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At least 199 records · Page 11Linked to original sources
PubMed1983

Calcium/calmodulin-regulated guanylate cyclase of the excitable ciliary membrane from Paramecium. Dissociation of calmodulin by La3+: calmodulin specificity and properties of the reconstituted guanylate cyclase.

Ca2+-regulated guanylate cyclase in ciliary membranes from Paramecium contained tightly bound calmodulin. Antisera against calmodulin from Tetrahymena and soybean inhibited enzyme activity. EGTA did not easily release calmodulin; however, La3+ inhibited guanylate cyclase by dissociation of calmodulin. While La could not replace Ca in the activation of guanylate cyclase, it substituted for Ca2+ in the activation of calmodulin-dependent phosphodiesterase from pig brain independently of whether homologous or Paramecium calmodulin was used. After removal of endogenous calmodulin from guanylate cyclase, reconstitution was achieved with calmodulin from Paramecium, Tetrahymena, pig brain, and soybean. Ca2+-binding proteins lacking trimethyllysine like calmodulin from Dictyostelium, parvalbumin, and troponin C failed to restore enzyme activity. The properties of the native and reconstituted guanylate cyclase/calmodulin complex were compared. Reassociation of calmodulin with its target enzyme was weak since all calmodulin remained in the supernatant after a single centrifugation. While most enzyme characteristics remained unchanged in the reconstituted complex, the inhibition by Ca greater than 100 microM was of a mixed-type compared to noncompetitive inhibition in the native enzyme. The regulation of the enzyme by cations was also altered. Whereas Ca was the most potent and specific activator of the native enzyme, in the reconstituted system Sr was far more effective.

Animals↗
PubMed1983

Immunocytochemical localization of cyclic GMP, cGMP-dependent protein kinase, calmodulin and calcineurin in Paramecium tetraurelia.

The localization of cGMP, cGMP-dependent protein kinase, calmodulin and the calmodulin-binding protein calcineurin in Paramecium tetrauelia cells has been examined with immunocytochemical methods. These molecules appeared to be localized to a large extent in the cilia of this protozoan. To ascertain that antibodies had access to all cellular compartments we have used three different preparations for immunocytochemistry: (i) with 'whole cell' preparations immunofluorescent staining for the four molecules was mainly visible in the cilia; (ii) in 'deciliated' Paramecium, staining for cGMP and calmodulin was found in regular patterns on the cell surface most likely representing kinetosomes; (iii) using 'sectioned cells', additional cytoplasmic calmodulin appeared to be associated with glycogen particles as evidenced by the disappearance of the granular staining pattern after preincubation with alpha-amylase. In contrast, cGMP, cGMP-dependent protein kinase and calcineurin fluorescence was only very weak and diffuse in cell bodies. No nuclear fluorescence was detectable after staining with any of the antibodies. Because of the colocalization of cGMP, cGMP-dependent protein kinase, a guanylate cyclase-calmodulin-complex, and calcineurin in cilia from Paramecium, an involvement of these components in the regulation of ciliary activity is discussed.

Animals↗
PubMed1982

Participation of algal surface structures in the cell recognition process during infection of aposymbiotic Paramecium bursaria with symbiotic chlorellae.

The endosymbiotic unit green Paramecium shows a strong specificity of its partners. The aposymbiotic Paramecium bursaria forms a stable symbiotic unit only with a special strain of Chlorella sp. Algae suitable for symbiosis formation are enclosed in individual perialgal vacuoles whereas unsuitable algae are sequestered into food vacuoles. It is probable that algae are recognized by the ciliate because of specific surface structures rather than by their physiological properties. Experiments with synchronized algae demonstrate that autospores are taken up into perialgal vacuoles to a higher degree than mother cells, which have a different surface structure as shown by immunological techniques. Symbiotic algae treated with cellulase and pectinase or having been coated with specific antibodies or with lectins (concanavalin A or Ricinus communis agglutinin) are usually not recognized as suitable and are mostly sequestered into food vacuoles although they show the same physiological properties as untreated algae. These results indicate the participation of carbohydrate structures at the recognition sites of symbiotic chlorellae in Paramecium bursaria which interact during infection with special receptor molecules in the membrane of the ingestion vacuole of the ciliate.

Animals↗
PubMed1975

Symbiosis in Paramecium Bursaria.

