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PubMed1995

Elucidation of nucleus-cytoplasm interaction: change in ability of the nucleus to express sexuality according to clonal age in Paramecium.

The expression of sexual activity in Paramecium caudatum is repressed for about 50 fissions after conjugation. The ability of the macronucleus to affect the expression of sexual activity according to the age of clonal development was investigated using the macronuclear fusion-reorganization method. When a mature macronucleus was transplanted into an immature cell and fused with a macronucleus in the immature cell, the clones derived from the recipient showed sexual immaturity. In a reverse experiment, an immature macronucleus was transplanted into a mature cell, and the clones also showed sexual immaturity. The ability of the macronucleus to transform mature cells to immature cells was clonal age-dependent. The characteristics of the immature-mature hybrid macronucleus indicate that the immature macronucleus is dominant over the mature macronucleus with respect to the ability to express sexual activity. On the other hand, in cells of the early immaturity period, the micronucleus, known as the germ nucleus, shows the ability to undergo meiosis and eventually to produce progeny under control of the mature macronucleus. The expression of sexual activity is thought to be governed by the clonal age of the macronucleus and not by the clonal age of the micronucleus or cytoplasm. The macronucleus seems to determine the ability to express sexual activity by counting post-conjugation divisions and keeping track of the age of the clone.

Animals↗
PubMed1995

Rapid bulk replacement of acceptor membrane by donor membrane during phagosome to phagoacidosome transformation in Paramecium.

The extent to which a donor membrane will be retrieved, or if it is retrieved at all after it fuses with an acceptor membrane, is usually difficult to determine. We have studied the dynamics of membrane retrieval in the phagosome system of Paramecium multimicronucleatum using six monoclonal antibody markers. Our previous freeze-fracture and transmission electron microscopic studies have indicated that extensive changes take place in the membrane of the young phagosome as it progresses through its cycle. Using immunofluorescence and immunoelectron microscopy to determine the times of entry and exit of these individual antigens into the digestive vacuole system, we showed that two hydrophilic antigens, one located on the cytosolic and one on the lumenal side of the discoidal membrane (phagosome membrane precursor), were completely retrieved from the phagosome by tubulation within the first three minutes. At the same time that this membrane was retrieved, membrane from a second population of vesicles, the acidosomes, fused with the phagosome to produce the phagoacidosome. On the basis of immunogold localization on cryosections of a total of six antigens, the two specific for phagosome/discoidal vesicle membrane as well as four specific for the acidosome/phagoacidosome membrane, this replacement is total. We also showed that in the presence of the actin-active drug cytochalasin B, this replacement was essentially prevented. However, when vacuole acidification was neutralized by ammonium chloride, this replacement process continued unaffected after a lag. Consequently, acidification, per se, is not required to trigger the replacement of the phagosome membrane. We conclude, on the basis of these studies as well as our previous freeze-fracture studies that during phagoacidosome formation most of the acceptor membrane is retrieved and is replaced by the donor membrane. This shows that at least one cell type possesses the mechanisms needed to substantially replace the membrane of a phagosomal compartment when radical and rapid changes are needed to modulate the digestive and absorptive processes.

Ammonium Chloride↗
PubMed1997

Imaging of Ca2+ transients induced in Paramecium cells by a polyamine secretagogue.

In Paramecium tetraurelia cells analysis of transient changes in Ca2+ concentration, [Ca2+]i, during aminoethyldextran (AED) stimulated synchronous (<1 second) trichocyst exocytosis has been hampered by various technical problems which we now have overcome. While Fura Red was found appropriate for quantitative double wavelength recordings, Fluo-3 allowed to follow, semi-quantitatively but with high time resolution, [Ca2+]i changes by rapid confocal laser scanning microscopy (CLSM). Resting values are between 50 and 70 nM in the strains analysed (7S wild type, as well as a non-discharge and a trichocyst-free mutant, nd9-28 degrees C and tl). In all strains [Ca2+]i first increases at the site of AED application, up to 10-fold above basal values, followed by a spillover into deeper cell regions. This might: (i) allow a vigorous Ca2+ flush during activation, and subsequently (ii) facilitate re-establishment of Ca2+ homeostasis within > or =20 seconds. Because of cell dislocation during vigorous trichocyst exocytosis, 7S cells could be reasonably analysed only by CLSM after Fluo-3 injection. In 7S cells cortical [Ca2+]i transients are strictly parallelled by trichocyst exocytosis, i.e. in the subsecond time range and precisely at the site of AED application. Injection of Ca2+ is a much less efficient trigger for exocytosis. Ca2+-buffer injections suggest a requirement of [Ca2+]i >1 to 10 microM for exocytosis to occur in response to AED. In conclusion, our data indicate: (i) correlation of cortical [Ca2+]i transients with exocytosis, as well as (ii) occurrence of a similar signal transduction mechanism in mutant cells where target structures may be defective or absent.

