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PubMed1989

Microinjection of plasmid DNA encoding the A surface antigen of Paramecium tetraurelia restores the ability to regenerate a wild-type macronucleus.

Strain d48 of Paramecium tetraurelia contains the A i-antigen gene in the micronucleus, but the gene is lost when micronuclear products develop into the macronucleus. It has recently been shown that when injected into d48, macronucleoplasm from the wild type transforms d48 cells to wild type. It is shown here that wild-type cytoplasm can also bring about transformation, with a marked stage-specific sensitivity for both donor and recipient. It was also found that a plasmid containing the cloned A gene could transform d48 to wild type. Injection of nucleoplasm from animals in the vegetative stage of the cell cycle into the cytoplasm of recipients at various stages of autogamy caused high-frequency transformation of cells able to express the A serotype both before and after the next autogamy. Injection of nucleoplasm into vegetative macronuclei produced over 70% transformants able to express the A serotype after the next autogamy. The ability of nucleoplasm to transform was acquired at the second cell cycle after autogamy and was maintained throughout the vegetative stage. When cytoplasm was obtained from donors during autogamy and injected into the cytoplasm of recipients 1 to 2 h after the sensitive period, quite high frequencies of stable revertants were found when tested both before and after the next autogamy. Cells that were injected into the macronucleus with the cloned A plasmid expressed the A serotype after five fissions in over 20% of the lines and maintained this ability through successive fissions; all transformants except one stably expressed the A serotype even after the next autogamy.

Animals↗
PubMed1986

Accumulation of DNA fragments in aging Paramecium tetraurelia in axenic and nonaxenic media.

The accumulation of DNA fragments in aging Paramecium tetraurelia after 15 days (or approximately 60 generations) of clonal growth in both axenic and nonaxenic media was assayed by alkaline elution assay. This sensitive technique permits measurement of single-strand breaks in double-stranded DNA. The results obtained indicate that P. tetraurelia aged in axenic medium accumulate more apurinic/apyrimidinic lesions, strand breaks, or single-strand gaps than do P. tetraurelia aged in nonaxenic medium. This implies that cells grown in axenic medium have a shorter lifespan than do cells grown in nonaxenic medium. The accumulation of DNA fragments may be the basic mechanism of aging in P. tetraurelia.

Carbon Radioisotopes↗
PubMed1987

Circadian rhythmicity within single cells of Paramecium bursaria.

Cell populations of Paramecium bursaria show mating reactivity in the light period, but not in the dark period, when exposed to a light-dark cycle (LD 12:12). After they are transferred to constant-light (LL) conditions (1,000 lux), they continue to show a circadian rhythm of mating reactivity. The rhythm gradually dampens in LL so that mating reactivity in populations becomes arrhythmic in LL within 2 weeks. We wanted to know whether the arrhythmicity of this population was due to the absence of circadian rhythmicity within each individual cell, or merely due to asynchrony of a population of individually rhythmic cells. Therefore, single cells were isolated randomly from an arrhythmic population that had been in LL for a long time. Then the mating reactivity of these single cells was individually tested every 3 hr for 2 days. Each single cell showed a circadian mating rhythm in LL. This shows that the disappearance of the mating rhythm in cell populations under LL is not caused by disappearance of circadian rhythmicity within individual cells, but is due to desynchronization among cells in a population. When an arrhythmic population in LL is darkened for 9 hr, the mating reactivity rhythm of the cell population reappears. This occurs by resynchronization of the rhythms among individual cells, as can be shown by exposing single cells to pulses of 9 hr of darkness. This dark treatment causes phase shifts of single-cell rhythms, and a phase response curve is obtained for this stimulus. This phase-shifting behavior explains the efficacy of 9-hr dark pulses in restoring the population's rhythm.

Animals↗
PubMed1987

The Paramecium circadian behavioral rhythm: light phase response curves and entrainment.

