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PubMed1991

Photoadaptation Alters the Ingestion Rate of Paramecium bursaria, a Mixotrophic Ciliate.

Bacteriovorous protozoa harboring symbiotic algae are abundant in aquatic ecosystems, yet despite a recent interest in protozoan bacterivory, the influence of light on their ingestion rates has not been investigated. In this study, Paramecium bursaria containing endosymbiotic Chlorella was tested for the effect of light on its ingestion rate. P. bursaria was grown for 4 to 6 days under five different light fluxes ranging from 1 to 90 microeinsteins s m. Ingestion rates were determined by using 0.77-mum-diameter fluorescent microspheres. 4',6-Diamidino-2-phenylindole dihydrochloride-labeled Enterobacter cloacae was used in one experiment to confirm differences in uptake rates of bacteria by P. bursaria. Unlike phagotrophic phytoflagellates, the ciliates demonstrated different ingestion rates in response to different light intensities. Although symbionts contribute carbon to their host via photosynthesis, the paramecia of the present study fed faster after exposure to higher light intensities, whereas their aposymbiotic counterparts (lacking endosymbionts) were unaffected. Light-induced changes in ingestion rates were not immediate, but corresponded to the period of time required for endosymbiont populations to change significantly. This strongly suggests that the symbionts, stimulated by higher light levels, may dictate the feeding rates of their hosts. Thus, light, apart from temperature, may influence the impact of certain protists on natural bacteria and may affect laboratory-based determinations of protistan feeding rates.

Journal Article↗
PubMed2001

RNA triphosphatase component of the mRNA capping apparatus of Paramecium bursaria Chlorella virus 1.

Paramecium bursaria chlorella virus 1 (PBCV-1) elicits a lytic infection of its unicellular green alga host. The 330-kbp viral genome has been sequenced, yet little is known about how viral mRNAs are synthesized and processed. PBCV-1 encodes its own mRNA guanylyltransferase, which catalyzes the addition of GMP to the 5' diphosphate end of RNA to form a GpppN cap structure. Here we report that PBCV-1 encodes a separate RNA triphosphatase (RTP) that catalyzes the initial step in cap synthesis: hydrolysis of the gamma-phosphate of triphosphate-terminated RNA to generate an RNA diphosphate end. We exploit a yeast-based genetic system to show that Chlorella virus RTP can function as a cap-forming enzyme in vivo. The 193-amino-acid Chlorella virus RTP is the smallest member of a family of metal-dependent phosphohydrolases that includes the RNA triphosphatases of fungi and other large eukaryotic DNA viruses (poxviruses, African swine fever virus, and baculoviruses). Chlorella virus RTP is more similar in structure to the yeast RNA triphosphatases than to the enzymes of metazoan DNA viruses. Indeed, PBCV-1 is unique among DNA viruses in that the triphosphatase and guanylyltransferase steps of cap formation are catalyzed by separate viral enzymes instead of a single viral polypeptide with multiple catalytic domains.

Acid Anhydride Hydrolases↗
PubMed1994

COMPARISON BETWEEN THERMORECEPTOR AND MECHANORECEPTOR CURRENTS IN PARAMECIUM CAUDATUM

1. A voltage-clamped Paramecium produced an inward membrane current upon thermal or mechanical stimulation of its anterior region, whereas it produced an outward membrane current upon similar stimulation of its posterior region. 2. Anterior thermo- and mechanoreceptor currents decreased when the membrane potential was shifted in a positive direction, showing sign reversal at a positive membrane potential, whereas posterior thermo- and mechanoreceptor currents decreased when the membrane potential was shifted in a negative direction, showing sign reversal at a membrane potential more negative than the resting potential. 3. The reversal potential for both anterior receptor currents shifted in a positive direction when external [Ca2+] was increased, whereas those for both posterior receptor currents shifted in a positive direction when external [K+] was increased. 4. External [Mg2+], [Mn2+], [Na+], [Rb+] and [TEA+] had similar effects on the thermo- and mechanoreceptor currents. 5. Thermoreceptor currents decreased whereas mechanoreceptor currents increased as the ambient temperature was raised. 6. When a mechanical stimulus was applied to the membrane where a thermoreceptor current was being produced, an algebraic summation of these receptor currents occurred. 7. It is concluded that thermoreceptor currents are dependent on ion channels different from those responsible for the mechanoreceptor currents, although the ionic pores for the channels are similar to each other in various respects. 8. A possibility that a thermoreceptor mechanism exclusively shares a Ca2+ pore in the anterior membrane, or a K+ pore in the posterior membrane, with a mechanoreceptor mechanism is discussed.

