The role of cell contact in the life cycle of some dinoflagellate species

It is suggested, based on published and experimental data, thatinter- and intra-specific cell contact plays an important rolein the life cycle of some dinoflagellate species.     

Nutritional suitability of the dinoflagellate Ceratium furcoides for four copepod species

The nutritional suitability of Ceratium furcoides for Eudiaplomusgracilis, Mesocyclops leuckarti, Thermocyclops oithonoides andCyclops abyssorum was studied by observing ingestion and assimilation.The dinoflagellate was ingested by female adults of all species.The calanoid copepod E.gracilis could not utilize Ceratium;mortality was high and no egg production was observed when Ceratiumwas the only food source. Mortality was low for the adult cyclopoidson this food and reproduction indicated that Ceratium was assimilated.The first two copepodite instars of M.leuckarti were not ableto handle Ceratium, while older stages preyed on them. The dinoflagellatewas not ingested by female M.leuckarti when its densities werelow. Advanced copepodite stages of C.abyssorum developed intoadults on a diet of Ceratium only. Mesocyclops leuckarti femalesingested Ceratium when offered a mixed food source of Ceratiumand the rotifer Brachionus rubens, but the rotifer was positivelyselected, even if its density was low. The results show thatCeratium provides a suitable food source for advanced copepoditeinstars and adult cyclopoid copepods, although it is not a preferredfood source.     

Distribution of cell surface saccharides on pancreatic cells

We describe here a simple, general procedure for the purification of a variety of lectins, and for the preparation of lectin-ferritin conjugates of defined molar composition and binding properties to be used as probes for cell surface saccharides. The technique uses a “universal” affinity column for lectins and their conjugates, which consists of hog sulfated gastric mucin glycopeptides covalently coupled to agarose. The procedure involes: (a) purification of lectins by chromatography of aqueous extracts of seeds or other lectin-containing fluids over the affinity column, followed by desorption of the desired lectin with its hapten suge; (b) iodination of the lectin to serve as a marker during subsequent steps; (c) conjugation of lectin to ferritin with glutaraldehyde; (d) collection of active lectin-ferritin conjugates by affinity chromatography; and (e) separation of monomeric lectin-ferritin conjugates from larger aggregates and unconjugated lectin by gel chromatography. Based on radioactivity and absorbancy at 310 nm for lectin and ferritin, respectively, the conjugates consist of one to two molecules of lectin per ferrritin molecule. Binding studies of native lectins and their ferritin conjugates to dispersed pancreatic acinar cells showed that the conjugation procedure does not significantly alter either the affinity constant of the lectin for its receptor on the cell surface or the number of sites detected.     

Lectin histochemistry of mouse vagina during the estrous cycle

Estrous cycle-related histochemical changes in the vaginal epithelium of sexually mature female mice were studied with 30 fluorescein isothiocyanate (FITC)-labeled lectins. On the basis of the staining pattern the lectins were divided into five groups: I, seventeen lectins that reacted with mucinous surface layer of proestrus. This group comprised two subgroups: Ia, seven lectins that reacted exclusively with the mucinous layer, and Ib, ten lectins that reacted with mucinous cells and the underlying squamous epithelium of proestrus; II, two lectins that reacted with squamous epithelium of proestrus only but were unreactive with mucinous cells; III, three lectins that reacted in a phase-specific manner with squamous epithelium; IV, six lectins that showed increased luminal surface reactivity in diestrus and/or metestrus; and V, eleven lectins that were unreactive with vaginal epithelium. These data indicate that the cyclic changes in the morphology of the vaginal epithelium are accompanied by distinct lectin reactivity patterns.     

Ultrastructural labeling of cell surface lectin receptors during the cell cycle.

The distribution of specific surface receptors in the course of the cell cycle has been studied on two transformed cell lines by means of ultrastructural labelling techniques employing Concanavalin A (ConA) and wheat germ agglutinin (WGA). Synchronized cultures of Cl₂TSV₅, an SV40-transformed hamster cell line and of CHO cells were labelled as monolayers or in suspension in the different phases of the cell cycle. In cells labelled in monolayers, a moderately discontinuous pattern of surface labelling was present during G 1, S, and G 2. On cells in mitosis, however, this pattern changes strikingly and becomes very discontinuous. These results indicate that the degree of receptor clustering is greater in mitosis than in interphase. In cells labelled in suspension, the differences in pattern between mitosis and interphase were absent. Colcemid treatment did not modify the distribution of the label, either in interphase or in mitosis. Moreover, cells in mitosis collected by Colcemid treatment and labelled at a moment in which parallel unblocked cultures had completed mitosis and passed into G 1 showed an interphase-type labelling pattern; this indicates that a certain dissociation exists between surface events and nuclear events during mitosis. These results are discussed in terms of several factors that may contribute to the production of receptor clustering, namely, direct lectin action, surface movement and membrane flow, participation of cytoplasmic structures and, finally, attachment of cells to a substratum.     