Paramecium bursaria normally appears green dut to several hundred symbiotic Chlorella which are dispersed throughout its cytoplasm. The symbionts are situated within individual vacuoles and these alga-vacuole complexes grow and divide at a rate compatible with that of the paramecium. The symbiotic units also persist through conjugation and the subsequent reorganization of the host. Studies of the benefit of the symbiosis to the ciliate hosts have shown that they are able to grow and survive better than aposymbiotic animals in environments deficient in bacteria. The symbionts are also able to extract nourishment from the host when it is well fed and they are deprived of light. The biochemical nature of these exchanges has not been determined. Potential symbionts usually enter the host in food vacuoles. If they are ingested in sufficient numbers, they are able to interfere with the normal course of host digestion, perhaps by preventing the release of digestive enzymes into the food vacuole. All natural symbionts of P. bursaria appear able to reinfect aposymbiotic cells. Some freeliving strains of Chlorella and related algae are also infective, but these associations are relatively unstable and provide little evident benefit to the host. Host susceptibility to infection by certain strains of free-living algae is invariably lost with time. This loss is specific and often rapid, but it does not occur simultaneously in subcultures derived from the original susceptible culture. The basis for these susceptibility changes is still unknown, but they may be related to long-lasting effect of the previous symbionts on the digestive efficiency of the paramecium host.

Bacteria↗
PubMed1994

Temperature-dependent expression of ciliary GPI-proteins in Paramecium.

Many eukaryotic membrane proteins have now been found to be anchored to the plasma membrane via a glycosylphosphatidylinositol membrane anchor (GPI-anchor). In Paramecium aurelia, a free-living ciliated protozoan, the major membrane protein, the surface antigen (SAg), is a GPI-anchored protein. This surface protein belongs to a multigene family, the expression and antigenic variation of which is controlled by environmental conditions. In order to screen for other Paramecium GPI-anchored proteins to identify those whose expression is also variable and influenced by external factors, we established a protocol permitting the rapid identification of GPI-proteins. The protocol is based upon the property of bacterial PI-PLCs to specifically release GPI-proteins. To overcome the resistance displayed by living paramecia to exogenous PI-PLCs, we used cilia purified from 35S-labeled cells obtained from various geographical strains grown under similar or different conditions. We observed a temperature-dependent variation in the electrophoretic patterns, as revealed by autoradiography of ciliary PI-PLC-releasable proteins from strain 513 of Paramecium primaurelia. In addition to a high molecular mass band corresponding to SAg molecules, three bands varying in apparent molecular mass from 30 to 50 kDa were observed at 23 degrees C. At 32 degrees C only one band of about 45 kDa was observed. Biosynthetic labeling experiments and the detection of the cross-reacting determinant after PI-PLC treatment (results reported elsewhere) provided definitive proof that these ciliary PI-PLC releasable proteins were actually GPI-proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1997

Differential distribution of calcium stores in paramecium cells. Occurrence of a subplasmalemmal store with a calsequestrin-like protein.

We have analyzed in Paramecium cells the occurrence and intracellular distribution of the high capacity/low affinity calcium-binding proteins, calsequestrin (CS) and calreticulin (CR) using antibodies against CS from rat skeletal muscle and against CR from rat liver, respectively. As revealed by Western blots, a CS-like protein isolated by affinity chromatography from Paramecium cells comigrated with CS isolated from rat skeletal muscle. The immunoreactivity of this 53 kDa protein band was blocked when the antibodies had been preadsorbed with purified rat CS. A band of identical molecular size was shown to bind 45Ca in overlays. By immunofluorescence and immunogold labeling this CS-like protein was localized selectively to the extended subplasmalemmal calcium stores, the "alveolar sacs", which cover almost the entire cell surface. Concomitantly the 53 kDa 45Ca-binding band became increasingly intense in overlays as we increasingly enriched alveolar sacs. Antibodies against rat CR react with a 61 kDa band but do not cross-react with CS-like protein in Paramecium. These antibodies selectively stained intracellular reticular structures, identified bona fide as endoplasmic reticulum.

Animals↗
PubMed2001

Growth kinetics of algal populations exsymbiotic from Paramecium bursaria by flow cytometry measurements.