Animals↗
PubMed1999

A key function of non-planar membranes and their associated microtubular ribbons in contractile vacuole membrane dynamics is revealed by electrophysiologically controlled fixation of Paramecium.

The contractile vacuole complex of the fresh water protozoan Paramecium multimicronucleatum exhibits periodic exocytotic activity. This keeps cytosolic osmolarity at a constant value. The contractile vacuole, the central exocytotic vesicle of the complex, becomes disconnected from its surrounding radial arms and rounds before its fluid content is expelled. We previously proposed a hypothesis that the rounding of the contractile vacuole corresponds to an increase in its membrane tension and that a periodic increase in membrane tension governs the exocytotic cycle. We also proposed a hypothesis that transformation of excess planar membrane of the contractile vacuole into 40 nm diameter tubules, that remain continuous with the contractile vacuole membrane, is a primary cause for the tension development in the planar membrane. In order to investigate tension development further, we have examined electron microscopically the contractile vacuole membrane at the rounding phase. To do this, we developed a computer-aided system to fix the cell precisely at the time that the contractile vacuole exhibited rounding. In this system a decrease in the electrical potential across the contractile vacuole membrane that accompanied the vacuole's rounding was monitored through a fine-tipped microelectrode inserted directly into the in vivo contractile vacuole. A decrease in membrane potential was used to generate an electric signal that activated an injector for injecting a fixative through a microcapillary against the cell at the precise time of rounding. Subsequent electron micrographs of the contractile vacuole membrane clearly demonstrated that numerous approximately 40 nm membrane-bound tubules formed in the vicinity of the vacuole's microtubule ribbons when the vacuole showed rounding. This finding suggested that membrane tubulation was the cause for topographical isolation of excess membrane from the planar membrane during the periodic rounding of the contractile vacuole. This together with stereo-pair images of the contractile vacuole complex membranes suggested that the microtubule ribbons were intimately involved in enhancing this membrane tubulation activity. Electron micrographs of the contractile vacuole complexes also showed that decorated tubules came to lie abnormally close to the contractile vacuole in these impaled cells. This suggested that the contractile vacuole was capable of utilizing the smooth spongiome membrane that lies around the ampullae and the collecting canals to increase its size.

Animals↗
PubMed2001

Growth and form of secretory granules involves stepwise assembly but not differential sorting of a family of secretory proteins in Paramecium.

Paramecium trichocysts are voluminous secretory vesicles consisting of a spindle-shaped body surmounted by a tip that serves to anchor them at exocytotic sites in the plasma membrane. This constrained shape is conferred by the proteins stored in the vesicles, which form an insoluble three-dimensional crystalline array. The constituent polypeptides (Trichocyst Matrix Proteins, TMPs), which assemble during trichocyst biogenesis, are produced by proteolytic processing of soluble proproteins encoded by a large multigene family. In order to investigate the functional significance of the TMP multigene family, which assures the synthesis of a mixture of related polypeptides, we have designed synthetic genes for heterologous expression of three different mature polypeptides, which were used to obtain sequence-specific rabbit antisera. We used these antisera to carry out immunolocalization experiments with wild-type trichocysts at different stages of development and found that the trichocyst matrix consists of two concentric layers containing different TMPs, and that the assembly of each layer corresponds to a distinct phase of trichocyst growth. Examination of mutant trichocysts created by targeted gene silencing of different TMP genes showed that the layer containing the products of the silenced genes is specifically affected, as are all subsequently assembled parts of the structure, consistent with an ordered assembly pathway. This stepwise assembly is not controlled by differential sorting of the TMPs, as single and double label experiments provided evidence that the different TMPs are delivered together to post-Golgi vesicles and developing trichocysts. We present a model for trichocyst biogenesis in which TMP assembly is controlled by protein processing.

Amino Acid Sequence↗
PubMed2002

Osmoregulation in Paramecium: in situ ion gradients permit water to cascade through the cytosol to the contractile vacuole.