The population of a ciliate protozoan, Paramecium multimicronucleatum, exhibits a circadian rhythm as measured by the number of the cells traversing an observation point ("traverse frequency," or TF). The present study examined phase shifting of the TF rhythm by administering 2-hr light pulses at different phases of the circadian cycle to cultures free-running in constant darkness (DD). The results were summarized in a phase response curve (PRC), categorized as Type 1. This PRC indicated a relatively narrow phase zone insensitive to the light pulse ("dead zone"). Entrainment of the rhythm to light pulses repeated at 24-hr intervals was also examined, and it was found that the rhythm gradually reached a steady state, following several transient cycles, with the pulses falling at a phase corresponding to the narrow dead zone. Such a steady-state rhythm, with a minimum at approximately 3 hr after the pulse and a maximum at approximately 12 hr after the pulse, was mathematically simulated by superimposing a response function to the pulse on a sinusoidal function representative of the free-running rhythm in DD.

Animals↗
PubMed1981

Quantitative data on peroxidatic markers for electron microscopy. With a note on actin identification in Paramecium cells.

Several important points of heme-peptide cytochemistry were quantitatively analyzed, with particular regard to their use in electron microscopic immunocytochemistry. A simple procedure is presented for the preparation of heme-octapeptide (H-8-P) microperoxidase. H-8-P, hemenonapeptide (H-9-P), and various horseradish peroxidase (HRP) isoenzymes were used for coupling with immunoglobulin (Ig)G or the papain-cleavage fragments from IgG (Fab) molecules. Ultracentrifugation and spectrophotometric analyses revealed the following characteristics of the conjugates: a) They are of a uniform size class; b) their diameters were calculated, and ranged from 5.6 (Fab-H-8-P; H-9-P) to 10.5 (IgG-HRP); c) the persistence of antigen binding capacity was ascertained; d) the deactivation of the marker peroxidase activity due to coupling was as low as 20-30%; e) optimal conditions for use of the electron microscopic (EM) with 3,3'-diaminobenzidine media were elaborated (with a pH optima somewhat different from some standard methods in current use); and f) on the basis of the quantitative data presented, an optimal compromise (either in favor of higher peroxidase activity with HRP conjugates or of smaller size with microperoxidase-Fab conjugates) can be achieved. Finally, the identification of isolated purified actin and of actin in cortical microfilament bundles and ciliary basal bodies of Paramecium cells served as a test object for the usefulness of conjugation products and optimized assay conditions for EM immunocytochemistry.

Actins↗
PubMed1986

Calmodulin in Paramecium tetraurelia: localization from the in vivo to the ultrastructural level.

Monospecific polyclonal antibodies against Paramecium tetraurelia calmodulin were prepared and labeled for calmodulin localization on different levels of resolution: by microinjection into living cells; with isolated cell surface complexes (cortices); on the ultrastructural level, using Lowicryl sections of non-permeabilized cells (with colloidal gold-protein A labeling of antibodies bound); or using permeabilized and gently fixed cells for incubation with peroxidase- or microperoxidase-tagged antibodies. Sites selectively labeled above cytoplasmic background largely coincided, irrespective of the method used, although sensitivity, resolution, and liability to redistribution of antigen were quite different. (The methodological diversification applied allowed for their mutual control.) Nonspecific binding can be largely excluded, since all these methods gave negative results with pre-immune sera. We reached the following conclusions on sites with selective calmodulin binding (above cytoplasmic background level) in P. tetraurelia cells. A pool of calmodulin co-localized with F-actin, not only in the cortex (including fibrous materials around ciliary basal bodies) but also around food vacuoles (phagosomes) and, to a lesser degree, around the buccal cavity. Trichocyst docking sites on the cell membrane, and coated pits also displayed calmodulin labeling, thus indicating the potential involvement of calmodulin in exo-endocytosis processes. Calmodulin was also enriched on membranes of compartments with presumable ion (possibly Ca2+) transport capacity, such as trichocysts and the osmoregulatory system. Not selectively labeled were nuclei, mitochondria, and some small lysosomal organelles (as identified in vivo by rhodamine 123 or acridine orange fluorescence, respectively).