BACKGROUND: The ciliate Paramecium bursaria normally exists as a green paramecium system because each animal cell carries several hundred, unicellular, green, algal cells in its cytoplasm. One of the remarkable and poorly understood pecularities of this system is the steady state in the number of algae per protozoan cell. A major point in the study of mechanisms governing the persistence of symbiont numbers is adequate understanding of the algal life cycle. METHODS: Asynchronously growing cell populations of several algal strains (SA-1, SA-3, and SA-9) exsymbiotic from P. bursaria were characterized by flow cytometry. Algal endogenous chlorophyll and DNA contents were monitored to analyze cell growth kinetics at logarithmic and stationary culture phases. Cell sorting visualized the morphology of algae corresponding to the hyperhaploid (2C and 4C) DNA peaks. RESULTS: Cell-division cycle-dependent changes in chlorophyll and DNA content distributions were most dramatic in logarithmically growing algal populations (an increase in the number of S-phase cells and cells with more chlorophyll), which are thought to be associated with accelerated DNA and chlorophyll metabolism in log-phase algal cultures. Upon reaching the stationary phase of growth, algal populations distinctly showed, in addition to one haploid (1C) DNA peak, two hyperhaploid peaks (2C and 4C) corresponding mainly to cells with two and four nuclei, respectively. CONCLUSIONS: Growth characteristics of algae exsymbiotic from P. bursaria monitored by flow cytometry provide valuable information for the analysis of the algal life cycle, which is important for understanding the regulation mechanisms of symbiont numbers.

Animals↗
PubMed2000

Effect of a 60 Hz magnetic field on the behavior of Paramecium.

Paramecium multimicronucleatum was used as a model cell to study the effects of 60 Hz magnetic fields on swimming behavior. When exposed to a vertical field of 0.6 T, the cells accumulated at the upper end of the cuvette. An analysis of the swimming behavior revealed that the exposure to the field increased the number of cells swimming upwards maximally at 1 min after onset of the exposure. This effect of the magnetic field was transient, disappearing within a few minutes during the exposure. It is suggested that the magnetic field may amplify to a large extent the negative gravitaxis of Paramecium. Effects of an induced electric field on the swimming behavior are also discussed.

Animals↗
PubMed1985

Effects of an extremely low frequency electromagnetic field on the cell division rate and plasma membrane of Paramecium tetraurelia.

The eukaryotic protozoan, Paramecium, was examined as a model for effects of pulsated electromagnetic fields (PEMF) on cells. A 72-Hz PEMF similar to fields employed clinically increased cell division rates in Paramecium by 8.5%. Two calcium transport mutants of these organisms showed differential responses to the same field. Verapamil, a calcium channel blocker, abolished any effect of PEMFs on cell division rates. A fluorescent probe that is thought to sense changes in membrane potential also manifested an altered response in the PEMF-exposed cells whereas a fluorescent lipid bilayer fluidity probe produced evidence of decreased membrane fluidity in the exposed cells. An effect of PEMFs on ion transport mediated by either a direct or indirect effect on the cell membrane is suggested by these studies.

Animals↗
PubMed2002

My favorite cell--Paramecium.

A Paramecium cell has a stereotypically patterned surface, with regularly arranged cilia, dense-core secretory vesicles and subplasmalemmal calcium stores. Less strikingly, there is also a patterning of molecules; for instance, some ion channels are restricted to certain regions of the cell surface. This design may explain very effective and selective responses, such as that to Ca(2+) upon stimulation. It enables the cell to respond to a Ca(2+) signal precisely secretion (exocytosis) or by changing its ciliary activity. These responses depend on the location and/or type of signal, even though these two target structures co-exist side-by-side, and normally only limited overlap occurs between the different functions. Furthermore, the patterning of exocytotic sites and the possibility of synchronous exocytosis induction in the sub-second time range have considerably facilitated analyses, and thus led to new concepts of exocytotic membrane fusion. It has been possible to dissect complicated events like overlapping Ca(2+) fluxes produced from external sources and from internal stores. Since molecular genetic approaches have become available for Paramecium, many different gene products have been identified only some of which are known from "higher" eukaryotes. Although a variety of basic cellular functions are briefly addressed to demonstrate the uniqueness of this unicellular organism, this article focuses on exocytosis regulation.

Animals↗
PubMed1990

Genetic dissection of Ca2(+)-dependent ion channel function in Paramecium.

The ciliated protozoan, Paramecium, broadcasts the activity of its individual ion channel classes through its swimming behaviour. This fact has made it possible to isolate mutants with defective ion currents, simply by selecting individuals with abnormal swimming patterns. At least four of Paramecium's ion currents are activated by rising intracellular calcium concentration, including two K+ currents and a Na+ current. A variety of cell lines with defects in these Ca2(+)-dependent currents have been isolated: in several cases, the defects have been traced to mutations in the structural gene for calmodulin. Sequence analysis of calmodulins from these and other Ca2(+)-dependent ion-current mutants may enable a detailed mapping of putative channel interaction domains on the surface of the calmodulin molecule.

Amino Acid Sequence↗
PubMed1984

Trifluoperazine-induced changes in swimming behavior of paramecium: evidence for two sites of drug action.