Development of cell surface saccharides on embryonic pancreatic cells

Using a battery of seven lectin-ferritin conjugates as probes for cell surface glycoconjugates, we have studied the pattern of plasmalemmal differentiation of cells in the embryonic rat pancreas from day 15 in utero to the early postpartum stage. Our results indicate that differentiation of plasmalemmal glycoconjugates on acinar, endocrine, and centroacinar cells is temporally correlated with development and is unique for each cell type, as indicated by lectin-ferritin binding. Specifically, (a) expression of adult cell surface saccharide phenotype can be detected on presumptive acinar cells as early as 15 d in utero, as indicated by soybean agglutinin binding, and precedes development of intracellular organelles characteristic of mature acinar cells; (b) maturation of the plasmalemma of acinar cells is reached after intracellular cytodifferentiation is completed, as indicated by appearance of Con A and fucoselectin binding sites only at day 19 of development; conversely, maturation of the endocrine cell plasmalemma is accompanied by "loss" (masking) of ricinus communis II agglutinin receptors; and (c) binding sites for fucose lectins and for soybean agglutinin are absent on endocrine and centroacinar cells at all stages examined. We conclude that acinar, centroacinar, and endocrine cells develop from a common progenitor cell(s) whose plasmalemmal carbohydrate composition resembles most closely that of the adult centroacinar cell. Finally, appearance of acinar lumina beginning at approximately 17 d in utero is accompanied by differenetiation of apical and basolateral plasmalemmal domains of epithelial cells, as indicated by enhanced binding of several lectin-ferritin conjugates to the apical plasmalemmal, a pattern that persists from this stage through adult life.     

Circadian rhythms of cell cycle processes in the marine dinoflagellate Gonyaulax polyedra

The circadian expression of several growth properties was examined in the dinoflagellate Gonyaulax polyedra under constant light and light-dark conditions. The cell concentration, mean cell volume and rate of DNA synthesis varied in a circadian rhythm, with the primary maximum of cytokinesis and DNA synthesis at about dawn. High rates of cell mortality also occurred during phases related to events of cytokinesis, and may be important in the expression of the other rhythms and in "red tide" generation. Flow-cytofluorimetric analysis indicated that cells of a population contain either a relatively high or a low amount of DNA, but the proportion of cells in each of these classes and the absolute amount of DNA in each cell varied rhythmically depending on the circadian time. This DNA-distribution pattern was unlike the usual G1-S-G2+M pattern typical of eukaryotic cell populations. Isotopically labelled thymidine, used as a marker of DNA synthesis, was continuously incorporated; but the incorporation rate fluctuated in a regular pattern that repeated each circadian period.     

Lectin binding to neural crest cells. Changes of the cell surface during differentiation in vitro

To examine possible changes in cell surface carbohydrates, fluorescent lectins were applied at various times during differentiation of neural crest cells in vitro. The pattern and intensity of binding of several lectins changed as the crest cells developed into melanocytes and adrenergic cells. Considerable amounts of concanavalin A (Con A) and wheat germ agglutinin (WGA) bound to all unpigmented cells throughout the culture period. Melanocytes, however, bound much less of these lectins. Soy bean agglutinin (SBA), unlike Con A and WGA, only bound later in development to unpigmented cells at about the time when catecholamines were detected histochemically. Binding of SBA could be induced in younger cultures by pretreating the cells with neuraminidase. Melanocytes, however, did not bind detectable amounts of SBA even if treated with neuraminidase. The SBA-binding sites were often concentrated on cytoplasmic extensions and on contact points between neighboring cells, even when receptor mobility was restricted by prefixation of the cells or adsorption of lectin at 0 degrees C. All three lectins bound to cell processes resembling nerve fibers in particularly high amounts.     

