Paramecium Cells Research Articles

Longevity in space; Experiment on the life span of Paramecium cell clone in space

Life span is the most interesting and also the most important biologically relevant time to be investigated on the space station. As a model experiment, we proposed an investigation to assess the life span of clone generation of the ciliate Paramecium. In space, clone generation will be artificially started by conjugation or autogamy, and the life span of the cell populations in different gravitational fields (microgravity and onboard 1 x g control) will be precisely assessed in terms of fission age as compared with the clock time. In order to perform the space experiment including long-lasting culture and continuous measurement of cell division, we tested the methods of cell culture and of cell-density measurement, which will be available in closed environments under microgravity. The basic design of experimental hardware and a preliminary result of the cultivation procedure are described.

Calcium current activated by cooling in Paramecium

The cooling of deciliated Paramecium cells induced a transient Ca current and its amplitude depended on the rate of the temperature drop. The amplitude of the Ca current was increased by the addition of Ca2+ to the bath solution in a concentration-dependent manner, whereas Ni2+, Co2+, Mn2+ and Mg2+ each reversibly inhibited the Ca current in a concentration-dependent manner with apparent dissociation constants of 0.52, 0.66, 0.67 and 2.17 mmol · l−1, respectively. The Ca current was also inhibited reversibly by amiloride, with a dissociation constant of 0.32 mmol · l−1. The Ca current was desensitized by repetitive cooling. The amplitude of the Ca current at the second cooling was smaller than that at the first cooling when the interval was short, but recovered as the interval increased. Replacing extracellular Ca2+ with equimolar Sr2+ or Ba2+ did not significantly affect the amplitude of the current response to cooling, but it accelerated the rate of recovery from desensitization and slowed the decay of the current response. These results suggest that the desensitization and the inactivation of the Ca current may involve a Ca2+-dependent pathway.

Regional Differentiation of Cortical Structures and Their Reorganization during Cell Division in Paramecium trichium

We observed the cell surface of Paramecium trichium using three different methods. A non‐dividing paramecium's cell surface consisted of three major regions outside of the oral apparatus: a) an oral groove region, with 2‐cilia‐2‐basal‐body (2C‐2BB) units; b) a posterior region, occupying 1/4 to 1/5 of the cell surface, with 1‐cilium‐l‐basal‐body (1C‐1BB) units; c) the remainder, with l‐cilium‐2‐basal‐body (1C‐2BB) units. Five kinds of region‐specific cortical reorganization occurred prior to cytokinesis: the 2C‐2BB and 1C‐1BB units were not duplicated, while the 1C‐2BB units were reorganized to 2C‐2BB, 1C‐2BB or 1C‐1BB units. These reorganizations of the cell surface progressed from the fission line to the anterior in the prospective anterior daughter cell, and to the posterior in the prospective posterior daughter cell, and bilaterally from the old and also newly developing oral apparatus in both daughter cells. In contrast, the development of cilia and their associated structures in each of the cortical units always progressed from posterior to anterior. The present work also showed that two fission lines began to develop bilaterally from the oral primordium, and then they joined to become a single fission line at the dorsal surface.

Immunolocalization of the exocytosis-sensitive phosphoprotein, PP63/parafusin, in Paramecium cells using antibodies against recombinant protein.

We have localized a structure-bound fraction of the exocytosis-sensitive phosphoprotein, PP63/parafusin (PP63/pf), in Paramecium cells by widely different methods. We combined cell fractionation, western blots, as well as light and electron microscopy (pre- and post-embedding immunolabeling), applying antibodies against the recombinant protein. PP63/pf is considerably enriched in certain cortical structures, notably the outlines of regular surface fields (kinetids), docking sites of secretory organelles (trichocysts) and the membranes of subplasmalemmal Ca2+-stores (alveolar sacs). From our localization studies we tentatively derive several potential functions for PP63/pf, including cell surface structuring, assembly of exocytosis sites, and/or Ca2+ homeostasis.

