphoproteins in photosensitive Blepharisma japonicum may also participate in the regulation of the efficiency of sensory transduction, resulting in the motile photophobic response in this cell.     

Endosymbiotic bacteria and their relationship with chlorella in the ciliate Climacostomum virens

Structure of cytoplasmic bacterial symbionts of chlorella-free ciliate Climacostomum virens has been investigated. It is shown that ciliates are not able to support simultaneously growth and duplication of two different symbionts--bacteria and chlorella. Cells of C. virens lost bacterial symbionts after an artificial infection with chlorella by microinjection. Competitive relationships between two endopionts are discussed.     

Behavioural changes induced by the conjugation-inducing pheromones,gamone 1 and 2, in the ciliate Blepharisma japonicum

Preconjugant interactions between complementary mating-type cells in ciliates occur before sexual reproduction. The interactions include retardation of swimming behaviour, courtship dancing, chemoattraction, nuclear activation, cell division, or cell agglutination, depending on ciliate species. In Blepharisma japonicum, chemoattraction of mating-type I by mating-type II has been reported previously. It has been shown that chemoattraction here is caused by a conjugation-inducing substance called gamone 2 secreted by mating-type II cells. In this study, we show that mating-type II cells accumulate near the site where gamone 1 secreted by mating-type I cells is present at a high concentration. We also show that the behaviour of individual cells changes when exposed to the complementary mating-type gamone; cells begin to rotate and swim slowly, thus shortening their minimum path length (final displacement of a cell from its origin). These results suggest that gamones 1 and 2 induce behavioural changes in type II and I cells, respectively, and that gamone-stimulated cells may accumulate at the site with the highest activity of the complementary gamone, after repetition of swimming changes in the gradient of gamone concentration. This reciprocal induction of the changes in behaviour may increase the probability of sexual encounters for conjugation.     

Observations on the Fine Structure of the Nuclear Apparatus of Blepharisma intermedium Bhandary (Ciliata: Spirotricha)

SYNOPSIS. An electron-microscope study of the macronucleus and the micronucleus of Blepharisma intermedium Bhandary has been made. Sections show that the macronucleus is bounded by a double membrane. Inside, there are two types of bodies: (a) small irregular bodies, from 0.05 to 0.2 μ in diameter, and (b) larger bodies, from 0.4 to 0.6 μ in diameter. The former are intrepreted as cut ends of long, branching filaments traversing the nuclear cavity in all directions. They correspond to the DNA filaments obtained by centrifugation and KCN action on the macronucleus. Each filament is made of fibrils aboue 150 Å thick. The large bodies correspond to the nucleoli; they also show a fibrillar structure. They offer the added interest of displaying dense particles, from 100 to 800 Å in size, whose nature and significance are obscure. The micro-nucleus has a double membrane, and the contents are divisible into an electron-dense network and a material of low density which fills the interstices of the network.     

A thermal denaturation study of macronuclear chromatin in Blepharisma japonicum (Protozoa, Ciliophora, Heterotrichida)

The macronuclear chromatin of the ciliate Blepharisma japonicum, in two starvation states, was studied by thermal denaturation analysis. The behaviour of B. japonicum chromatin, native and reconstituted with Tetrahymena pyriformis H1 histone, was analysed. The data obtained are consistent with the hypothesis that B. japonicum macronuclear chromatin contains a H1-like peptide associated with the linker DNA, although this peptide is reduced in amount and/or chromatin stabilising ability when compared to Tetrahymena macronuclear H1.     

ATP and the autonomy of the contractile vacuole in Amoeba proteus

Contractile vacuole function in amoebae treated with immobilizing (5 mM) and nonimmobilizing (0.125 mM) concentrations of ATP has been studied. In ATP-immobilized amoebae, most vacuolar parameters are accelerated, especially the rate of output which passes from 30 to 70 micron3/sec. This favors the concept of an autonomous vacuole, fully functional in the absence of any bulk contribution to it from remote points of the cell. A lower concentration of ATP (0.125 mM), which does not inhibit movement, causes a still greater acceleration of vacuolar function. Work is in progress to elucidate the site and mode of action of exogenous ATP on Amoeba.     

