Ultrastructure and phylogeny of colpodellids (Colpodellida,Alveolata)

The structure of cells of the predatory flagellates Colpodella pseudoedax and C. unguis was studied. The cell was found to contain two heterodynamic flagella, three-membrane pellicle, micropores, subpellicular microtubules, microtubular open conoid, roptries, micronemes, extrusive organelles (trichocysts), and mitochondria with tubular cristae. Upon discharging, trichocysts form cross-striated bands. The thin-walled cylinder lies in the transitional zone of the flagella. Cells reproduce by means of longitudinal binary fission. The similarities between given species and other colpodelids and such between perkinsea, sporozoans, and dinoflagellates are discussed.     

The morphology of predatory flagellate Colpodella pseudoedax Mylnikov et Mylnikov, 2007 (Colpodellida, Alveolata)

The structure of the predatory freshwater flagellate Colpodella pseudoedax was studied. The cell was found to contain two heterodynamic flagella, three-membrane pellicle, micropores, subpellicular microtubules, microtubular open-side conoid, roptries, micronemes, extrusive organelles (trichocysts), and mitochondria with vesicular and tubular cristae. Upon discharge, trichocysts form cross-striated bands. A thin-walled cylinder lies in the transitional zone of the flagella. Cells reproduce by means of longitudinal binary fission. This species differs from similar C. edax by their smaller cell size and lack of reproduction cyst. Similarities between C. pseudoedax and other colpodelids, as well as between colpodellids and perkinseids and sporozoans, are discussed.     

Secretory organelle docking at the cell membrane of Paramecium cells: Dedocking and synchronized redocking of trichocysts

We present the first evidence that secretory organelle docking at the cell membrane can be reversed in vivo. In nondischarge (nd) mutants of Paramecium tetraurelia all trichocysts can be detached from the cell surface within 2–3 h by different means, including cytochalasin B (but not D), high cell density, or Ca²⁺ ionophores. Considering the well-established ultrastructural differences between nd and wild-type (wt) cells, one can conclude that trichocyst docking at the cell periphery involves two docking sites (I, II): Site I ties the organelles to the epiplasm, and site II is the connection to the cell membrane at the fusogenic zone (expressed only in wt cells); both sites are close to the cell surface and only 150 nm apart. When the trigger for detachment of cortically docked trichocysts (high cell density, cytochalasin B) is relieved, trichocysts are synchronously reattached at the cell membrane, within 40–50 min, with a rate of 20–40 organelles/min, which far exceeds spontaneous docking rates. This is therefore also the first report on synchronization of secretory organelle docking. It is shown by radioactive leucine labeling that the same organelles are redocked, because trichocyst biogenesis is minimal under the conditions of de/redocking used. Surprisingly not only redocking but also detachment of trichocysts from the cell surface can be abolished by inhibitors of protein synthesis. Since Ca²⁺ ionophores mimic the effects of other conditions sufficient to detach trichocysts from the cell surface, we assume that a protein-dependent mechanism sensitive to Ca²⁺ (or other ions in exchange) may operate in trichocyst detachment. The precise mechanism involved in attachment or detachment of trichocysts remains to be elucidated.     

Secretory proteins in a ciliate cell: Identification of epitopes common to numerous polypeptides

Secretory vesicles of the ciliate Pseudomicrothorax dubius, called trichocysts, are separated into > 40 proteins by two-dimensional gel electrophoresis. The trichocyst, composed of a shaft and four arms, is in a condensed state when docked in the cell cortex, and it elongates into an extended state during exocytosis. Monoclonal antibodies (mAbs) were raised against trichocyst proteins. Their reactivities were analysed: I) on Western blots of extended, isolated trichocysts by immunolabeling; 2) on entire cells and extended trichocysts by indirect immunofluorescent binding assay (IFA); 3) on semi-thin sectioned cells by IFA; and 4) on ultra-thin sections of cells by immunogold labeling. mAb IV 4E5 labels major trichocyst proteins at 15–19, 22 and 24 kDa, pI 4.6?6.6. The epitope recognized by mAb IV 4E5 is common to as many as 30 proteins and suggests a family of proteins with possible sequence homology. By IFA, the shafts of extended trichocysts are labeled. The shafts of condensed trichocysts are labeled on both semi-thin sections in Lowicryl and ultrathin sections. On semi-thin Epon sections, the part of the trichocyst which is labeled is arm-like. mAb VI 2D12 labels three major trichocyst proteins at 31 kDa, pI 5.0?5.4. The arms of extended trichocysts are labeled by IFA, but are only weakly labeled on ultrathin sections. The shaft of extended trichocysts is labeled by IFA, and the shaft of condensed trichocysts is labeled on ultrathin sections.     