Structural changes in fully hydrated Chilomonas Paramecium exposed to copper

Summary The aim of this study was to determine structural changes induced by the free copper ion in fully hydrated cells of the flagellate protozoon Chilomonas paramecium , a common inhabitant of polluted waters, and often exposed to fluctuating levels of external metals. Light microscopy showed that C. Paramecium was sensitive to copper; the cells rounded up within minutes of exposure to 2.5 mg/1 copper, and were almost spherical within 20 minutes. However, they were able to tolerate the increased copper level, and remained in this altered state for weeks. Soft X-ray microscopy showed that compartments formed and ejectosomes were released in response to initial copper exposure. All cells exposed for 28 hours had a posteriorly positioned indentation that could not be correlated to the vestibulum. Some cells had regenerated their flagella. Quantified data pertaining to changes in the pellicle width after copper exposure of live, fully hydrated cells with natural specimen contrast are given.

Succinate dehydrogenase (SnH) activity in single Paramecium caudatum cells

We measured in situ the activity of succinate dehydrogenase (SDH), one of the mitochondrial marker enzymes, in single Paramecium cells. SDH activity was detected with nitroblue tetrazolium (Nitro BT). Images of cells were captured every 30 sec at 590 nm, nearly the isosbestic wavelength of two reduction products of Nitro BT, by using a microphotometric system for image analysis. Activity was estimated by the slope of linear regression lines representing the relationship between total absorbance of the processed image and delta reaction time (real reaction time minus 30 sec). To investigate individual differences in Paramecium cell populations, SDH activity was measured in cells at various succinate concentrations. Paramecium SDH showed bimodal activity distribution patterns at three of four succinate concentrations tested. This result suggests that there are two groups of Paramecium populations with different SDH activity under control culture conditions. On the basis of the relationship between SDH activity and succinate concentration, mean Vmax and apparent Km values were estimated. A Km of 3.2 mM was found for Paramecium.

Caffeine-induced Ca2+ transients and exocytosis in Paramecium cells. A correlated Ca2+ imaging and quenched-flow/freeze-fracture analysis.

Caffeine causes a [Ca2+]i increase in the cortex of Paramecium cells, followed by spillover with considerable attenuation, into central cell regions. From [Ca2+]resti approximately 50 to 80 nm, [Ca2+]acti rises within </=3 sec to 500 (trichocyst-free strain tl) or 220 nm (nondischarge strain nd9-28 degrees C) in the cortex. Rapid confocal analysis of wildtype cells (7S) showed only a 2-fold cortical increase within 2 sec, accompanied by trichocyst exocytosis and a central Ca2+ spread during the subsequent >/=2 sec. Chelation of Ca2+o considerably attenuated [Ca2+]i increase. Therefore, caffeine may primarily mobilize cortical Ca2+ pools, superimposed by Ca2+ influx and spillover (particularly in tl cells with empty trichocyst docking sites). In nd cells, caffeine caused trichocyst contents to decondense internally (Ca2+-dependent stretching, normally occurring only after membrane fusion). With 7S cells this usually occurred only to a small extent, but with increasing frequency as [Ca2+]i signals were reduced by [Ca2+]o chelation. In this case, quenched-flow and ultrathin section or freeze-fracture analysis revealed dispersal of membrane components (without fusion) subsequent to internal contents decondensation, opposite to normal membrane fusion when a full [Ca2+]i signal was generated by caffeine stimulation (with Ca2+i and Ca2+o available). We conclude the following. (i) Caffeine can mobilize Ca2+ from cortical stores independent of the presence of Ca2+o. (ii) To yield adequate signals for normal exocytosis, Ca2+ release and Ca2+ influx both have to occur during caffeine stimulation. (iii) Insufficient [Ca2+]i increase entails caffeine-mediated access of Ca2+ to the secretory contents, thus causing their decondensation before membrane fusion can occur. (iv) Trichocyst decondensation in turn gives a signal for an unusual dissociation of docking/fusion components at the cell membrane. These observations imply different threshold [Ca2+]i-values for membrane fusion and contents discharge.