In vivo K(+), Na(+), Ca(2+) and Cl(-) activities in the cytosol and the contractile vacuole fluid of Paramecium multimicronucleatum were determined in cells adapted to a number of external osmolarities and ionic conditions by using ion-selective microelectrodes. It was found that: (1) under standardized saline conditions K(+) and Cl(-) were the major osmolytes in both the cytosol and the contractile vacuole fluid; and (2) the osmolarity of the contractile vacuole fluid, determined from K(+) and Cl(-) activities only, was always more than 1.5 times higher than that of the cytosol. These findings indicate that excess cytosolic water crosses the contractile vacuole complex membrane osmotically. Substitution of choline or Ca(2+) for K(+) in the external solution or the external application of furosemide caused concomitant decreases in the cytosolic K(+) and Cl(-) activities that were accompanied by a decrease in the water segregation activity of the contractile vacuole complex. This implies that the cytosolic K(+) and Cl(-) are actively coimported across the plasma membrane. Thus, the osmotic gradients across both the plasma membrane and the membrane of the contractile vacuole complex ensure a controlled cascade of water flow through the cell that can provide for osmoregulation as well as the possible extrusion of metabolic waste by the contractile vacuole complex.

Adaptation, Physiological↗
PubMed1975

Ciliary granule plaques: membrane-intercalated particle aggregates associated with Ca2+-binding sites in paramecium.

In Paramecium nine rectangular aggregates of membrane-intercalated particles surround the freeze-cleaved membrane of the ciliary base. These "ciliary granule plaques" occur independently of the "ciliary necklace" which is observed in a more basal position on some cilia. In each individual plaque the approximately 10 nm large granules are arranged in a square grid pattern: the granules of one plaque invariably form 3 vertical rows (number of horizontally arranged particles per row: Nh = 3) and 3-6 horizontal rows (mean value fos intrahepatic tributaries then can be approached under direct vision in a relatively bloodless field for safe, accurate liquation and/or reconstruction.

Animals↗
PubMed1978

The maintenance of kappa during macronuclear reorganization in Paramecium tetraurelia.

Kappa particles were injected into stock-51 sensitives (KK) of Paramecium tetraurelia to investigate the maintenance of kappa by gene K during macronuclear reorganization. Injection into exconjugants and autogamous cells followed by macronuclear regeneration, induced by microsurgical removal of macronuclear Anlagen, resulted in maintenance of kappa particles by a considerable proportion of the clones descended from the recipient paramecia. Injection into animals that underwent normal reorganization however usually failed to yield particle-maintaining paramecia.

Animals↗
PubMed1978

Genetic aspects of intracellular motility: cortical localization and insertion of trichocysts in Paramecium tetraurelia.

Wild-type and selected mutants of Paramecium tetraurelia were subjected to detailed analysis of the trichocyst phenotype. The characteristics of morphology, saltatory motility, and cortical insertion of trichocysts were found to be genetically distinguishable. In mutants with defective trichocyst saltatory motility, other intracellular motility systems (i.e. mitochondrial saltatory motility, and cyclosis) appeared to be unaffected. Saltatory motility appears to be an essential intermediate step in the functional development of the trichocyst, since it serves to transport the trichocyst from its site of assembly, deep in the cytoplasm, to the cortex, where it is inserted and becomes discharge-competent. If trichocyst saltatory motility is lost, as in the mutants ndA and tam8, then trichocysts are not transported to the cortex and are therefore not inserted. However, the successful transport of a trichocyst to the cortex does not guarantee the proper insertion of that trichocyst, as in the case of the mutant am. Apparently there are independent steps involved specifically with trichocyst insertion. The gross morphology of the trichocyst appears to be independent of its saltatory motility, and to a degree, independent of cortical insertion.

Animals↗
PubMed1978

Adenosinetriphosphate, calcium and temperature requirements for the final steps of exocytosis in Paramecium cells.

In Paramecium cells a synchronized discharge of trichocysts (which involves only the final exocytosis steps of membrane fusion, content discharge and membrane resealing) was achieved with ATPase-blockers, Ca2+-ionophores, lipid solvents (including lysolecithin), polyethyleneglycol, anaesthetics (Dibucain) and cationic detergents (cetyltrimethylammonium bromide (CTMAB) and cetylpyridinium chloride (CPC). Only Dibucain--and to some extent cationic detergents--can trigger exocytosis independently of extracellular Ca2+, possibly by mobilizing intracellular Ca2+. The internal free [Ca2+] necessary for exocytosis can be estimated to be greater than 10(-6) to 10(-4) M. Membrane-free trichocyst contents were isolated by density gradient centrifugation; they are converted from the contracted to the expanded state by Dibucain, CTMAB and CPC, and also by exogenous ATPase (Apyrase). Thus, it is possible to de-couple the discharge (stretching) process from membrane-related phenomena. Since only the latter are inhibited by low temperature (0 degrees C), membrane lipids probably have to be in a fluid state for exocytosis to occur. At least 2 steps appear to be involved: when membrane fusion is initiated, an independent matrix-bound system is activated for the synchronized stretching process. The energy requirement for one discharge event is estimated to be about 14 X 10(6) ATP molecules.