Actins↗
PubMed1986

Filamentous actin in Paramecium cells: mapping by phalloidin affinity labeling in vivo and in vitro.

In living Paramecium cells, microinjected rhodaminyl (R)-phalloidin rapidly labels a thin cortical layer. This can be more clearly resolved with microinjected and fixed cells (allowing for better resolution) as well as with isolated pellicles (surface membrane complexes with trichocysts, microfilaments, and mitochondria attached). Labeling of a longitudinal and perpendicular pattern, reflecting the relief of the cell surface, and labeling of ciliary basal bodies then becomes clearly visible. Other structures labeled by R-phalloidin are the surfaces of food vacuoles of different sizes and, although inconsistently, the borders of the buccal cavity. Small acidic compartments (as identified by acridine orange fluorescence vital staining), probably representing acidosomes and small lysosomes, were not labeled. F-actin on food vacuole surfaces may somehow be involved in intracellular transport or fusion processes. No labeling was observed in association with the osmoregulatory system (contractile vacuoles and their ampullae and radial canals). The specificity of in vivo labeling obtained was supported by the abolition of R-phalloidin labeling when isolated pellicles were pretreated with unlabeled phalloidin or with DNAse I. It was also possible to discriminate among different layers of R-phalloidin binding in the cortex by detaching different layers of the surface complex from each other. Since localization of F-actin in ciliates has raised a considerable amount of dispute in the past, we also repeated all these experiments with RITC-labeled HMM, but we obtained essentially the same labeling pattern as with R-phalloidin. Ciliary basal bodies therefore clearly contain some F-actin. Our data shed some light on aspects of surface structuring and motility in these cells.

Actin Cytoskeleton↗
PubMed1986

Filamentous actin in paramecium cells: functional and structural changes correlated with phalloidin affinity labeling in vivo.

Rhodaminylated (R)-phalloidin microinjected into Paramecium tetraurelia cells at a final concentration of greater than or equal to 20 micrograms/ml produces considerable functional and structural changes. F-actin bundles (with 20 micrograms/ml phalloidin within 15 min) are formed, which subsequently (greater than 30 min) are sequestered into autophagic vacuoles; simultaneously, the originally intense fluorescence of a narrow cortical layer becomes more and more diminished. When such microinjected cells are processed for electron microscopy, they display concomitant ultrastructural alterations, namely, the formation of transcellular bundles of 5-7 nm-thick filaments, which subsequently appear in autophagosomes, as well as a considerable reduction of filamentous materials in the cortex. This, in turn, entails a considerable restructuring of the cortex, enabling free access of various structural components to the cortex. Higher doses of R-phalloidin abolish cytoplasmic streaming (e.g., 50 micrograms/ml after 20-30 min); although the cells may survive, new secretory organelles (trichocysts) are no longer docked to the cell membrane. In contrast, exocytosis of docked trichocysts (as well as subsequent membrane resealing and retrieval) is not impaired under any conditions. Cortical F-actin may account for the cytoplasmic streaming that may normally guarantee the delivery of new trichocysts to free docking sites at the cell membrane. When docking is inhibited by high R-phalloidin doses, excess free trichocysts are sequestered into autophagosomes (crinophagy). One of the most sensitive cell functions is food vacuole formation (assayed by prelabeling with India ink), which correlates with the presence of R-phalloidin labeling in the cytostomal region and around food vacuoles. The main conclusions from this work are that filamentous actin may be involved in structuring of the cortex and in cytoplasmic streaming, and may therefore influence the formation, and possibly the transcellular transport (cyclosis), of food vacuoles, as well as the docking of trichocysts, whereas it does not play a role in exocytosis per se or in the steps immediately following.

Actin Cytoskeleton↗
PubMed1992

Secretory organelles of Paramecium cells (trichocysts) are not remarkably acidic compartments.