A calcium-activated, large conductance and non-selective cation channel in Paramecium cell

A non-selective cation channel was found in mutant Paramecium cells (K115). This cell had been selected as a resistant mutant in a high-K + solution. In patch clamp studies of these cells in the inside-out configuration, this channel was activated by bath applications of elevated Ca 2+ concentrations. The channels became very active when the Ca 2+ concentration was above 3.2 μM. The channel was also activated by depolarization. The voltage dependency was steep upon depolarization, whereas upon hyperpolarization the channel activity barely changed. This channel had poor selectivity for monovalent alkali cations. Using the Goldman–Hodgkin–Katz equation for the reversal potential, the permeability ratios with respect to K + for Na +, Rb +, Cs + and Li + were nearly 1. Although the permeability ratios were similar for each cation, the single channel conductances differed. The single channel conductances were 467 pS with K + as the charge carrier, 406 pS with Na +, 397 pS with Rb +, 253 pS with Cs + and 198 pS with Li + upon depolarization in 100 mM cation solutions. A similar calcium-activated large conductance channel was observed in the wild-type (G3) Paramecium cells but was very rare.

The Paramecium circadian clock: synchrony of changes in motility, membrane potential, cyclic AMP and cyclic GMP

The behavior of a ciliate protozoan, Paramecium, is known to represent the electrical state of the cell membrane, and regulation of the membrane potential and ciliary motion are known to involve cAMP and cGMP. The present study shows the synchrony of circadian changes in motility, resting membrane potential and cyclic nucleotides in P. multimicronucleatum. Using an automated system for tracking isolated single microorganisms, the isolated Paramecium cells are confirmed to swim fast and straight during the day (and subjective day) and slowly, with frequent turning, at night (and subjective night). The resting membrane potential is more negative during the day than at night. cAMP and cGMP concentrations oscillate in a manner, such that both cAMP and cGMP are higher during the day (or subjective day) than at night (or subjective night). The ratio of cGMP to cAMP during the light and dark cycle (LD) fluctuates, paralleling the fluctuation of the resting membrane potential measured during the LD. These results suggest that the Paramecium will provide an excellent model to explore daily and circadian orchestration of second messengers mediating signals from ambient light/dark cycles and circadian pacemaker to ion channels and cilia, directly involved in daily and circadian cellular outputs of resting membrane potential and motility.

Dynamics of calcium regulation in Paramecium and possible morphogenetic implication.

This paper is the first report of the use of a fluorescent indicator (Dextran-coupled calcium green-1) for imaging of cytosolic free calcium in ciliate cells. Using this technique in Paramecium, we show that a very transient increase in the mean intracellular calcium concentration accompanied exocytosis. It has long been postulated based on indirect experimental evidence, that a calcium wave which would spread across the cortex at the time of cell division, would be the primary event that triggers morphogenesis in these species. We theoretically show that a unifying interpretation can be given for the possible occurrence of a single wave and that of multiple oscillations of cytosolic calcium: both of which correspond to two different behaviors of the same dynamic system. Experimental conditions allowing the visualization of possible calcium periodicities in the interphase Paramecium cell are much more easily fulfilled than those permitting the observation of a single wave at the time of cell division. Hence, experiments were performed on interphase cells. After microinjection of calcium indicator into a mutant strain which is defective in exocytosis, we observed Ca2+ oscillations with a period close to 2 minutes. Hence, we conclude that Paramecium possesses all the dynamic elements required to generate, at the time of cell division, a morphogenetic calcium wave.

