Identification of the basal bodies and kinetodesmal fibers in living cells of Paramecium tetraurelia Sonneborn, 1975 and Paramecium sonneborni Aufderheide, Daggett & Nerad, 1983

Critical use of Nomarski DIC optics and a rotocompressor permits basal bodies and kinetodesmal fibers to be visualized in the cortices of living Paramecium tetraurelia and Paramecium sonneborni. The identification of these structures is confirmed by the correspondence of the images obtained by DIC optics of living cells and by brightfield optics of fixed cells stained by the Fernández-Galiano silver technique. Examination of cells carrying cortical inversions (portions of the cortex rotated 180 degrees) shows that inverted regions may be identified and distinguished from normal regions by the orientation of the kinetodesmal fibers of the kinetids (cortical units) within the kineties (ciliary rows). This demonstrates that both the asymmetry and the polarity of each cortical unit may be assessed in the living cell. This technique has useful applications in the study of morphogenesis and patterning in living cells and for the screening of mutations and variants conferring altered cortical phenotypes.     

Protein phosphorylation and dynamics of cytoskeletal structures associated with basal bodies in Paramecium

The presence of phosphorylated proteins associated with microtubule organizing centers in tissue culture cells during mitosis has been demonstrated by the use of monoclonal antibodies raised against mitotic HeLa cells [Vandre et al., Proc. Natl. Acad. Sci. U.S.A. 81:4439-4443, 1984]. We report here that in Paramecium two of the mitosis specific antibodies, MPM-1 and MPM-2, decorate throughout the cell cycle all the microtubule organizing centers (MTOCs) located in the cortex and in the oral apparatus (gullet). Immuno-electron microscopy showed that these antibodies labeled the electron-dense material surrounding basal bodies from which several microtubule networks as well as kinetodesmal fibers originate. During mitosis, these antibodies also stained other cortical cytoskeletal structures, the kinetodesmal fibers (MPM-1 and MPM-2) and the epiplasm (MPM-1). Among the different polypeptides recognized by the antibodies on immunoblots, three major ones of 60, 63, and 116 kDa were found to be common to the cortex (where several thousand ciliary basal bodies are anchored) and the oral apparatus (which comprises several hundred basal bodies around which various arrays of cytoplasmic microtubules are organized). Alkaline phosphatase treatment abolished the immunoreactivity of the polypeptides and the labeling observed by immunofluorescence. These results demonstrate that phosphorylated proteins are associated with all the known active microtubule organizing centers present in the cortex throughout the cell cycle of Paramecium. Furthermore they indicate that in Paramecium phosphorylation of proteins could also be involved in the cell cycle dependent dynamics of cortical cytoskeletal structures other than microtubules.     

The Ultrastructure of Specimens of Paramecium multimicronucleatum Impregnated with Silver by the Fernández-Galiano Method1

Transmission and scanning electron microscopy of specimens of Paramecium multimicronucleatum treated with the Rio-Hortega silver-impregnation method as modified by Fernández-Galiano demonstrate that considerable deposition of silver occurs around the kinetosomes, especially at the level of the basal plate and also at the proximal end of the kinetosome. In addition, silver is heavily deposited within the kinetodesmal fibers, in the fibrous matrix that surrounds the postciliary and transverse microtubules, in the connective structures observed between the two kinetosomes of a pair and between the kinetodesmal fiber and the anterior kinetosome, and in the trichocysts. Differences and similarities in sites of deposit when other methods of silver impregnation are employed are discussed and the particular value of the present technique in studies of ciliate systematics and phytogeny is stressed.     