``Frustrated Exocytosis' — A Novel Phenomenon: Membrane Fusion without Contents Release, Followed by Detachment and Reattachment of Dense Core Vesicles in Paramecium Cells

The lipophilic fluorescent dye, FM1-43, as now frequently used to stain cell membranes and to monitor exo-endocytosis and membrane recycling, induces a cortical [Ca²⁺] _i transient and exocytosis of dense core vesicles (``trichocysts') in Paramecium cells, when applied at usual concentrations (≤10 μm) in presence of extracellular Ca<sup>2+</sup> ([Ca<sup>2+</sup>] <sub>o</sub> = 50 μm). When [Ca<sup>2+</sup>] <sub>o</sub> is kept at 30 nm (<[Ca<sup>2+</sup>]<sup>rest</sup> <sub>i</sub> ), in about one third of the population of extrudable trichocysts docked at the cell membrane, FM1-43 induces membrane fusion, visible by FM1-43 fluorescence of the vesicle membrane. However, in this system extrusion of secretory contents cannot occur in absence of any sufficient Ca<sup>2+</sup> <sub>o</sub> . Upon readdition of Ca<sup>2+</sup> <sub>o</sub> or some other appropriate Me<sup>2+</sup> <sub>o</sub> at 90 μm, secretory contents can be released (complete exocytosis). Resulting ghosts formed in presence of Ca<sup>2+</sup>, Sr<sup>2+</sup> or Mn<sup>2+</sup> are vesicular, but when formed in presence of Mg<sup>2+</sup>, for reasons to be elucidated, they are tubular, though both types are endocytosed and lose their FM1-43 stain. In contrast, in presence of [Mg<sup>2+</sup>] <sub>o</sub> = 3 mm (which inhibits contents release), the exocytotic openings reseal and intact trichocysts with labeled membranes and with still condensed contents are detached from the cell surface (``frustrated exocytosis') within ∼15 min. They undergo cytoplasmic streaming and saltatory redocking, with a half-time of ∼35 min. During this time, the population of redocked trichocysts amenable to exocytosis upon a second stimulus increases with a half-time of ∼35 min. Therefore, acquirement of competence for exocytotic membrane fusion may occur with only a small delay after docking, and this maturation process may last only a short time. A similar number of trichocysts can be detached by merely increasing [Mg²⁺] _o to 3 mm, or by application of the anti-calmodulin drug, R21547 (calmidazolium). Essentially we show (i) requirement of calmodulin and appropriate [Me²⁺] to maintain docking sites in a functional state, (ii) requirement of Ca²⁺ _o or of some other Me²⁺ _o to drive membrane resealing during exo-endocytosis, (iii) requirement of an ``empty' signal to go to the regular endocytotic pathway (with fading fluorescence), and (iv) occurrence of a ``filled' signal for trichocysts to undergo detachment and redocking (with fluorescence) after ``frustrated exocytosis'. Received: 20 January 2000/Revised: 5 May 2000     

Regulation of Cortical Proteins in Euplotes*

SYNOPSIS. A method of isolating cortical organelles from single specimens of Euplotes eurystomus, involving lysis in an induced electric current, is described. The isolation technic was coupled with radioautography to study the patterns of incorporation and conservation of labeled proteins in the membranellar band (MB). Isolated single cells of known age were followed thru one or more divisions. A method of distinguishing between daughter cells (proters and opisthes) at division was utilized in some experiments. The old MB, which is apparently morphostatic, incorporates significant amounts of labeled proteins. The pattern of incorporation in total cell proteins at the 1st and 2nd divisions after pulse-chase indicates that the levels of incorporation among daughter cells is equivalent. However, at the 1st division after pulse-chase, the new (opisthe) MB is generally more heavily labeled than the old (proter) MB, and at the 2nd and 3rd divisions new MBs incorporate less label as their development is farther removed in time from the beginning of the chase. The higher level of incorporation in the MB of the 1st division opisthe is maintained thru subsequent divisions, indicating that in Euplotes proteins of the MB are relatively stable.     