Buoyancy control of four species of eleotrid fishes during aquatic surface respiration

Synopsis Four species of Australian Eleotridae from hypoxic habitats were examined in the laboratory to study buoyancy control in hypoxic water (<10 torr) when performing aquatic surface respiration (ASR; irrigating gills with upper millimeter of surface water). A conflict can arise here because O₂ can be reabsorbed from the swimbladder (reducing buoyancy) at a time when additional lift may be required to perform ASR. Three species were negatively buoyant and initially performed ASR while resting on the bottom in shallow water. After 24 h swimbladder lift increased to nearly neutral and ASR was performed while fish were pelagic. The fourth species remained pelagic at near neutral buoyancy in hypoxic water. With sudden exposure to hypoxia these physoclists reabsorbed between 5–27% (depending on species) of swimbladder volume (standard pressure) during the initial 30–90 min exposure to hypoxia. Additional experiments on one species (Hypseleotris galii) showed such loss to occur at O₂ tensions below 68 torr and when O₂ declined rapidly (2.17 torr min^-1). Secretion of gas compensated for losses under slower, natural rates of nocturnal O₂ decline. Eleotrids appear to reduce the conflict between respiration and buoyancy control in hypoxia by restricting gas reabsorbtion from the swimbladder and by rapidly secreting gases into the swimbladder.     

Microtubular spindle and centrosome structures during the cell cycle in a dinoflagellate Crypthecodinium cohnii B.: an immunocytochemical study

In order to determine if a mitotic spindle organizing center is present in dinoflagellate cells, we used a library of 12 monoclonal antibodies obtained by immunizing mice with isolated human centrosomes. When tested by immunofluorescence on cryosections of the dinoflaggelate Crypthecodinium cohnii B., a positive labeling was obtained with three of these antibodies. In interphase cells, the anti-centrosome antibodies labeled structures located either in the cell periphery, corresponding probably to both basal bodies (i.e. kinetosomes) and in the perinuclear area. In the latter case, two punctate structures were observed near the nuclear envelope. They have never been described, either in light, or in electron microscopic studies of dinoflagellates. We have designated them as centrosome-like structures. A microtubular desmose reacting positively with anti-tubulin Ab was also visible, linking kinetosomes and centrosome-like structures. During mitosis, the double punctate structures were observed at the poles of the nucleus. Double immunolabeling with tubulin and anti-centrosome Ab was also carried out and strongly suggested that in mitotic cells, centrosome-like structures, located at the poles of the mitotic spindle, were associated with microtubular bundles and probably organize and polarize them. These data indicate the existence of centrosome-like structures in C. cohnii cells and the strong conservation of some centrosomal epitopes from dinoflagellates to human. One of the antibodies (CTR 210) recognized by immunoblotting, a single protein band at 72 kDa from a total protein extract. The direct demonstration that this protein is located at the centrosome-like structures and at the kinetosomes deserves further study.     

Simultaneous cell surface phenotype and cell cycle analysis of lymphocytes by flow cytometry

A method for the simultaneous measurement of cell surface components and nucleic acids (DNA and RNA) of human lymphocytes by flow cytometry has been developed, thereby providing a means of analyzing cell surface changes during the various phases of the cell cycle. Unfixed cells were coated with fluorescein-conjugated concanavalin A (F Con A) or surface antigen-specific antibody, fixed sequentially with paraformaldehyde and methanol, treated with specific nucleases, and then stained with propidium iodide. Neither portion of the procedure (cell surface staining, nucleic acid staining) interfered significantly with the other. Cell cycle phases of phytohemagglutinin-stimulated human lymphocytes as determined by this method were comparable with those identified by acridine orange staining. Cell cycle-specific blocking agents were used to additionally demonstrate the specificity of the staining procedure. Simultaneous measurement of cell cycle phase and detection of surface receptors for Con A and T lymphocyte surface determinants was performed with this method.     

Effects of light and nitrogen limitation on the cell cycle of the dinoflagellate Amphidinium carteri

Cell cycle phase durations of cultures of Amphidinium carteriin light- or nitrogen-limited balanced growth were determinedusing flow cytometry. For both types of growth rate limitation,the increases in generation time caused by increasing degreesof limitation were due solely to expansion of the G₁ phase ofthe cell cycle. The durations of the S and G₂ + M phases wereindependent of growth rate. Furthermore, when cells were deprivedcompletely of light and nitrogen, they arrested in the G₁ phaseof the cell cycle. The results indicate that light- and nitrogen-dependentprocesses are heavily concentrated in the early part of thecell cycle, while DNA replication and cell division, once initiated,are independent of light or nitrogen supply.     

Morphometrical analysis of Sertoli cell ultrastructure during the seminiferous epithelial cycle in rats

Stage-dependent variations of Sertoli cell organelles during the seminiferous epithelial cycle were analyzed morphometrically in rats by use of a point-counting method. Cyclic changes in volume and surface area of various organelles were observed. Mitochondria and rough endoplasmic reticulum increased at stages VII-VIII and stage VII, respectively. Lipid droplets accumulated markedly after spermiation, decreased rapidly after meiotic division, and remained at low levels for stages IV-VIII. The most prominent change was a topographic alteration of the Golgi apparatus. It was usually located exclusively in the basal cytoplasm, but shifted upwards to the mid and apical cytoplasm at stages VII-VIII. This shift may be implicated in an increase of plasma membrane and lysosomes in these regions. Consecutive increases of primary and secondary lysosomes were observed twice in the basal and mid cytoplasm. The first peak of the primary lysosomes at stage IV was followed by the first peak of the secondary lysosomes at stage VI; and the second peak of the primary lysosomes at stages VII-IX was followed by the second peak of the secondary lysosomes at stage IX. These consecutive increases may indicate that Sertoli cells anticipate the increase of structures to be removed and accordingly produce primary lysosomes before their appearance.     