CORTICAL ULTRASTRUCTURE OF PARAMECIUM AURELIA : Studies on Isolated Pellicles

Two methods have been devised for the isolation of large quantities of purified pellicles (cortical layers) of Paramecium aurelia. Pellicles isolated by both procedures, when examined by electron microscopy, were found to contain ciliary basal bodies, two types of cortical membranes, ribbons of microtubules, kinetodesmal fibers, and elements of the infraciliary lattice system. By electron microscopy, the extent of preservation of the various cortical structures when pellicles are isolated by each method has been characterized. Pellicles isolated in both ways have been utilized to investigate cortical morphology of Paramecium. Both phase-contrast and electron microscopic observations have been made. Many new ultrastructural features were observed and are reported herein. An interesting result of this study is the discovery in stock CD that the structure of cortical territories (the territory is the functional unit of cortical morphogenesis and physiology) may vary within a single organism. Features which show variation include number of parasomal sacs, microtubular ribbons, and basal bodies (and therefore cilia) per territory, number of microtubules per ribbon, and length of kinetodesmal fibers. The possible significance of these variations, with respect to territory replication, is discussed. In addition, preliminary observations on the solubility of various cortical organelles in the presence of a number of protein-denaturing agents are reported.     

Die stoffwechselphysiologischen Beziehungen zwischen Paramecium bursaria Ehrbg. und Chlorella spec. in der Paramecium bursaria-Symbiose

Zusammenfassung Infektionsexperimente algenfreier Paramecium bursaria mit aus diesen isolierten und unter Stickstoffmangel-Bedingungen vorkultivierten Algen deuten darauf hin, daß die Versorgung der endosymbiontischen Algen mit stickstoffhaltigen Verbindungen durch ihren Wirt in einem zu gutem Wachstum und Vermehrung der Alge ausreichendem Maße möglich ist. Die Bedeutung dieser stoffwechselphysiologischen Beziehung für die Symbiosepartner wird diskutiert.Die Vergiftung der Photosynthese der endosymbiontischen Chlorella durch 3-(3,4-Dichlorphenyl)-1,1-dimethylharnstoff (DCMU) führt in grünen Paramecium bursaria durch Beeinflussung des Kohlenstoff-Stoffwechsels zu einer Entkoppelung des symbiontischen steady state-Systems und damit zur Auflösung der Symbiose. Eine ausreichende heterotrophe Ernährung der Alge durch das Paramecium ist in der Symbiose offenbar nicht möglich.Die Anwendung von 3-(3,4-Dichlorphenyl)-1,1-dimethylharnstoff (DCMU) kann als neue Methode zur Züchtung algenfreier Paramecium bursaria dienen.

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The metabolic interactions between Paramecium bursaria Ehrbg. and Chlorella spec. in the Paramecium bursaria-symbiosisI. The nitrogen and the carbon metabolism

Symbiotic Chlorellae have been isolated from Paramecium bursaria Ehrbg. and cultivated under conditions of nitrogen deficiency. Reinfection of Chlorella-free Paramecium bursaria with these nitrogen-deficient algae resulted in a complete regeneration and multiplication of the algae within the host cells. The endosymbiotic algal cells of the Paramecium bursaria-symbiosis can be supplied by their host with nitrogen.The inhibition of photosynthesis by 3-(3,4-Dichlorophenyl)-1,1-dimethylurea (DCMU) leads in green Paramecium bursaria to a breakdown of the symbiotic steady state-system resulting in a loss of algal cells. Obviously the endosymbiotic algae cannot be fed heterotrophically by their host to such an extent that a stable symbiosis is maintained.The application of 3-(3,4-Dichlorophenyl)-1,1-dimethylurea (DCMU) can be used as a new method for culturing Chlorella-free Paramecium bursaria.

     

PARTIAL PURIFICATION AND PHOSPHOTUNGSTATE SOLUBILIZATION OF BASAL BODIES AND KINETODESMAL FIBERS FROM TETRAHYMENA PYRIFORMIS

Previously devised methods for the isolation of basal bodies from ciliate protozoans were found to be inadequate for chemical analysis. We have modified and expanded these procedures and developed a method which gives preparations containing mainly basal bodies and kinetodesmal fibers. This procedure involved fixation of cells in 30% ETOH followed by digitonin or Triton X-100 solubilization and homogenization with a Brinkmann Polytron. This is followed by sucrose gradient centrifugation. Negative staining and thin sectioning revealed these preparations to be substantially more pure than those of previous workers. It was also found that neutralized phosphotungstate (PTA) solubilized many of the components present in fixed Tetrahymena. Neutralized 1.0% PTA solubilized axonemes, cortical, axonemal, and basal body microtubules as well as kinetodesmal fibers. These results have been confirmed by both electron microscope observations and gel electrophoresis of 100,000 g supernatants of the PTA extracts. A solution of 0.1% PTA did not affect the fibers but did solubilize basal bodies. Running 1.0% PTA extracts from our basal body fractions on sodium dodecyl sulfate (SDS) polyacrylamide gels allowed us to tentatively identify the peptides of basal bodies and kinetodesmal fibers. The latter structures appear to consist of a single 21,000 mol wt peptide. These results also suggest that great caution should be taken in interpreting PTA images, especially of microtubules and axonemes.     