Adenosine Triphosphate↗
PubMed1979

Specificity of the immaturity substances in Paramecium.

Cytoplasm of Paramecium caudatum syngen 3 in the period of sexual immaturity was effective in inducing immaturity when injected into mating-reactive cells of other syngens of P. caudatum. When immature cytoplasm of P. caudatum was injected into mature cells of P. multimicronucleatum, immaturity was also induced in the latter cells. When cytoplasm of P. bursaria in the immaturity period was injected into mature cells of the same species, the latter became immature. But immature cytoplasm of P. caudatum was ineffective when injected into mature cells of P. bursaria. When immature cytoplasm of P. busaria was injected into mature cells of P. caudatum, no difference from the control was observed. These results suggest that a difference in specificity of the immaturity substances exists bewteen paramecia of the 'aurelia' group and those of the 'bursaria' group.

Animals↗
PubMed1979

Immaturity substances in Paramecium primaurelia and their specificity.

Cells of Paramecium primaurelia in the immaturity period of the life cycle contain substances which repress the expression of mating ability when introduced into mature cells. When the immature cytoplasm of P. primaurelia was injected into mating-reactive cells of P. tetraurelia, P. caudatum and P. multimicronucleatum, immaturity was also induced in them. But the immature cytoplasm of P. primaurelia was ineffective when injected into mature cells of P. bursaria. When the immature cytoplasm of P. bursaria was injected into mating-reactive cells of P. primaurelia and P. tetraurelia, no difference from the control was observed. When the immature cytoplasm of P. caudatum was injected into mating-reactive cells of P. primaurelia and P. tetraurelia, immaturity was induced in cells of the latter 2 species.

Animals↗
PubMed1980

Clonal death associated with the number of fissions in Paramecium caudatum.

Contrary to an earlier report suggesting clonal immortality in Paramecium caudatum, the clones of the same species in this study terminated inevitably with a maximal life-span of 658 fissions and showed, prior to clonal death, decreased fission rate, increased probability of the appearance of non-dividing or dead cells after cell division, and increased frequency of morphological abnormality and of division asynchrony. Clonal senescence and death after a limited number of fissions was reproducible even if subclones derived from the original clone of a known fission age were examined again after a lag of 468 days. These results indicate that clones of P. caudatum are mortal and that they use fissions, not days, to measure life-span. Possible causes for the discrepancies between the earlier report and the present one are discussed.

Animals↗
PubMed1980

Abnormal microtubule deployment during defective macronuclear division in a Paramecium mutant.

The tam 8 mutant of Paramecium tetraurelia is a representative of a class of mutants characterized by abnormal nuclear divisions during binary fission and the failure of trichocysts to attach to the plasma membrane. Compared with wild-type organisms the following abnormalities occur in tam 8 individuals. (I) The spherical interphase macronucleus is not positioned near the oral apparatus; it is randomly located in the cytoplasm of interfission organisms. (2) The macronucleus does not migrate towards the anterior dorsal cortex as its division starts, nor is it dorsally and subcortically positioned as it elongates. (3) Elongating macronuclei exhibit variable and irregular shapes. (4) This elongation is delayed and reduced. (5) Longitudinally oriented microtubules assemble in the nucleoplasm of dividing macronuclei but their spatial deployment is abnormal. (6) Unequal segregation of micronuclei between daughter organisms occurs during binary fission. The abnormal arrangement of nucleoplasmic microtubules provides support for the proposal that a microtubule sliding mechanism is involved during the elongation of dividing macronuclei. The extent to which macronuclear division may be controlled by the cell cortex is considered in relation to tthe pleiotropic effects of the tam 8 mutation.

Animals↗
PubMed1980

Membrane recycling and endocytosis in Paramecium confirmed by horseradish peroxidase pulse-chase studies.