Cytoskeleton-secretory vesicle interactions during the docking of secretory vesicles at the cell membrane in Paramecium tetraurelia cells

Stationary-phase cells of Paramecium tetraurelia have most of their many secretory vesicles ("trichocysts") attached to the cell surface. Log-phase cells contain numerous unoccupied potential docking sites for trichocysts and many free trichocysts in the cytoplasm. To study the possible involvement of cytoskeletal elements, notably of microtubules, in the process of positioning of trichocysts at the cell surface, we took advantage of these stages. Cells were stained with tannic acid and subsequently analyzed by electron microscopy. Semithin sections allowed the determination of structural connections over a range of up to 10 micrometer. Microtubules emanating from ciliary basal bodies are seen in contact with free trichocysts, which appear to be transported, with their tip first, to the cell surface. (This can account for the saltatory movement reported by others). It is noteworthy that the "rails" represented by the microtubules do not directly determine the final attachment site of a trichocyst. Unoccupied attachment sites are characterized by a "plug" of electron-dense material just below the plasma membrane; the "plug" seems to act as a recognition or anchoring site; this material is squeezed out all around the trichocyst attachment zone, once a trichocyst is inserted (Westphal and Plattner, in press. [53]). Slightly below this "plug" we observed fasciae of microfilaments (identified by immunocytochemistry using peroxidase labeled F(ab) fragments against P. tetraurelia actin). Their arrangement is not altered when a trichocyst is docked. These fasciae seem to form a loophole for the insertion of a trichocyst. Trichocyst remain attached to the microtubules originating from the ciliary basal bodies--at least for some time--even after they are firmly installed in the preformed attachment sites. Evidently, the regular arrangement of exocytotic organelles is controlled on three levels: one operating over a long distance from the exocytosis site proper (microtubules), one over a short distance (microfilament bundles), and one directly on the exocytosis site ("plug").     

The Trichocysts of Chilomonas paramecium*

SYNOPSIS Axenic cultures of Chilomonas paramecium were grown in media lacking a C-source, resulting in breakdown in autophagosomal vesicles of large numbers of trichocysts. Return of the starved organisms to complete media was followed by a wave of trichocyst formation. Stages in the degeneration and subsequent reformation of trichocysts are described as well as attempted labeling of the developing organelles with ³H-thymidine. A modification of the method of Anderson et al. (2) was used for isolating quantities of exploded trichocysts from Chilomonas. Attempts at isolation of the trichocyst in its coiled state were unsuccessful. Isolated trichocysts mounted on electron microscope grids were subjected to various types of enzymatic digestions.     

Tubular trichocysts in a species ofPyramimonas (P. grossii Parke)

Summary Pyramimonas grossii is shown to possess up to 4 tubular trichocysts per cell each composed of a rolled ribbon 50Å thick and 0.5 wide wound into a flat spiral of 15–25 turns before discharge, and after discharge pushed out into a hollow tube 0.1 wide and up to 35 long, tapered at both ends. The distribution on the cell and various stages of discharge are illustrated. Comparisons are possible with the larger and more complex trichocysts previously known in the Cryptophyceae.Attention is drawn to the need for further information regarding the taxonomic incidence of such trichocysts among other pigmented flagellates before phyletic conclusions can be drawn. Their functional significance is unknown.Dedicated to Prof. Dr.Lothar Geitler on the occasion of his 70th Birthday.     