Analysis of the mechanism of dinoflagellate flagella contraction-relaxation cycle

Summary— Dinoflagellates possess two flagella. One of them, the longitudinal flagellum, retracts from time to time in some species, such as Ceratium and Peridinium. Additional structures which run along the axoneme seem to be responsible for this particular behaviour. The retraction which is rapid (less than 60 ms) may be subdivided into several steps: i) the undulating movement stops; ii) the flagellum appears then as a jagged line during 20 ms; iii) finally a rapid retraction (20 ms) takes place, the flagellum being folded 20 times inside the cylindrical flagellar pocket. The measurements on video-records suggest that the R-fibre shortens to 30% of its original length. The contraction and relaxation mechanism of nanofilaments is proposed to be through coiling and uncoiling dependent on Ca²⁺ concentration.     

Cellulose synthesis is coupled to cell cycle progression at G1 in the dinoflagellate Crypthecodinium cohnii

Cellulosic deposition in alveolar vesicles forms the "internal cell wall" in thecated dinoflagellates. The availability of synchronized single cells, the lack of secondary deposition, and the absence of cellulosic cell plates at division facilitate investigation of the possible roles of cellulose synthesis (CS) in the entire cell cycle. Flow cytograms of cellulosic contents revealed a stepwise process of CS in the dinoflagellate cell cycle, with the highest rate occurring at G(1). A cell cycle delay in G(1), but not G(2)/M, was observed after inhibition of CS. A cell cycle inhibitor of G(1)/S, but not G(2)/M, was able to delay cell cycle progression with a corresponding reduction of CS. The increase of cellulose content in the cell cycle corresponded well to the expected increase of surface area. No differences were observed in the cellulose to surface area ratio between normal and fast-growing G(1) cells, implicating the significance of surface area in linking CS to the coupling of cell growth with cell cycle progression. The coupling of CS to G(1) implicates a novel link between CS and cell cycle control, and we postulate that the coupling mechanism might integrate cell wall integrity to the cell size checkpoint.     

Colocalization of the cyclin B homologue P56 and β-tubulin during the cell cycle in a unicellular eucaryote dinoflagellate

We provide evidence for an unusual behavior of the cyclin B homologue, p56, in the dinoflagellate Crypthecodinium cohnii. p56, of which we previously demonstrated the presence in this original eukaryotic protist, is present all along the cell cycle progression, and is exclusively cytoplasmic as revealed after immunofluorescence labeling with anti-p56 Ab and counterstaining with Dapi. It was never found in the nucleus as is the case in higher eukaryotic cells. During motosis, p56 was essentially associated with the mitotic apparatus: centrosomes and mitotic spindle, as shown after double immunofluorescence labeling with anti p56 and anti β-tubulin Ab. Using high pressure freeze fixation, we clearly detected in transmission electron microscopy (TEM) the localization of p56 cyclin B homologue and β-tubulin: single immunogold labeling demonstrated that p56 is localized along the whole cell cortex, along the cleavage furrow of anaphase to cytokinesis cells and into cytoplasmic channels passing throughout the mitotic nucleus where is located the mitotic spindle. Double immunogold labeling realized with anti-p56 and anti-β-tubulin antibodies confirm that p56 antigens colocalize with β-tubulin in many sites. The significance of the exclusively cytoplasmic localization of the cyclin B homologue is discussed.     

Membranes of animal cells. V. Biosynthesis of the surface membrane during the cell cycle

KB cells grown in suspension culture were synchronized by using a double thymidine block. At various times throughout the life cycle aliquots of cells were pulsed with ¹⁴C-L-leucine, ¹⁴C-D-glucosamine and ¹⁴C-choline for one hour periods. Surface membranes, cell particulates and soluble proteins were isolated and their ¹⁴C specific activities were determined. It was found that there was a marked increase in the rate of incorporation into surface membrane just after division. The pattern of incorporation was the same for all three isotopic precursors. The rate of incorporation of isotopic precursors into soluble proteins was constant throughout the cycle. Some increase in rate of incorporation of isotope into the particulate fraction was observed during division.