Paramecium caudatum cells were pulse-chased in horseradish peroxidase (HRP) using pulses of 30 s to follow endosome (endocytotic vesicle) formation from defaecating digestive vacuoles and 3-min pulses to follow the movement and fate of these vesicles in the cell. Endosomes formed during the 30 s in HRP are heavily labelled with HRP reaction product and are mostly flattened. Some align along microtubules that point toward the cytopharynx. Diskoidal vesicles at the cytopharynx are unlabelled in unchased cells. Cells exposed to a 3-min HRP pulse contain varied amounts of HRP-labelled diskoidal vesicles at the cytopharynx but no labelled vesicles near their closed cytoprocts. Labelled vesicles are also found aligned along the cytopharyngeal microtubular ribbons. Diskoidal vesicles are no longer labelled after a 47-min chase. Use of pulse-chase HRP cytochemistry supports the hypothesis that the membrane of digestive vacuoles retrieved at the cytoproct is moved along microtubular ribbons directly to the cytopharynx where the membrane enters the diskoidal vesicle pool. This membrane appears to enter neither the Golgi nor lysosomal systems in its passage. HRP reaction product is also found in vesicles near the parasomal sacs, in some secondary lysosomes and in small spherical vesicles that are probably trichocyst membrane fragments. Possibly some membrane from parasomal sacs or condensing digestive vacuoles may also enter the diskoidal vesicle pool but apparently not membrane from trichocysts.

Animals↗
PubMed1980

Genetic dissection of the final exocytosis steps in Paramecium tetraurelia cells: cytochemical determination of Ca2+-ATPase activity over performed exocytosis sites.

In different Paramecium tetraurelia strains the occurrence of a Ca2+-ATPase (or p-nitro-phenylphosphatase) activity at the preformed attachment and exocytosis sites of the secretory vesicles (trichocysts) was analysed by electron-microscopic cytochemistry and X-ray microanalysis. In conjunction with freeze-fracture studies it was found that only those strains, which contain rosette particles, display this Ca2+-ATPase activity (7S, K 40I, nd 9 (18 degrees C), while other strains (nd 6, nd 9 (27 degrees C), tam 38) are devoid of both these characteristics. The presence (absence) of rosette particles and of Ca2+-ATPase activity at the preformed exocytosis sites is correlated with the capability (incapability) of performing exocytosis in these strains. We discuss several possible interpretations of this structure-function correlation.

Animals↗
PubMed1980

Genetic dissection of the final exocytosis steps in Paramecium tetraurelia cells: trigger analyses.

A variety of trigger procedures were applied to analyse the exocytotic capability of different Paramecium tetraurelia strains. 7,S K 40I, kin 24I, and 9 (18 degrees C) are capable of exocytosis (permissive strains), in contrast to nd 6, nd 7, nd 9 (27 degrees C), tam 38 and ftb A, although all procedures used enhance [Ca2+]i in the cytoplasm of all strains tested and although strains nd 6, nd 7 and nd 9 (27 degrees C) contain a full set of morphologically normal trichocysts attended to the cell membrane. The results show that only those strains are permissive which were shown previously to contain a rosette of membrane-integrated particles and a Ca2+-ATPase activity in the cell membrane over the trichocyst attachment (exocytosis) sites. The results from trigger experiments with permissive and non-permissive strains would be compatible with a dual function of rosette particles as Ca2+ pumps and Ca2+ channels. Nevertheless, the latter aspect remains uncertain since we show that experiments along these lines published by others (introducing a Ca2+ ionophore from the outside) involve a solvent-induced artifact (pseudoexocytosis: matrix stretching in the absence of membrane fusion). In all strains, except for tam 38 and ftb A (which have abnormal trichocysts incapable of being attached to the cell membrane), the isolated trichocyst matrix can be transferred from the contracted to the expanded state in vitro with certain trigger procedures. Our data clearly show that an increase of [Ca2+]i in the cytoplasm is not sufficient for exocytosis to occur and that non-permissiveness is somehow due to an inability to perform membrane fusion. It remains open whether the lack of rosettes and Ca2+-ATPase activity at trichocyst attachment sites are primary cause of non-permissiveness.

Animals↗
PubMed1982

Age-associated change in macronuclear DNA content in Paramecium caudatum.

Macronuclear DNA content in Paramecium caudatum was found to be almost unchanged with a mean of about 400C during the earlier two-thirds of the life span in terms of the number of fissions and then it dropped rapidly to about one-fifth of the initial content. The age when rapid DNA decline occurred corresponded to that when the characteristics of senescence appeared. This decreasing pattern of macronuclear DNA content contrasted with earlier observations in P. tetraurelia, P. bursaria and Tetrahymena thermophila. The data suggested that in P. caudatum the distribution pattern of macronuclear DNA to daughter cells changed from an almost equal distribution in younger cells to an unequal distribution in older cells, while the relative volume of the macronucleus to the whole cell remained almost constant throughout the life cycle.

Aging↗