Motility Events of Trichocyst Insertion in Paramecium tetraurelia*

SYNOPSIS. Following electroshock-induced extrusion of its inserted trichocysts, Paramecium tetraurelia rapidly begins replacement of the population of lost organelles. Light microscopy of the cortical insertion of new trichocysts reveals a series of characteristic motility activities. An uninserted trichocyst in the cyclotic flow of the cell appears to be “captured” and removed to the noncyclotic, subcortical regions. The trichocyst then makes a series of saltatory motions which apparently serve to transport it to the cortex, with proper orientation (tip first) for insertion. Trichocyst saltations end with either cortical insertion of the organelle, or return to cyclosis. If the trichocyst is inserted, it makes a series of unique pivoting movements around the motionless tip. This form of motility, termed “wobble,” continues for a short period of time. After cessation of wobble, the insertion of the trichocyst is apparently complete, since no further motility is observed. With the aid of these observations it was possible to identify saltatory motility as the means for transporting trichocysts to the cortex for insertion, and also to observe a motility of unknown significance (wobble) apparently associated with the process of cortical insertion.     

Accumulation of DNA damages in aging Paramecium tetraurelia

Summary Paramecium tetraurelia cells of ages 4, 15, and 27 days were labeled with [¹⁴C]-thymidine. In addition, cells were grown clonally for 27 days (108 generations) and labeled with [¹⁴C]-thymidine in the presence of 0.5 or 7.5 g/ml of mitomycin-C (MMC) or no MMC. These cells were gently deposited on a filter membrane, which impedes the passage of DNA strands. The cells were then lysed with detergents and the cellular components washed through the filters, leaving double-stranded DNA intact on the surface. Proteinase K was used to remove histone or DNA-bound proteins. The DNA was then eluted under alkaline conditions, which denatures double-stranded DNA and converts apurinic/apyrimidinic sites into single-strand breaks. The results obtained with the cells of ages 4, 15, and 27 days (16, 60, and 108 generations, respectively) indicate that as Paramecium tetraurelia ages during asexual reproduction, apurinic/apyrimidinic lesions, strand breaks or single-strand gaps accumulate. This accumulation may be the basic mechanism of aging in such cells. In the MMC-treated cells of 27 days (108 generations), the MMC reduced elution of DNA fragments more at the higher than at the lower pH's used; random MMC cross-links should occur more often in longer strands than in shorter strands. The reductions in elution preferentially at higher pH, at which longer single strands would be eluted, confirmed the pH-versuslength relationship for Paramecium DNA eluted under our conditions.     

Aminotransferase and the Production of Alanine During Hyperosmotic Stress in Paramecium calkinsi

When Paramecium calkinsi encounter hyperosmotic stress, intracellular free alanine increases. In vivo assays indicate that the reaction catalyzed by alanine aminotransferase contributes to the build up of alanine in response to hyperosmotic shock. ¹⁴C-pyruvate is converted to ¹⁴C-alanine in cells grown axenically at 200 mosm. When shifted to 600 mosm, the rate of conversion of pyruvate to alanine increases, and conversion at either 200 or 600 mosm is blocked by 1 mM aminooxyacetic acid (AOA), an inhibitor of aminotransferase. Intracellular free alanine increase is partially inhibited by AOA, and AOA prevents cells living in fresh water from acclimating to higher salinities, an indication that the increase in intracellular alanine is physiologically significant.     