Manipulation,by nutrient limitation,of the biosynthetic activity of immobilized cells of Capsicum frutescens Mill. cv. annuum

The relationship between the synthesis and accumulation of protein and capsaicin was investigated in cultured cells of Capsicum frutescens Mill. cv. annuum immobilized in reticulate polyurethane. Cells were cultured in media containing reduced concentrations of essential nutrients, in an attempt to manipulate the rates of protein synthesis. Cells cultured in the absence of orthophosphate for 7 d demonstrated no reduction in the incorporation of l-[U-¹⁴C]phenylalanine into soluble protein or an increase in incorporation into capsaicin, compared with controls supplied with orthophosphate. By day 15 of culture, however, a differential incorporation of label was observed. Over a 21-d culture period the intracellular phosphate did not completely disappear. Cells cultured in the absence of nitrate and phosphate combined, however, exhibited some reduction in incorporation of [¹⁴C]phenylalanine into protein and an increased incorporation into capsaicin after 7 d of culture, but the differences were greater at day 15, when increases in the total capsaicin content of the cultures were apparent. There was observed a relationship between the intracellular nitrate concentration, the culture growth index, and the incorporation of [¹⁴C]phenylalanine into soluble protein — each of these factors was inversely related to the incorporation of label into capsaicin and the total capsaicin content of the cultures.Abbreviations HPLC high-performance liquid chromatography - Phe phenylalanine     

Fluorescence staining of living cells with fluorescamine

Summary The interaction of fluorescamine with living plant and animal cells was investigated to determine which subcellular structures and molecular species might react with the dye and to assess its effects on cell viability and function.Plasma and nuclear membranes ofXenopus erythrocytes, mitochondria of sea urchin sperm, growing apices of Timothy root hairs, and various organelles ofNitella andEuglena were labelled as judged by fluorescence microscopy. Cytoplasmic fluorescence was particulate inNitella and easily displaced by moderate centrifugal fields in sea urchin eggs. Chloroplasts and nuclei isolated from cells labelledin vivo exhibited fluorescamine dependent fluorescence.Reaction seemed to have little or no effect on cell viability (Euglena) photoautotrophic growth (Euglena), cell motility (sperm), fertilizability (sperm or egg), embryonic development (sea urchin), or cytoplasmic streaming (Nitella, Timothy).Quantitative fluorometric analysis of thein vivo reactants in sperm indicated a reaction preference for phospholipid over protein compared to control cells dissociated in SDS prior to labelling. The bulk of labelled lipid was phosphatidylethanolamine.These results suggest that fluorescamine is a true vital dye which can label the cell surface as well as penetrate deeply within cells to label a variety of organelles. The distribution of fluorescence and results of chemical analysis suggest thatin vivo the dye may preferentially react with membrane.     

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

 We have localized a structure-bound fraction of the exocytosis-sensitive phosphoprotein, PP63/parafusin (PP63/pf), in Paramecium cells by widely different methods. We combined cell fractionation, western blots, as well as light and electron microscopy (pre- and postembedding immunolabeling), applying antibodies against the recombinant protein. PP63/pf is considerably enriched in certain cortical structures, notably the outlines of regular surface fields (kinetids), docking sites of secretory organelles (trichocysts) and the membranes of subplasmalemmal Ca²⁺-stores (alveolar sacs). From our localization studies we tentatively derive several potential functions for PP63/pf, including cell surface structuring, assembly of exocytosis sites, and/or Ca²⁺ homeostasis. Accepted: 4 December 1997     

d-arabinose metabolism byBacteroides fragilis strain 2044

During growth in the presence of 1-14C-d-arabinose,Bacteroides fragilis strain 2044 formed labeled succinic, acetic, and propionic acis. Degradation of the acids by the Schmidt reaction revealed that at least 89% of the succinate radioactivity was found in the methylene carbons and that 75% and 84% of the label in propionate and acetate were found in the noncarboxyl carbons of these molecules. No label was found in acetate, propionate, or succinate during growth of strain 2044 in the presence of 5-3H-d-arabinose. Strain 2044 converted radioactivity from 1- or 2-labeled glycolic acid and glycine to succinate by a mechanism involving cleavage of the glycine and glycolic acid carbon skeletons. Label from 1- or 2-labeled glycine and 2 but not 1-labeled glycolic acid was found in acetate. Uniformly labeled 14C-glyoxalate gave rise to labeled acetate, but not succinate.Bacteroides fragilis strain 2044 metabolizesd-arabinose by a mechanism involving a 32 cleavage of the molecule.     