Acridine orange (AO) trapping in conjunction with fluorescence microscopy was applied to Paramecium cells. Trichocysts were not labeled when analyzed with an image intensification system (as opposed to a lysosomal population). Only with increasing intensity of ultraviolet light (UV) did trichocysts (and to some extent the cytosol) exhibit orange fluorescence, both effects being paralleled by increasing cell damage. Therefore, in comparison with the reported cytosolic pH (6.8), trichocysts cannot be considered as essentially acidic compartments. This is supported by experiments in vitro, using isolated cortex fragments or isolated fractions of membrane-bounded trichocysts (greater than or equal to 90% non-leaky). Again, during UV illumination orange fluorescence was observed even in the absence of ATP and Mg2+. Furthermore, this AO fluorescence and the condensation state of trichocyst contents were not affected by NH3 or by any of the widely differing ion- and H(+)-exchange inhibitors or ionophores tested. Decondensation of trichocyst contents occurred only when Ca2+ ionophore A23187 or X537A was incorporated into trichocyst membranes and when Ca2+ was then added. In this case all trichocysts partially decondensed within their intact membranes. We conclude that AO might be trapped in trichocysts by the abundant acidic secretory components during observation with UV light, rather than by acidic luminal pH.

Acridine Orange↗
PubMed1993

Ultrastructural and antigenic preservation of a delicate structure by cryopreparation: identification and immunogold localization during biogenesis of a secretory component (membrane-matrix connection) in Paramecium trichocysts.

Ultrastructure and antigenicity of the "mesh-like sheath" (MLS), a very delicate structure connecting the membrane and the paracrystalline matrix of Paramecium trichocysts, are well preserved after cryofixation (rapid freezing followed by freeze-substitution in methanol and embedding in Lowicryl K11M at 213K). The MLS is labeled by colloidal gold-bound antibodies (Ab-Au10nm) with primary antibody (Ab) against trichocyst components obtained by recloning hybridoma cells twice. We prepared Western blots from reduced gels obtained from subfractionated trichocysts. Trichocyst membranes displayed reactive bands of 68-70, 63-66, 43, 40 (strongest), and 57 and 54 KD, with a weak band of 38 KD. One of the most abundant protein bands of soluble secretory components (56-57 KD) was also strongly stained on blots. On ultra-thin sections pre-trichocysts display Ab-reactive material concentrated below the trichocyst membrane before the MLS can be recognized as a structural entity. Quantitative evaluation of Ab-Au10-labeled ultrathin sections also revealed passage of MLS materials through the very inconspicuous Golgi apparatus. This was substantiated by Ab-peroxidase labeling. We conclude that MLS components (whose ultrastructure is difficult to preserve) are largely membrane-associated, partly soluble proteins. They form a connection (released during exocytosis) between the abundant paracrystalline matrix components and the organelle membrane. MLS might thus maintain a peripheral aqueous space of functional importance.

Animals↗
PubMed1996

Immunolocalization of protein phosphatase type 1 in Paramecium cells using antibodies against recombinant protein and peptides.

We localized protein phosphatase Type 1 (PP1) in Paramecium cells using antibodies (specified on Western blots) against recombinant protein, amino- or carboxy-terminal peptides, or peptide segments containing both terminals and an intermediate segment. Cell fractionation and ELISA revealed high PP1 concentrations in cilia, corresponding to observations by immunofluorescence and immunogold labeling analyses. We compared ELISA results obtained with MnCl2- or detergent-mediated deciliation and immunolocalizations obtained with digitonin and saponin- or detergent-mediated permeabilization. We observed that detergents at too high concentrations can displace the antigen from its original position. Quantitative evaluation of immunogold labeling revealed a predominant localization of PP1 in cilia, notably in the narrow space between the membrane and the outer microtubule doublets, as ascertained by immunogold labeling of Lowicryl sections obtained after rapid freezing and freeze-substitution. This localization to the periphery of cilia is compatible with previous suggestions of PP1 involvement in ciliary beat regulation, notably of cilia on the free cell surface. Immunolabeling occurs along the entire length of surface cilia. Despite much higher PP1 concentrations in cilia, ELISA values for absolute PP1 content were considerably higher in deciliated cells. This may indicate still other functional aspects of PP1. Along these lines, we also discuss the differences observed when immunochemical and enzymatic data are compared.