A novel, calcium-inhibitable casein kinase in Paramecium cells

This is the first identification of a Ca 2+-inhibitable casein kinase (CPK) which we have isolated from the 100 000× g supernatant of Paramecium cell homogenates. The 1000-fold enriched CPK activity depends on millimolar Mg 2+ and is inhibited by low concentrations of heparin or by ≥100 μM Ca 2+. Enzyme activity is stimulated by polylysine or polyarginine with either casein or with specific casein kinase-2 (CK-2) peptide substrates (RRRDDDSDDD and RREEETEEE). The enzymic properties are similar with GTP instead of ATP. CPK does not undergo autophosphorylation. In gel kinase assays, enzyme activity is associated with a 36 kDa band. Calmodulin as another characteristic substrate for mammalian CK-2 has not been phosphorylated by this protein kinase. Besides casein, CPK phosphorylates in vitro the catalytic subunit of bovine brain calcineurin (CaN), a typical substrate of type 1 mammalian casein kinase (CK-1) in vitro. Again this phosphorylation is significantly reduced by Ca 2+. Thus, CPK combines aspects of different casein kinases, but it is clearly different from any type known by its Ca 2+ inhibition. Since CPK also phosphorylates the exocytosis-sensitive phosphoprotein, PP63, in Paramecium, which is known to be dephosphorylated by CaN, an antagonistic Ca 2+-effect during phosphorylation/dephosphorylation cycles may be relevant for exocytosis regulation.

Cold-sensitive Ca2+ influx in Paramecium.

The concentration of intracellular calcium, [Ca2+]i, in Paramecium was imaged during cold-sensitive response by monitoring fluorescence of two calcium-sensitive dyes, Fluo-3 and Fura-Red. Cooling of a deciliated Paramecium caused a transient increase in [Ca2+]i at the anterior region of the cell. Increase in [Ca2+]i was not observed at any region in Ca(2+)-free solution. Under the electrophysiological recording, a transient depolarization of the cell was observed in response to cooling. On the voltage-clamped cell, cooling induced a transient inward current under conditions where K+ currents were suppressed. These membrane depolarizations and inward currents in response to cooling were lost upon removing extracellular Ca2+. The cold-induced inward current was lost upon replacing extracellular Ca2+ with equimolar concentration of Co2+, Mg2+ or Mn2+, but it was not affected significantly by replacing with equimolar concentration of Ba2+ or Sr2+. These results indicate that Paramecium cells have Ca2+ channels that are permeable to Ca2+, Ba2+ and Sr2+ in the anterior soma membrane and the channels are opened by cooling.

Thigmotaxis in Paramecium caudatum is induced by hydrophobic or polyaniline-coated glass surface to which liver cells from rat adhere with forming multicellular spheroids

Summary Paramecium caudatum shows thigmotaxis. Paramecium cells touch the object with the tips of their oral groove cilia and raise their posterior ends when they show thigmotactic behavior, inducible by changing the ionic condition of the surrounding medium. We found that the thigmotaxis of Faramecium could also be induced by changing the surface character of the chamber, e. g. by coating with highly hydrophobic groups or with doped-polyaniline, which is one of the best materials for the cultivation of hepatocytes from rat, that is hepatocytes adhere to the material with forming spheroids rapidly and keep up a high activity, such as secretion of albumin. We also found that hepatocytes reacted in this way to the hydrophobic surface of the chamber used here. Our results might suggest that Paramecium cells and hepatocytes show similar preferences for surface character in adhesion.