Method for the Simultaneous Establishment of Many Axenic Cultures of Paramecium*†

SYNOPSIS. A method is described for the simultaneous treatment of 42 (or more) stocks of Paramecium, and their adaptation to growth in axenic culture. Samples of dense cultures of these ciliates growing with Enterobacter aerogenes are rendered bacteria-free by migration through 2 sets of tubes containing Adaptation Medium (Peters' salts solution, stigmasterol, vitamins, and autoclaved E. aerogenes). The 2nd set of tubes contains Adaptation Medium plus antibiotics. Bacteria-free samples containing ～ 100 animals are then transferred to test tubes containing Adaptation Medium without antibiotics. This medium also serves as a growth medium. It supports indefinite growth of all Paramecium stocks tested. After adaptation to this medium, the ciliates can be grown in the axenic medium developed by Soldo, Godoy & van Wagtendonk. On a single trial at least half of the stocks can be expected to produce axenic cultures within 5 to 10 days by these procedures. The method has been applied successfully to several of the species of the Paramecium aurelia complex, to all syngens of Paramecium multimicronucleatum, to several stocks of Paramecium jenningsi, and to 1 stock each of Paramecium caudatum and Paramecium calkinsi. A modification of the method also works for Didinium nasutum.     

Directed positioning of nuclei in living Paramecium tetraurelia: Use of the laser optical force trap for developmental biology

We have constructed a laser optical force trap (“laser tweezers”) by coupling an Nd:YAG laser to an optical microscope with a high numerical aperture objective. The laser beam (approximately 0.1 W power) is focused to a diffraction-limited spot at the specimen plane of the objective: the wavelength chosen (1,064 nm) is not strongly absorbed by most biological materials and is thus not ablative. Because the intensity of the laser beam increases towards the center of the focal spot, small particles brought near the spot will be attracted to the center and held there. Movement of the laser beam will tend to move any trapped particles with it. The laser tweezers can permit precise, nondestructive repositioning of small structures inside a living cell, without recourse to micromanipulators. Initial work has involved the use of laser tweezers on cells of Paramecium tet-raurelia held by a rotocompressor. We have been able to trap and reposition small organelles, especially the highly refractile structures known as crystals. Using a trapped crystal as a “tool”, we have been able to push micronuclei and other structures for many micrometers to virtually any desired location in a cell. In spite of extended exposure of specific structures and of individual cells to the laser beam, no damage has been detectible. Exposed cells, which were removed from the rotocompres-sor and cultured, showed complete viabilty. The laser tweezers technique shows tremendous potential for applications to the study of many fundamental cellular and developmental phenomena in paramecia and other ciliates. For example, we intend to use this technique to investigate temporal and spatial characteristics of nuclear determining regions during sexual reorganization in Paramecium. © 1992 Wiley-Liss, Inc.     

Lithium-Induced respecification of pattern in Paramecium

The long-known teratogenic effects (dorsalisation) of lithium on amphibian embryos has recently raised renewed interest. As it is known that lithium blocks the polyphosphoinositide (PI) cycle, causing a depressed level of myo-inositol, and as injections of myo-inostiol have been shown to rescue the effects of Li⁺, it was postulated that Li⁺ causes a flattening of gradients of PI cycle activity underlying the developmental polarities. We have studied the effect of Li⁺ on the morphogenesis of the unicellular organism, Paramecium. We show (1) that exposure to 25 mM Li⁺ during division yields precise distorsions of the cortical pattern that can be explained by a uniformisation of surface growth i.e. partial suppression of the right/left and antero/posterior asymmetries and (2) that Li⁺ effects are rescued by injection of myo- inositol. These results suggest that spatially graded activity of the PI cycle (ensuring in turn a spatially graded distribution of secondary messengers directly involved in the morphogenetic processes) appeared early in evolution. © 1992 Wiley-Liss, Inc.     