Amino Acid Sequence↗
PubMed2001

Role of delta-tubulin and the C-tubule in assembly of Paramecium basal bodies.

BACKGROUND: A breakthrough in the understanding of centriole assembly was provided by the characterization of the UNI3 gene in Chlamydomonas. Deletion of this gene, found to encode a novel member of the tubulin superfamily, delta-tubulin, results in the loss of the C-tubule, in the nine microtubule triplets which are the hallmark of centrioles and basal bodies. Delta-tubulin homologs have been identified in the genomes of mammals and protozoa, but their phylogenetic relationships are unclear and their function is not yet known. RESULTS: Using the method of gene-specific silencing, we have inactivated the Paramecium delta-tubulin gene, which was recently identified. This inactivation leads to loss of the C-tubule in all basal bodies, without any effect on ciliogenesis. This deficiency does not directly affect basal body duplication, but perturbs the cortical cytoskeleton, progressively leading to mislocalization and loss of basal bodies and to altered cell size and shape. Furthermore, additional loss of B- and even A-tubules at one or more triplet sites are observed: around these incomplete cylinders, the remaining doublets are nevertheless positioned according to the native ninefold symmetry. CONCLUSIONS: The fact that in two distinct phyla, delta-tubulin plays a similar role provides a new basis for interpreting phylogenetic relationships among delta-tubulins. The role of delta-tubulin in C-tubule assembly reveals that tubulins contribute subtle specificities at microtubule nucleation sites. Our observations also demonstrate the existence of a prepattern for the ninefold symmetry of the organelle which is maintained even if less than 9 triplets develop.

Amino Acid Sequence↗
PubMed1989

5-Azacytidine affects the programming of expression of the somatic nucleus of Paramecium.

This report introduces a new system in the study of programming of genomic function during development of the somatic nucleus of Paramecium tetraurelia. Previous works have established a definite, but replaceable, role of the germ nuclei (micronuclei) in oral development in the asexual cycle; their removal from the cell generates viable amicronucleate cell lines, which characteristically suffer a transient period of growth depression marked by abnormal oral development. Such cell lines gradually recover, showing that a compensatory mechanism is activated in the absence of the germ nuclei to bring the cell back to near-normal. To test the notion that the somatic nucleus (macronucleus) is involved in this compensation, cells possessing micronuclei were treated with 5-azacytidine during sexual reproduction when new somatic nuclei develop. These cells were then propagated asexually for a number of fissions in the absence of the drug, and thereafter micronuclei were removed from them. The amicronucleate cell lines generated in this manner clearly did not suffer a depression as severe as the untreated controls did in terms of growth rate and oral development, and they recovered much sooner. This supports the notion that the somatic nucleus is the physical basis of the compensatory mechanism. This study suggests that the stomatogenic sequences in question normally become repressed in the somatic nucleus developing in sexual reproduction, and that 5-azacytidine administered to the cells at this time could alter this programme which then persists during subsequent asexual propagation. The possibility that the somatic nucleus is programmed by methylation of cytosine at the 5' position is discussed.

Animals↗
PubMed1998

Uncoupling of basal body duplication and cell division in crochu, a mutant of Paramecium hypersensitive to nocodazole.