Distribution of amino acids in the cellular fractions ofStaphylococcus aureus

WhenStaphylococcus aureus cells were labeled with a single radioactive amino acid for 20 minutes, the highest activity, except for alanine, leucine, and glycine, was found in the free pool. Significant amounts of the above amino acids and also valine and methionine were incorporated into the protein — cell wall fraction.Cells previously labeled with a single amino acid underwent a net loss of radioactivity when transferred to buffer, glucose, or complete medium. An exception was glycine. The greatest loss in activity occurred in the free pool.While some amino acids (alanine, cystine) were transferred from the free pool to the protein — cell wall fraction under all conditions tested, others (glutamic acid, proline) were transferred only under conditions of growth.Cells labeled with certain single amino acids and then transferred to a complete medium lost a significant portion of the label. The most extreme case noted was proline, but other amino acids also effluxed from the cell under these conditions.     

Evidence for the Transfer of Rhizobial Metabolites to the Polyploid Nuclei of Young Clover Nodules

Autoradiography was used to provide evidence for the transfer of Rhizobium produced moieties to the host nuclei of young clover nodule cells. Cells of Rhizobium trifolii Dangeard were labeled with ³H-adenine or 4,5-³H-l -leucine and 3,4-³H-l .-proline, washed free of external label, and allowed to nodulate young seedlings of white clover (Trifolium repens L. cv. White Dutch). Sections (0.5–1.0 μm) of young nodules up to four days old were autoradiographed using the dipping technique. Grain counts indicated movement of tritium from the leucine-proline labeled rhizobia to the polyploid nuclei of two day old clover nodule cells. Acetylene reduction was not detected until approximately 24 hours after the transfer of tritium was observed. No transfer of tritium was observed with ³H-adenine labeled rhizobia. It is hypothesized that nodulating rhizobia may induce clover nodule cells to initiate leghemoglobin synthesis by the transfer of a bacterially produced inducer.     

Physiologische Untersuchungen an einer ungewöhnlich säureresistenten Chlorella

Summary The influence of the hydrogen-ion concentration on the growth and metabolism of a highly acid-resistant green alga, Chlorella ellipsoidea (strain Marburg St), was studied. Chlorella pyrenoidosa (Emerson strain) served as a normal control organism. Growth of Chlorella ellipsoidea occurs in the entire range from Ph 2.0 to Ph 10, whereas for Chlorella pyrenoidosa the limits were found to be Ph 3.5 and Ph 10. Respiration is much less sensitive to hydrogen-ion concentration in the acid-resistant as compared to the normal strain. Thus an increase in acidity from Ph 4.0 to Ph 2.0 increases the respiratory oxygen uptake by 120% in Chlorella pyrenoidosa and by 25% in Chlorella ellipsoidea. In addition, only the less resistant Chlorella pyrenoidosa shows an accumulation of nitrite in the dark in acid culture media, indicating a disturbance of the normal course of nitrate reduction under these conditions. On the other hand, the rate of photosynthesis of both organisms was found to be almost independent of acidity between Ph 4.0 and Ph 2.0. At the acid and alkaline limits of growth in both algae, an inhibition of cell division leads to an increase of cell size and dry weight per cell, frequently connected with the occurrence of bizarre giant cells. — In addition, adaptation phenomena were found to play a role in determining the acid limit of growth. Cells of Chlorella ellipsoidea, after inoculation from normal medium (Ph about 6) into a solution of Ph 2.0, begin growth at a high rate only after a lag of about two weeks. Cells grown previously in an acid medium, however, immediately resume growth upon inoculation into a medium of Ph 2.0. This adaptation involves a considerable reduction of cell size.     