Defensive function of trichocysts in Paramecium against the predatory ciliate Monodinium balbiani

Summary Defensive function of trichocysts in Paramecium was recently verified against the predatory ciliate Dileptus margaritifer. Until then, however, their defensive function had been repeatedly questioned based on the observation that trichocysts are of little use against Didinium nasutum . We tested whether trichocysts can defend Paramecium against Monodinium , a predatory ciliate closely related to Didinium . Cells of trichocyst-non-discharge (tnd) mutants, artificially induced trichocyst-deficient cells, and intact wild-type cells of Paramecium were compared as prey for M. balbiani . In both P. caudatum and P. tetraurelia , mutant cells were much more vulnerable than wild-type cells (killed 12–40 times more often by the predator under a certain condition). Cells of P. tetraurelia with reduced numbers of trichocysts, obtained by treating wild-type cells with lysozyme, were almost as vulnerable as mutant cells. On the contrary, when tested against D. nasutum , wild-type and mutant cells of P. tetraurelia were equally vulnerable. We conclude that discharge of trichocysts defends Paramecium against M. balbiani but not against D. nasutum . These results strongly suggest that trichocysts in Paramecium function as defensive organelles against many toxicyst-bearing predatory ciliates with a few exceptions such as D. nasutum .

Participation of GTP-binding protein in the photo-transduction of Paramecium bursaria.

Analysis of the flow of light information in Paramecium, especially the participation of GTP-binding protein, was carried out. Injection of GDP-beta-S into Paramecium cell abolished the light-induced inward current. The partially purified rhodopsin-like protein (RLP) that we obtained activated frog rod outer segments (ROS) GTPase. A GTP-binding protein, with Mw 57,000 was detected by immunostaining with anti-rat G alpha rabbit IgG. These results suggest that the light information flows from the light-captured RLP to the GTP-binding protein.

Monoclonal and polyclonal antibodies detect a new type of post-translational modification of axonemal tubulin.

Polyclonal (PAT) and monoclonal (AXO 49) antibodies against Paramecium axonemal tubulin were used as probes to reveal tubulin heterogeneity. The location, the nature and the subcellular distribution of the epitopes recognized by these antibodies were, respectively, determined by means of: (i) immunoblotting on peptide maps of Paramecium, sea urchin and quail axonemal tubulins; (ii) immunoblotting on ciliate tubulin fusion peptides generated in E. coli to discriminate antibodies directed against sequential epitopes (reactive) from post-translational ones (non reactive); and (iii) immunofluorescence on Paramecium cells, using throughout an array of antibodies directed against tubulin sequences and post-translational modifications as references. AXO 49 monoclonal antibody and PAT serum were both shown to recognize epitopes located near the carboxyl-terminal end of both subunits of Paramecium axonemal tubulin, whereas the latter recognized additional epitopes in alpha-tubulin; AXO 49 and a fraction of the PAT serum proved to be unreactive over fusion proteins; both PAT and AXO 49 labelled a restricted population of very stable microtubules in Paramecium, consisting of axonemal and cortical ones, and their reactivity was sequentially detected following microtubule assembly; finally, both antibodies stained two upward spread bands in Paramecium axonemal tubulin separated by SDS-PAGE, indicating the recognition of various alpha- and beta-tubulin isoforms displaying different apparent molecular masses. These data, taken as a whole, definitely establish that PAT and AXO 49 recognize a post-translational modification occurring in axonemal microtubules of protozoa as of metazoa. This modification appears to be distinct from the previously known ones, and all the presently available evidence indicates that it corresponds to the very recently discovered polyglycylation of Paramecium axonemal alpha- and beta-tubulin.

Induction of the thigmotaxis in Paramecium caudatum

Induction of thigmotaxis in Paramecium caudatum was examined. We succeeded in the induction by changing the ionic concentration and the Ja-value ([K+]/[Ca2+]1/2) of the surrounding medium. P. caudatum showed thigmotaxis stably in the solution containing a relatively high ionic concentration but a lower Ja-value. We found that the Paramecium cell touched the object with the tip of its oral groove and the cilia touching the object scarcely beat when the organism showed thigmotaxis.