In Paramecium the development of cell shape and surface pattern during division depends on a precise spatial and temporal pattern of duplication of the ciliary basal bodies which are the organizers of the cortical cytoskeleton. According to their localization, basal bodies will duplicate once, more than once or not all and this duplication is coupled with cell division, as is centrosomal duplication in metazoan cells. We describe here a monogenic nuclear recessive mutation, crochu1 (cro1), resulting in abnormal cell shape and cortical pattern and hypersensitivity to nocodazole. The cytological analysis, by immunofluorescence and electron microscopy, demonstrates that the mutation causes hyper duplication of basal bodies and releases both spatial and temporal control of duplication as basal bodies continue to proliferate in interphase and do so at ectopic locations, beneath the surface and in cortical territories where no duplication occurs in the wild type. However, the abnormal surface organization of cro1 cells does not affect the program of basal body duplication during division. By genetic analysis, no interaction was detected with the sm19 mutation which impairs basal body duplication. In contrast, the cro1 mutation suppresses the nocodazole resistance conferred by nocr1, a mutation in a beta-tubulin gene. This interaction suggests that the primary effect of the mutation bears on microtubule dynamics, whose instability, normally increased during division, would persist throughout the interphase and provide a signal for constitutive basal body duplication.

Animals↗
PubMed2006

A broad spectrum of actin paralogs in Paramecium tetraurelia cells display differential localization and function.

To localize the different actin paralogs found in Paramecium and to disclose functional implications, we used overexpression of GFP-fusion proteins and antibody labeling, as well as gene silencing. Several isoforms are associated with food vacuoles of different stages. GFP-actin either forms a tail at the lee side of the organelle, or it is vesicle bound in a homogenous or in a speckled arrangement, thus reflecting an actin-based mosaic of the phagosome surface appropriate for association and/or dissociation of other vesicles upon travel through the cell. Several paralogs occur in cilia. A set of actins is found in the cell cortex where actin outlines the regular surface pattern. Labeling of defined structures of the oral cavity is due to other types of actin, whereas yet more types are distributed in a pattern suggesting association with the numerous Golgi fields. A substantial fraction of actins is associated with cytoskeletal elements that are known to be composed of other proteins. Silencing of the respective actin genes or gene subfamilies entails inhibitory effects on organelles compatible with localization studies. Knock down of the actin found in the cleavage furrow abolishes cell division, whereas silencing of other actin genes alters vitality, cell shape and swimming behavior.

Actin Cytoskeleton↗
PubMed1991

Multiple families of proteins in the secretory granules of Paramecium tetraurelia: immunological characterization and immunocytochemical localization of trichocyst proteins.

Paramecium tetraurelia has thousands of secretory granules (trichocysts), which release their protein contents by regulated exocytosis. The secretory proteins that fill the granule comprise the condensed trichocyst matrix (ctmx), a paracrystalline structure that, upon exocytosis, expands about eightfold in length within milliseconds. The resulting needle-like extended trichocyst matrix (xtmx), also paracrystalline, is released outside the cell. Both ctmx and xtmx are composed of 35 or more small (Mr 14-25 x 10(3)), acidic (pI 4.4-5.8) proteins. We used monoclonal antibodies (mAbs) raised against proteins of the xtmx to study the relationship among these proteins, and to determine their locations within the paracrystalline ctmx and xtmx. The antibodies defined four distinct protein groups. Group I proteins (defined by mAb A1-3 and B5-5) showed a relatively wide range of pI values, and existed in xtmx as disulfide-linked heterodimers. They were distributed throughout the matrix of condensed and extended trichocysts, as judged by electron-microscopic immunocytochemistry. Group II proteins (defined by mAb B4-4 and B3-5) were more acidic, also present as heterodimers and specifically localized in a 150 nm wide cortex in ctmx and in a much thinner cortex in xtmx. In xtmx, antibodies against group II proteins stained the outer surface on the regions between the electron-dense striations with 55 nm intervals. However, these regions were not accessible to antibody B4-4 in ctmx. Group III proteins (defined by mAb B7-4) are monomeric proteins; group IV are two subunits of heterodimers. Proteins of groups IV are two subunits of heterodimers. Proteins of groups III and IV were localized in the core of ctmx, but were distributed uniformly in xtmx. Our results show that these very similar tmx proteins are not structurally equivalent. Within the highly regular structures of condensed and extended tmx, immunologically distinct families of tmx proteins occupy specific and different positions in the paracrystalline array. One family of tmx proteins (group II) is buried in the condensed tmx and only becomes accessible to antibodies upon trichocyst extension. Our results suggest that the 150 nm cortex of condensed tmx expands lengthwise, while decreasing in the thickness, to form the outer shell of extended tmx, and the core expands in length without decreasing in diameter to form the inside structure of the extended tmx.