Journal Article↗
PubMed1994

RECONSTITUTION OF METACHRONAL WAVES IN CILIATED CORTICAL SHEETS OF PARAMECIUM - WAVE STABILITIES

We reconstituted metachronal waves on ciliated cortical sheets prepared from detergent-extracted Paramecium multimicronucleatum cells. Ciliary movements of the cortical sheet, whose intracellular side adhered to a glass coverslip, were reactivated by perfusion of a basic reactivation medium containing ATP. In this condition, the ciliary field showed only unstable localised ripples. Addition of either cyclic AMP or cyclic GMP to the basic reactivation medium generated propagating metachronal waves characteristic of each nucleotide. In order to estimate the stability of the metachronal waves, autocorrelation coefficients were calculated from images of an 8 µm diameter region within the reactivated ciliary field. The decay time for the correlation coefficient to decrease to 0.5 was only 0.04 s in the basic reactivation medium, but was increased to 0.4 or 0.9 s by the addition of cyclic AMP or cyclic GMP, respectively. The decay time was dependent not only on the concentration of cyclic nucleotide but also on the wave frequency. In order to test whether cyclic-nucleotide-dependent phosphorylation affected the generation of waves, the ciliated cortical sheets were thiophosphorylated by incubation in ATP-gamma-S (adenosine-5'-o-3-thiotriphosphate) medium containing either cyclic AMP or cyclic GMP. Following this, perfusion with the basic reactivation medium generated metachronal waves only after cyclic GMP treatment. The effect of cyclic GMP is probably related to phosphorylation of ciliary proteins.

Journal Article↗
PubMed1994

RECONSTITUTION OF METACHRONAL WAVES IN CILIATED CORTICAL SHEETS OF PARAMECIUM - ASYMMETRY OF THE CILIARY MOVEMENTS

In conditions in which ciliated cortical sheets prepared from detergent-extracted Paramecium multimicronucleatum cells adhered to glass coverslips on a microscope stage, perfusion of a reactivation medium containing ATP plus cyclic AMP or cyclic GMP generated metachronal waves. An analysis of the ciliary movements that generate these metachronal waves yielded the following results. During the generation of metachronal waves, there were phase differences in the ciliary orientation of adjacent cilia in the direction of wave propagation. Addition of cyclic AMP or cyclic GMP increased the rotational angular velocities during the effective stroke of ciliary beating, but did not increase the rotational angular velocity of the recovery stroke. When the ATP concentration in the cyclic GMP reactivation medium was increased, the rotational angular velocity during the effective stroke rose steeply and saturated at 0.8 mmol l-1 ATP, whereas that during the recovery stroke rose gradually. Addition of cyclic nucleotides caused a single cilium isolated from neighbouring cilia on the cortical sheet to incline almost parallel to the cortical surface during the recovery stroke. Addition of cyclic GMP increased the amplitude of bending of cilia detached from the cortical sheet. From these results, it was concluded that increases in the asymmetrical movement of individual cilia, caused by the addition of cyclic nucleotides, create the ciliary interaction that generates the metachronal waves.

Journal Article↗
PubMed1994

GRAVIRESPONSES IN PARAMECIUM CAUDATUM AND DIDINIUM NASUTUM EXAMINED UNDER VARIED HYPERGRAVITY CONDITIONS

The swimming behaviours of two species of ciliates characterized by different mechanosensory and ciliary motor properties were investigated under hypergravity up to 5.4 g. The experiments were designed to examine large numbers of cells using video recording, digital data processing and statistics for the documentation of the rates and orientations of swimming. The gravikinetic responses (change in active swimming rates) were calculated from (1) the velocities of vertical swimming in the gravity field, (2) sedimentation of Ni2+-immobilized cells and (3) the intrinsic rate of propulsion, independent of gravity. Propulsion was determined from the intersection of regression lines of the gravity-dependent upward and downward swimming velocities. The rates of swimming and sedimentation, and consequently the gravikineses, were linear functions of gravitational acceleration. Comparisons of cell populations from different cultures suggest that there is an age-dependent change in gravikinesis. In starved Paramecium caudatum (7-day cultures), the kinetic responses antagonizing sedimentation (negative gravikinesis) increased with acceleration. In Didinium nasutum, negative gravikinesis was documented at 1 g in downward-swimming specimens only, which agrees with the mechanosensory organization of this cell. Hypergravity induced the gravikinesis of Didinium to change sign. In both species, and at all accelerations tested, a neutral gravitaxis was documented. Such behaviour incorporates distinct acceleration-dependent orientational and velocity responses, keeping populations of cells stationary in the gravity field (taxis coefficients close to zero).

Journal Article↗
PubMed1997

The contractile vacuole fluid discharge rate is determined by the vacuole size immediately before the start of discharge in Paramecium multimicronucleatum

The precise relationship between the rate of contractile vacuole fluid discharge and the vacuole diameter at the start of systole was determined in cells of Paramecium multimicronucleatum subjected to various external conditions. The rate of discharge was higher when the diameter was larger. When the rate of discharge was plotted against the diameter, the points fell around a single parabolic line passing through the origin and were independent of the external conditions employed. This implies that the rate of discharge is proportional to the square of the vacuole diameter. We have previously proposed a hypothesis in which membrane tension in the contractile vacuole is altered as its planar membrane becomes tubular or as tubules become planar membrane (termed the membrane area-proportional tension hypothesis). We propose here that it is this change in membrane tension which determines the vacuole pore shape and sets the subsequent rate of fluid discharge.