The Effects of Altering Extracellular Potassium Ion Concentration on the Membrane Potential and Circadian Clock of Paramecium Bursaria

In some neural models of circadian rhythmicity, membrane potential and transmembrane flux of potassium and calcium ions appear to play important roles in the entrainment and central mechanisms of the biological clock. We wondered whether these cellular variables might be generally involved in circadian clocks, even non-neural clocks. Therefore, we tested the impact of changing extracellular potassium level on the circadian rhythm of photoaccumulation of Paramecium cells, whose membrane potential responds to changes of extracellular potassium in a manner similar to that of neurones. We found that pulse or step changes of extracellular potassium concentration did not phase-shift the circadian clock of P. bursaria cells in a phase-specific manner. Furthermore, modifying the extracellular concentration of calcium did not affect the magnitude of light-induced phase resetting. Therefore, while membrane potential and calcium fluxes may be crucial components of the circadian clock system in some organisms, especially in neural systems that involve intercellular communication, the P. bursaria data indicate that membrane potential changes are not necessarily an intrinsic component of circadian organization at the cellular level.

Ca2+ release from subplasmalemmal stores as a primary event during exocytosis in Paramecium cells.

A correlated electrophysiological and light microscopic evaluation of trichocyst exocytosis was carried out the Paramecium cells which possess extensive cortical Ca stores with footlike links to the plasmalemma. We used not only intra- but also extracellular recordings to account for polar arrangement of ion channels (while trichocysts can be released from all over the cell surface). With three widely different secretagogues, aminoethyldextran (AED), veratridine and caffeine, similar anterior Nain and posterior Kout currents (both known to be Ca(2+)-dependent) were observed. Direct de- or hyperpolarization induced by current injection failed to trigger exocytosis. For both, exocytotic membrane fusion and secretagogue-induced membrane currents, sensitivity to or availability of Ca2+ appears to be different. Current responses to AED were blocked by W7 or trifluoperazine, while exocytosis remained unaffected. Reducing [Ca2+]o to < or = 0.16 microM (i.e., resting [Ca2+]i) suppressed electrical membrane responses triggered with AED, while we had previously documented normal exocytotic membrane fusion. From this we conclude that the primary effect of AED (as of caffeine) is the mobilization of Ca2+ from the subplasmalemmal pools which not only activates exocytosis (abolished by iontophoretic EGTA injection) but secondarily also spatially segregated plasmalemmal Ca(2+)-dependent ion channels (indicative of subplasmalemmal [Ca2+]i increase, but irrelevant for Ca2+ mobilization). The 45Ca2+ influx previously observed during AED triggering may serve to refill depleted stores. Apart from the insensitivity of our system to depolarization, the mode of direct Ca2+ mobilization from stores by mechanical coupling to the cell membrane (without previous Ca(2+)-influx from outside) closely resembles the model currently discussed for skeletal muscle triads.

Veratridine triggers exocytosis in Paramecium cells by activating somatic Ca channels

Paramecium tetraurelia wild-type (7S) cells respond to 2.5 mM veratridine by immediate trichocyst exocytosis, provided [Ca2+]o (extracellular Ca2+ concentration) is between about 10(-4) to 10(-3) M as in the culture medium. Exocytosis was analyzed by light scattering, light and electron microscopy following quenched-flow/freeze-fracture analysis. Defined time-dependent stages occurred, i.e., from focal (10 nm) membrane fusion to resealing, all within 1 sec. Veratridine triggers exocytosis also with deciliated 7S cells and with pawn mutants (without functional ciliary Ca channels). Both chelation of Ca2+o or increasing [Ca2+]o to 10(-2) M inhibit exocytotic membrane fusion. Veratridine does not release Ca2+ from isolated storage compartments and it is inefficient when microinjected. Substitution of Na+o for N-methylglucamine does not inhibit the trigger effect of veratridine which also cannot be mimicked by aconitine or batrachotoxin. We conclude that, in Paramecium cells, veratridine activates Ca channels (sensitive to high [Ca2+]o) in the somatic, i.e., nonciliary cell membrane and that a Ca2+ influx triggers exocytotic membrane fusion. The type of Ca channels involved remains to be established.