Effects of Actinomycin D on Generation Time and Morphogenesis in Paramecium*

SYNOPSIS. The sensitivity of Paramecium tetraurelia (=P. aurelia syngen 4) cells to pulse treatments with various doses of Actinomycin D (AMD) was estimated by comparing the generation times of treated and untreated sister cells. It was found that the delay of division in treated cells depended on the concentration of AMD, on their “age” at the time of the pulse treatment, and on their individual sensitivity. Sensitivity of Paramecium to AMD changes during the cell cycle in a predictable way. About 3 1/2 hr before the normally expected cell fission (total generation time ～ 5 1/2 hr) there is a decrease of sensitivity. Thereafter, the cell enters a new stage with a progressive increase of sensitivity. This 2nd phase ends at the “transition point” (～ 2 hr before cell division), when sensitivity drops abruptly. The division process itself may be altered and slowed down by high concentrations of AMD, even if the drug is applied after the transition point, but this process can never be completely annulled. The impairment of the division mechanism may lead to morphologic anomalies in the offspring. Resorption of oral anlagen in P. tetraurelia probably never occurs during the cell cycle after AMD treatment. The reason for individual variability of the cells, mechanisms controlling development, and the question of an obligate sequence of gene action in each cell cycle are discussed.     

Developmental expression of macronuclear specific antigen in Paramecium caudatum

We obtained a monoclonal antibody (MA-1) specific for macronuclei of the ciliate Paramecium caudotum and P. dubosqui. Immunoblotting showed that the antigen was a poly-peptide of 50 kilodalton (kDa). During the process of nuclear differentiation in P. caudatum, the MA-1 antigens appeared in the macronuclear anlagen immediately after four out of eight post zygotic nuclei differentiated morphologically into the macro-nuclear anlagen. Afterwards, the antigens could be detected in the macronucleus through the cell cycle, and disappeared when the macronucleus began to degenerate in exconjugant cells. These results suggest that the antigens may play a role in the differentiation and function of the macronucleus. © 1992 Wiley-Liss, Inc.     

Economic Mass Cultivation of Paramecium tetraurelia on a 200-Liter Scale1

ABSTRACT. A new and inexpensive medium is described for axenic mass cultivation of Paramecium tetraurelia stock 51s and the double mutant pawn A/pawn B. Skim milk powder is the major carbon and nitrogen source in this medium. Growth characteristics (proliferation rate, final cell density, and cell size) are similar to those observed with other axenic culture media. Cultures were run in one-liter erlenmeyer flasks, a 20-liter, and a 250-liter airlift bioreactor. The yield of a large bioreactor is 750 g (wet wt.) Paramecium or 5 × 10⁹ cells. This easy, economical culture technique will greatly facilitate the use of Paramecium as a model organism for extensive biochemical studies.     

An Inhibition and Kinetic Study of Acid Phosphatase in Paramecium caudatum and Paramecium tetraurelia1

ABSTRACT. Inhibition, inactivation, pH, and kinetic studies using both homogenates and purified lysosomal fractions of Paramecium caudalum and of P. tetraurelia were carried out to examine the lysosomal acid phosphatase (AcPase) and its relationship to p-nitrophenylphosphatase (pNPPase), glucose-6-phosphatase (G6Pase), and 5′-nucleotidase (AMPase). The results generally support the idea that Paramecium cells contain a distinct lysosomal AcPase with a broad substrate specificity. The hydrolysis of glucose-6-phosphate (G6P) and adenosine 5′-monophosphate (AMP) was shown to be due to this enzyme, suggesting that true G6Pase and AMPase may be lacking in these two species; however, some hydrolysis of AMP at pH 7.5 catalyzed by an unknown soluble enzyme distinct from alkaline phosphatase and Na⁺-K⁺-ATPase was observed. Since the hydrolysis of p-nitrophenylphosphate (pNPP) at acid pH was also shown to be due to AcPase alone, pNPPase could be used as a rapid assay for Paramecium AcPase. At an alkaline pH, however, this activity was catalyzed by an alkaline phosphatase located in the cytosol fraction. P. caudatum AcPase was shown to have kinetic properties similar to those of purified rat liver and human prostatic AcPase and to have relative substrate affinities in the order of G6P < β-glycerophosphate < pNPP < AMP. These different substrate affinities might account for the observed differences in the inhibition of the four lysosomal activities by NaF, L(+)-tartrate, and molybdate, all of which inhibited the hydrolysis of G6P, β-glycerophosphate, and pNPP competitively, but which exhibited a noncompetitive inhibition of a mixed type with the hydrolysis of AMP.     