Secretory organelle docking at the cell membrane of Paramecium cells: dedocking and synchronized redocking of trichocysts

We present the first evidence that secretory organelle docking at the cell membrane can be reversed in vivo. In nondischarge (nd) mutants of Paramecium tetraurelia all trichocysts can be detached from the cell surface within 2-3 h by different means, including cytochalasin B (but not D), high cell density, or Ca2+ ionophores. Considering the well-established ultrastructural differences between nd and wild-type (wt) cells, one can conclude that trichocyst docking at the cell periphery involves two docking sites (I, II): Site I ties the organelles to the epiplasm, and site II is the connection to the cell membrane at the fusogenic zone (expressed only in wt cells); both sites are close to the cell surface and only 150 nm apart. When the trigger for detachment of cortically docked trichocysts (high cell density, cytochalasin B) is relieved, trichocysts are synchronously reattached at the cell membrane, within 40-50 min, with a rate of 20-40 organelles/min, which far exceeds spontaneous docking rates. This is therefore also the first report on synchronization of secretory organelle docking. It is shown by radioactive leucine labeling that the same organelles are redocked, because trichocyst biogenesis is minimal under the conditions of de/redocking used. Surprisingly not only redocking but also detachment of trichocysts from the cell surface can be abolished by inhibitors of protein synthesis. Since Ca2+ ionophores mimic the effects of other conditions sufficient to detach trichocysts from the cell surface, we assume that a protein-dependent mechanism sensitive to Ca2+ (or other ions in exchange) may operate in trichocyst detachment. The precise mechanism involved in attachment or detachment of trichocysts remains to be elucidated.     

REJUVENATION OF MELOSIRA GRANULATA (BACILLARIOPHYCEAE) RESTING CELLS FROM THE ANOXIC SEDIMENTS OF DOUGLAS LAKE,MICHIGAN. I. LIGHT MIGROSCOPY AND 14C UPTAKE 1

Resting cells of Melosira granulate (Ehr.) Ralfs were collected from the anoxic sediments of Douglas Lake, Michigan. Sediment containing M. granulata was inoculated into distilled water and incubated in a growth chamber for one week during which observations were made on the cytological differentiation process. Cells classified as “condensed,” i.e. containing a dark brown cytoplasmic mass were identified as resting cells. The differentiation process consisted of a series of gradual cytological changes that included elongation of the cytoplasmic mass and recognition of definable organelles to the point where the cells were non-distinguishable from water column vegetative cells. Differentiating cells accumulated large polyphosphate and lipid granules. However, these granules disappeared just prior to cell division. The complete differentiation or rejuvenation sequence occurred in some cells in less than 24 h. However, not all dormant cells rejuvenated at the same time and it was observed that the lag period for rejuvenation increased with resting cell age (depth of burial in sediments). In the ¹⁴C uptake studies, label was initially observed in condensed state cells. The label gradually progressed to the more differentiated forms. Total carbon uptake during the rejuvenation process was initially lower in the rejuvenating cells, but roughly equal to water column populations after 8 h, indicating a period of high metabolic activity in the rejuvenating cells between 1 and 8 h.     

Sieve element specific labeling with an anti-idiotypic monoclonal antibody mimic of the phytotoxin dothistromin

A monoclonal antibody, 12C9, an anti-idiotypic mimic of dothistromin, a toxin produced by Dothistroma pini, was found to label the cell wall of sieve elements in a number of different plant tissues and species. The antibody labeled apple leaf tissue, tobacco leaf mid vein, leaf and meristem, and Coprosma robusta leaf mid vein. Labeling was restricted to cell walls of sieve elements and did not label the companion cells or the lumen of the cells. The antibody labeled over a wide range of dilutions. This antibody could be used to differentiate sieve elements from other types of phloem. It could also be used to co-localize sieve elements and microorganisms such as phytoplasmas stained with DAPI.     

A light and electron microscopic study of the quadriflagellate green algaSpermatozopsis exsultans

The green flagellateSpermatozopsis exsultans Korshikov has been studied in culture by light and electron microscopy. The organism is naked, bears four flagella and is conspicuously spirally twisted. The ultrastructure and location of cell organelles (except the flagellar apparatus) has been investigated in detail using an absolute configuration analysis. With the exception of a doubling of the flagella and of the secondary cytoskeletal microtubule system,S. exsultans has the exact same complement of organelles occupying the same relative positions as has been described forS. similis. The two species are therefore correctly placed in the same genus. The usefulness of absolute orientations of cell organelles for green algal taxonomy and phylogeny is stressed.Dedicated to Prof.M. Mix on the occasion of her 60th birthday.