Journal Article↗
PubMed1997

How does the contractile vacuole of Paramecium multimicronucleatum expel fluid? Modelling the expulsion mechanism

To examine the forces needed for discharge of the fluid contents from the contractile vacuole of Paramecium multimicronucleatum, the time course of the decrease in vacuole diameter during systole (the fluid-discharging period) was compared with that of various vacuole discharge models. The observed time course did not fit that predicted by a model in which contraction of an actin­myosin network surrounding the vacuole caused discharge nor that predicted by a model in which the surface tension of the lipid bilayer of the vacuole caused discharge. Rather, it fitted that predicted by a model in which the cell's cytosolic pressure was responsible for discharge. Cytochalasin B, an effective inhibitor of actin polymerization, had no effect on the in vivo time course of systole. An injection of a monoclonal antibody raised against the proton pumps of the decorated spongiomes (now known to be the locus of fluid segregation in P. multimicronucleatum) disrupted the decorated spongiomes and reduced the rate of fluid segregation, whereas it did not alter the time course of systole. We conclude that in P. multimicronucleatum the internal pressure of the contractile vacuole is caused predominantly by the cytosolic pressure and that the fluid-segregation mechanism does not directly affect the fluid-discharge mechanism. Elimination of this cytosolic pressure by rupturing the cell revealed the presence of a novel fluid-discharge mechanism, apparently centered in the vacuole membrane. The involvement of tubulation of the vacuole membrane as the force-generating mechanism for fluid discharge in disrupted cells is discussed.

Journal Article↗
PubMed1998

Membrane potential responses of paramecium caudatum to bitter substances: existence of multiple pathways for bitter responses

The membrane potential responses of Paramecium caudatum to the external application of bitter substances were examined by employing conventional electrophysiological techniques. Mutant cells defective in voltage-gated Ca2+ channels were used to record the potential responses in the absence of contamination by Ca2+ action potentials. The cells produced a transient depolarization followed by a transient hyperpolarization in response to a rapid whole-cell application of chloroquine, strychnine nitrate or brucine. Of these chemicals, chloroquine was the most potent. Cells produced a simple depolarization in response to a localized application of test chemicals to the anterior region, whereas they produced a transient hyperpolarization in response to an application to the posterior region. Membrane potential responses to an application of chloroquine declined with repeated application. The presence of chloroquine in the external bathing solution strongly inhibited the membrane potential responses to an application of brucine or strychnine. However, the presence of chloroquine did not affect the membrane potential responses to an application of quinine. It is suggested that chloroquine, strychnine and brucine share a common component of their transduction pathways, but that the transduction pathway for quinine is different.

Journal Article↗
PubMed1998

Electrophysiology of the in situ contractile vacuole complex of Paramecium reveals its membrane dynamics and electrogenic site during osmoregulatory activity.

In the freshwater protozoan Paramecium multomicronucleatum, excess cytosolic water, acquired osmotically, is segregated and expelled to the cell exterior through the activity of the contractile vacuole complex. This process keeps the cell volume constant. The electrophysiological parameters of the organelle were measured in situ using a fine-tipped microelectrode inserted into the contractile vacuole, the exocytotic vesicle of the organelle to which the segregated fluid is transported before being expelled to the exterior. The input capacitance decreased markedly immediately before fluid expulsion and regained its previous value when fluid filling resumed after fluid expulsion. This change in the capacitance proved that the contractile vacuole became disconnected from its radial arms, which project from the vacuole, before fluid expulsion occurred and then reconnected with the arms after fluid expulsion. A positive electrical potential was recorded from the contractile vacuole only when it was connected to the radial arms. This implies that the electrogenic mechanism resides exclusively in the radial arms and supports the idea that the decorated spongiomes, V-type proton-pump-covered terminal tubules of the radial arms that end blindly in the cytosol, are electrogenic. The linear current­voltage relationship of the contractile vacuole membrane also implies that few voltage-activated ion channels are present in the membrane. To explain the movement of water into the contractile vacuole complex, we favour the hypothesis that the potential generated across the decorated spongiome membrane can be used to drive counter-anions from the cytosol into the lumen of the complex. The anions could then act as an osmolite to pull cytosolic water into the lumen of the organelle.

Journal Article↗
PubMed2002

Behavioral responses of the ciliated protozoan Paramecium caudatum to 2,4-dichlorophenoxyacetic acid and its analogues.

We found that the ciliated protozoan Paramecium caudatum ON-1 was repelled by 2,4-dichlorophenoxyacetic acid (2,4-D). P. caudatum was also repelled by phenoxyacetic acid, while it was attracted to benzoic acid. No significant behavioral response was detected with 2,4-dichlorophenol (2,4-DP) and phenol. These results suggested that the substitution of-OH of phenol with -OCH2COOH, but not the chlorination of phenol, was important for eliciting the avoiding response to 2,4-D in P. caudatum. Electrophysiological analysis revealed that 2,4-D and phenoxyacetic acid were able to trigger the action potential in P. caudatum, whereas 2,4-DP and phenol failed to elicit electrical responses in this organism. Well test assays suggested that the avoiding reactions allowed P. caudatum to escape from the harm caused by this herbicide.

Journal Article↗