Trifluoperazine (TFP), a drug that binds to Ca2+-calmodulin (CaM) complexes, altered swimming behavior not only in living paramecia, but also in reactivated, Triton-extracted "models" of the ciliate. By comparing the responses of living cells and models, we have ascertained that two sites of drug action exist in paramecium cilia. Swimming movements were recorded in darkfield stroboscopic flash photomicrographs; this permitted accurate quantitation of velocities and body-shape parameters. When living paramecia were incubated in a standard buffer containing 10 microM TFP, their speed of forward swimming fell over several minutes and their bodies shortened. Untreated paramecia backed up repeatedly and frequently upon transfer to a solution containing barium ions (the "barium dance"), but cells preincubated in TFP did not "dance." Instead they swam forward slowly for long periods of time without reversing and occasionally then exhibited abnormally prolonged reversals. W7 effects on swimming mimicked low doses of TFP, and the analog W5 did not visibly alter normal swimming patterns. These results suggest that TFP induces a decrease in the intracellular pCa of living paramecia, perhaps by reducing the efficiency of a calmodulin-activated calcium pump in the cell membrane. Paramecia extracted with Triton X-100 and reactivated to swim forward (7 greater than or equal to pCa greater than or equal to 6) were not affected by addition of up to 40 microM TFP to the reactivation medium. We conclude that the main drug effect in living cells is probably not at the axoneme. However, at low pCa, TFP directly affected the ciliary axoneme to shift its behavior to one characteristic of a higher pCa: TFP inhibited backward swimming in models reactivated at pCa less than 6; instead they swam forward or rocked in place. The mechanism of ciliary reversal in paramecium may therefore depend on an axonemal Ca2+-sensor, possibly bound CaM, which is affected by TFP only at low pCa, as has been postulated for other types of cilia.

Animals↗
PubMed1984

Actin microfilaments in paramecium: localization and role in intracellular movements.

Using heavy meromyosin (HMM) or the fragment S1 of myosin as probes for actin microfilaments, we studied their organization in Paramecium both by fluorescence and electron microscopy. In interphasic cells, HMM decorates (a) most prominently the periphery of nascent and young food vacuoles and their route during the early phase of their intracellular transit; (b) a thin meshwork radiating from the gullet throughout the cytoplasm; (c) a small area beneath the pore of contractile vacuoles and beneath the cytoproct when open to release food residues. Most of these HMM-decorated structures are in close contact with microtubular arrays. All HMM decoration disappears in dividing cells and in cytochalasin-treated cells. In vivo, the drug immediately blocks food vacuole formation but does not affect cytokinesis, cyclosis, contractile vacuole pulsation, defecation, or nuclear movements. The data show that, as in the cells of other organisms, actin microfilaments form defined arrays that undergo physiologically controlled cycles of assembly/disassembly. These arrays contribute (at least in the phagocytotic process) to diverse types of movement: constriction, membrane fusion, and migration of food vacuoles. However, aside from their massive concentration along the phagocytotic tractus, actin microfilaments are neither major structural components of Paramecium cytoplasm nor the only cytoskeletal components ensuring motility or contractility processes.

Actins↗
PubMed1986

Regulation of ciliary motility by membrane potential in Paramecium: a role for cyclic AMP.

The membrane potential of Paramecium controls the frequency and direction of the ciliary beat, thus determining the cell's swimming behavior. Stimuli that hyperpolarize the membrane potential increase the ciliary beat frequency and therefore increase forward swimming speed. We have observed that 1) drugs that elevate intracellular cyclic AMP increased swimming speed 2-3-fold, 2) hyperpolarizing the membrane potential by manipulation of extracellular cations (e.g., K+) induced both a transient increase in, and a higher sustained level of cyclic AMP compared to the control, and 3) the swimming speed of detergent-permeabilized cells in MgATP was stimulated 2-fold by the addition of cyclic AMP. Our results suggest that the membrane potential can regulate intracellular cAMP in Paramecium and that control of swimming speed by membrane potential may in part be mediated by cAMP.

1-Methyl-3-isobutylxanthine↗
PubMed1989

In vitro phosphorylation of Paramecium axonemes and permeabilized cells.