Inducible defence against a ciliate grazer Pseudomicrothorax dubius,in two strains of Phormidium (cyanobacteria)

Experiments were done with two strain of filamentous, mat-forming Phormidium and their ciliate grazer Pseudomicrothorax dubius, to explain why the ciliates remain hungry in an apparent surplus of food, except for the first 24 hours after feeding. Under grazing pressure, both strains of cyanobacteria showed statistically significant increases in the number of filaments terminating in an empty sheath, compared to the control. Direct observations revealed that the mechanism behind this effect was active withdrawal of the trichomes inside the sheaths when disturbed by grazers. As P. dubius is unable to ingest trichomes with such endings, we conclude that cyanobacteria are not limited to chemical means of defence against grazers but can also defend themselves by means of movement and changes in filament morphology. This is apparently the first report on behavioural defence observed in cyanobacteria.     

Microtubules, Microfilaments, and Membranes in Phagocytosis: Structure and Function of the Oral Apparatus of the Ciliate Climacostomum virens

Climacostomum virens uses oral membranelles to drive suspended food particles into its buccal cavity. The cavity leads to a buccal tube which extends into the cell by as much as half a cell length. The inner end of this tube is delimited by a haplokinety (two rows of basal bodies). Internal to this zone is the cytostome and cytopharynx where food vacuoles form. The buccal tube is encircled by a ring of fibrous material, the cytostomal cord, in the region of the cytostome immediately below the haplokinety. Ribbons of postciliary microtubules extend from the kinetosomes of the haplokinety, attach to the cytopharyngeal membrane, and pass under the cytostomal cord. They become broader and expand into the cytoplasm. Cytopharyngeal vesicles pass between the microtubular ribbons and fuse with the cytopharyngeal membrane to generate membrane for forming food vacuoles. The cytopharyngeal vesicles contain a material which is secreted into the forming food vacuoles. Ciliates continue to feed after incubation in a medium containing cycloheximide, indicating that they draw on a pre-existing pool of membrane when forming the food vacuole.     

A Redescription of Climacostomum virens (Ehrenberg) Stein and Proposal of a New Heterotrich Ciliate Family, Climacostomidae fam. n.

SYNOPSIS. Structure of the vegetative and asexually dividing forms of the large ciliate Climacostomum virens is redescribed with emphasis on stomatogenesis. These findings are discussed in relation to the taxonomic and possible evolutionary position of Climacostomum among heterotrichous ciliates. Comparative considerations of the buccal and somatic structures as well as of the stomatogenic patterns in this and other closely related ciliate genera indicate the need for placing Climacostomum and Fabrea in a new family, CLIMACOSTOMIDAE . The morphologic evidence suggests that Climacostomum may represent a line linking Fabrea and Stentor.     

Sucrose uptake by pinocytosis in Amoeba proteus and the influence of external calcium

The relationship between Ca++ and pinocytosis was investigated in Amoeba proteus. Pinocytosis was induced with 0.01% alcian blue, a large molecular weight dye which binds irreversibly to the cell surface. The time-course and intensity of pinocytosis was monitored by following the uptake of [3H]SUCROSE. When the cells are exposed to 0.01% alcian blue, there is an immediate uptake of sucrose. The cells take up integral of 10% of their initial volume during the time-course of pinocytosis. The duration of pinocytosis in the amoeba is integral of 50 min, with maximum sucrose uptake occurring 15 min after the induction of pinocytosis. The pinocytotic uptake of sucrose is reversibly blocked at 3 degrees C and a decrease in pH increases the uptake of sucrose by pinocytosis. The process of pinocytosis is also dependent upon the concentration of the inducer in the external medium. The association between Ca++ and pinocytosis in A. proteus was investigated initially by determining the effect of the external Ca++ concentration on sucrose uptake induced by alcian blue. In Ca++-free medium, no sucrose uptake is observed in the presence of 0.01% alcian blue. As the Ca++ concentration is increased, up to a maximum of 0.1 mM, pinocytotic sucrose uptake is also increased. Increases in the external Ca++ concentration above 0.1 mM brings about a decrease in sucrose uptake. Further investigations into the association between Ca++ and pinocytosis demonstrated that the inducer of pinocytosis displaces surface calcium in the amoeba. It is suggested that Ca++ is involved in two separate stages in the process of pinocytosis; an initial displacement of surface calcium by the inducer which may increase the permeability of the membrane to solutes and a subsequent Ca++ influx bringing about localized increases in cytoplasmic Ca++ ion activity.