Paramecium sonneborni n. sp., a New Member of the Paramecium aurelia Species-Complex1

ABSTRACT. From an intermittent stream in College Station, Texas, a Paramecium was isolated that did not appear to belong to any recognized species. On the basis of nuclear and whole-body morphology, it can be assigned to the Paramecium aurelia species-complex, and it can be distinguished from other members of that complex on the basis of mating-type reactivity and isoenzyme patterns. These characteristics are felt sufficient to justify a new species assignment. The new species has been named Paramecium sonneborni n. sp. in honor of the late Dr. Tracy M. Sonneborn of Indiana University.     

Directed Positioning of Micronuclei in Paramecium tetraurelia with Laser Tweezers: Absence of Detectible Damage After Manipulation

Possible covert damage from the use of the laser optical force trap (laser tweezers) to reposition micronuclei in Paramecium tetraurelia was assessed by measuring proliferation rates and postautogamous survival and mutation rates of cells after laser manipulations. No differences in subsequent daily proliferation rates among laser manipulated and various control classes of cells were seen. Similarly, the rates of postautogamous lethality and of “slow growth mutations” after repositioning of both micronuclei were not different from such rates in unmanipulated controls. In spite of extensive manipulations of micronuclei by the laser tweezers, there is no evidence of any damage induced by these manipulations. The laser tweezers therefore appears to be a tool of benign effect upon living cells, with tremendous potential use in many cell and developmental biological investigations.     

Polyamine Triggering of Exocytosis in Paramecium Involves an Extracellular Ca2+/(Polyvalent Cation)-Sensing Receptor, Subplasmalemmal Ca-Store Mobilization and Store-Operated Ca2+-Influx via Unspecific Cation Channels

The polyamine secretagogue, aminoethyldextran (AED), causes a cortical [Ca²⁺] transient in Paramecium cells, as analyzed by fluorochrome imaging. Our most essential findings are: (i) Cortical Ca²⁺ signals also occur when AED is applied in presence of the fast Ca²⁺ chelator, BAPTA. (ii) Extracellular La³⁺ application causes within seconds a rapid, reversible fluorescence signal whose reversibility can be attributed to a physiological [Ca²⁺] _i transient (while injected La³⁺ causes a sustained fluorescence signal). (iii) Simply increasing [Ca²⁺] _o causes a similar rapid, short-lived [Ca²⁺] _i transient. All these phenomena, (i–iii), are compatible with activation of an extracellular ``Ca<sup>2+</sup>/(polyvalent cation)-sensing receptor' known from some higher eukaryotic systems, where this sensor (responding to Ca<sup>2+</sup>, La<sup>3+</sup> and some multiply charged cations) is linked to cortical calcium stores which, thus, are activated. In Paramecium, such subplasmalemmal stores (``alveolar sacs') are physically linked to the cell membrane and they can also be activated by the Ca²⁺ releasing agent, 4-chloro-m-cresol, just like in Sarcoplasmic Reticulum. Since this drug causes a cortical Ca²⁺ signal also in absence of Ca²⁺ _o we largely exclude a ``Ca<sup>2+</sup>-induced Ca<sup>2+</sup> release' (CICR) mechanism. Our finding of increased cortical Ca<sup>2+</sup> signals after store depletion and re-addition of extracellular Ca<sup>2+</sup> can be explained by a ``store-operated Ca²⁺ influx' (SOC), i.e., a Ca²⁺ influx superimposing store activation. AED stimulation in presence of Mn²⁺ _o causes fluorescence quenching in Fura-2 loaded cells, indicating involvement of unspecific cation channels. Such channels, known to occur in Paramecium, share some general characteristics of SOC-type Ca²⁺ influx channels. In conclusion, we assume the following sequence of events during AED stimulated exocytosis: (i) activation of an extracellular Ca²⁺/polyamine-sensing receptor, (ii) release of Ca²⁺ from subplasmalemmal stores, (iii) and Ca²⁺ influx via unspecific cation channels. All three steps are required to produce a steep cortical [Ca²⁺] signal increase to a level required for full exocytosis activation. In addition, we show formation of [Ca²⁺] microdomains (≤0.5 μm, ≤33 msec) upon stimulation. Received: 30 August 1999/Revised: 1 December 1999