This study seeks to identify phosphoproteins in axonemes from Paramecium tetraurelia whose phosphorylation responses to adenosine 3', 5'-cyclic monophosphate (cAMP) and Ca2+ parallel responses induced by these agents in ciliary behavior in this cell. In purified axonemes, over 15 bands ranging from Mr greater than 300 kDa to 19 kDa on SDS-PAGE incorporate 32P from adenosine 5'-gamma-[32P]triphosphate (gamma-32P-ATP) at pCa 7 in the absence of cAMP. A major band whose label turns over rapidly was identified at Mr 43 kDa. In the presence of 5 microM cAMP, more than eight bands, but not the Mr 43 kDa band, were labeled additionally or enhanced their labeling. These phosphoproteins and their kinases are structural components of the axoneme. Overall, some of the same major bands are labeled in the presence of cAMP in Triton X-100-permeabilized paramecia that retain their behavioral responses and in axonemes mechanically isolated from these cells. In particular, two major bands have been identified whose phosphorylation is greatly enhanced by cAMP at low concentrations: 1) a 29 kDa polypeptide whose cAMP-dependent phosphorylation is diminished at pCa 4 compared with pCa 7 and 2) a 65 kDa polypeptide whose phosphorylation is pCa insensitive. These polypeptides meet minimal criteria for signal-sensitive regulators of motility parameters in the Paramecium axoneme.

Animals↗
PubMed1993

Ciliary dynein of Paramecium tetraurelia: photolytic maps of the three heavy chains.

The ciliate Paramecium tetraurelia presents a powerful system to define the structural basis for dynein functional diversity within a single cell. This analysis will depend on the biochemical resolution of the dynein proteins. As an important first step, the three heavy chains of the ciliary outer arm dynein of paramecium were characterized. Sucrose density gradient centrifugation in a high salt buffer separated the dynein into a 22S species, which contained the alpha and beta heavy chains, and a 12S species, which contained the gamma chain as well as the inner arm dynein heavy chains. Both the 22S and 12S species retained enzymatic latency as indicated by stimulation of MgATPase activity by 0.1% Triton X-100. An unusual ATP-independent V1-like photolysis of only the beta chain provided the basis for estimating that the beta chain contributes almost half of the 22S MgATPase activity that is susceptible to V1 photolysis. The combination of the density gradient separation of the partially dissociated dynein and the ATP-independent V1-like photolysis of only the beta chain led to the unambiguous assignment of the V1 photolytic products to the appropriate parent heavy chains. An estimate of the molecular sizes of the three heavy chains was obtained. The photolytic peptide maps, which define the ATP-binding domains, were determined for the three heavy chains.

Amino Acid Sequence↗
PubMed1995

Protein substrates for cGMP-dependent protein phosphorylation in cilia of wild type and atalanta mutants of Paramecium.

In the ciliated protozoan Paramecium, swimming direction is regulated by voltage-gated Ca2+ channels in the ciliary membrane. In response to depolarizing stimuli, intraciliary Ca2+ rises, triggering reversal of the ciliary power stroke and backward swimming. One class of Ca(2+)-unresponsive behavioral mutants of Paramecium, atalanta mutants, cannot swim backward even though they have functional Ca2+ channels in their ciliary membrane. Several atalanta mutants were characterized with regard to several Ca(2+)-dependent activities, but no significant difference between wild type and the mutants was detected. However, one allelic group, atalanta A (initially characterized by Hinrichsen and Kung [1984: Genet. Res. Camb. 43:11-20]), showed a helical swimming path of opposite handedness from that of wild-type cells when detergent-permeabilized cells ("models") were reactivated with MgATP. When cGMP-dependent protein kinase purified from wild-type cells was added to atalanta A models, the handedness of the swimming path was reversed. Cyclic GMP stimulated in vitro phosphorylation of several proteins in isolated cilia, and the pattern of phosphoproteins was very similar for wild type and atalanta mutants, with one exception: a protein of 59 kDa was phosphorylated much less in the mutant ata A. When ciliary proteins were separated by gel electrophoresis and then phosphorylated "on blot" by purified cGMP-dependent protein kinase, phosphoprotein patterns were similar in wild type and ata mutants except that a 48 kDa protein (p48) from ata A3 was more heavily phosphorylated. This difference in p48 phosphorylation was also observed with cGMP-dependent protein kinase purified from ata A3 mutant cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Bacteriophage lambda DNA fragments replicate in the Paramecium macronucleus: absence of active copy number control.

We show that bacteriophage lambda DNA fragments microinjected into the macronucleus of the ciliated protozoan Paramecium can replicate as unit-length linear molecules. These linear DNA molecules are substrates for the addition of Paramecium telomeres by an endogenous telomerase. The linear DNA pieces can exist at copy numbers much higher than that of typical endogenous macronuclear chromosomes. We show that the copy number of injected DNA many fissions after microinjection reflects that of the original input copy number, suggesting that active control of copy number does not occur. Instead, the results suggest that injected DNA is replicated once per cell division.

Animals↗