Animals↗
PubMed1992

Endosomal system of Paramecium: coated pits to early endosomes.

A detailed morphological and tracer study of endocytosis via coated pits in Paramecium multimicronucleatum was undertaken to compare endocytic processes in a free-living protozoon with similar processes in higher organisms. Permanent pits at the cell surface enlarge, become coated and give rise to coated vesicles (188 +/- 41 nm in diameter) that enclose fluid-phase markers such as horseradish peroxidase (HRP). Both the pits and vesicles are labeled by the immunogold technique when a monoclonal antibody (mAb) raised against the plasma membrane of this cell is applied to cryosections. The HRP is delivered to an early endosome compartment, which also shares the plasma membrane antigen. The early endosome, as shown in quick-freeze deep-etch replicas of chemically unfixed cells, is a definitive non-reticular compartment composed of many individual flattened cisternal units of 0.2 to 0.7 microns diameter, each potentially bearing one or more approximately 80-nm-wide coated evaginations. These coated evaginations on the early endosomes contain HRP but are not labeled by the mAb. The coated evaginations pinch off to form a second group of coated vesicles (90 +/- 17 nm in diameter), which can be differentiated from those formed from coated pits by their smaller size, absence of plasma membrane antigen and their location somewhat deeper into the cytoplasm. This study shows a striking similarity between protozoons and mammalian cells in their overall early endosomal machinery and in the ability of early endosomes to sort cargo from plasma membrane components. The vesicles identified in this study form two distinct populations of putative shuttle vesicles, pre-endosomal (large) and early endosome-derived vesicles (small), which facilitate incoming and outgoing traffic from the early endosomes.

Acid Phosphatase↗
PubMed1993

Osmoregulation in Paramecium: the locus of fluid segregation in the contractile vacuole complex.

In a previous study, monoclonal antibody DS-1 was found to specifically label the decorated spongiome along the radial arms of the contractile vacuole complexes in Paramecium multimicronucleatum. Fluorescein isothiocyanate-conjugated DS-1, when injected into cells, labels the radial arms initially, but with increasing postinjection time both the intensity of fluorescence and the number of fluorescently labeled radial arms were reduced. When these cells were fixed after 45 minutes and probed indirectly using a second fluorochrome, little new label was seen on the already fluorescein-labeled radial arms. Thin sections showed that the amount of decorated tubules along some collecting canals decreased from the proximal to the distal end and vesicles, which were never seen in control cells, appeared next to the decorated spongiome. These results suggested that the decorated spongiome was undergoing disassembly and sequestration into one region of the cell. The injected DS-1 also reduced the expulsion frequency of the contractile vacuoles in a dose-, time- and site-dependent manner. The contractile vacuole complexes near the injection site were affected more than those farther from the site, but the sizes of both contractile vacuoles were only transiently affected so that fluid output per cell was reduced by approximately 60%. Beyond 45 minutes postinjection, both the expulsion frequency and total fluid output began to recover as the DS-1 was sequestered into one part of the cell. This region persisted in cells up to 18 hours but disappeared by 24 hours, which coincided with the full recovery of the expulsion frequency and of decorated spongiome along the radial arms. The contractile vacuole, the collecting canals and the smooth spongiome were morphologically unaffected. These results indicate that when the decorated spongiome is dissociated from the contractile vacuole complex, the complex's function is strongly inhibited, showing the decorated spongiome to be the site of fluid segregation.

Animals↗