Cell Surface Glycoconjugates of Euglena gracilis (Euglenozoa): Modifications under Potassium and Magnesium Deficiency Angelika Preisfeld*, Gabriele Scholten-Beck, Hans Georg Ruppel Universität Bielefeld, Fakultät für Biologie, Postfach 100131, D-33501 Bielefeld, Bundesrepublik Deutschland Z. Naturforsch. 52c, 33-41 (1997); received September 20/0ctober 29, 1996 Deficiency, Euglena gracilis, Lectin Assay, Mucilage Biochemical and ultrastructural examinations on the pellicle of autotrophically grown Eu­ glena gracilis were carried out after three days under potassium and magnesium deficiency. Cell-surface changes were detected by lectin assay. Compared to cells grown in complete medium, deficient cells become larger in shape, accompanied by rising carbohydrate, chloro­ phyll and protein content, bind more and other lectin molecules: an increase of mainly galac­ tose and N-acetylgalactosamine receptors was observed. Investigations with the mucilage stains alcian blue and ruthenium red indicated that mucilaginous material is released under deficient conditions, whereas the control cells show a strong precipitate of these stains well inside the cells beneath the pellicle. Introduction 12-17% lipids (Barras and Stone, 1965; Hofmann A model organism suitable for measuring the and Bouck, 1976; Nakano et al., 1987). influence of deficient nutrition on the cell, and Some surfaces of euglenoid cells have already here especially on the cell membrane, is the green been tested in regard to their lectin binding capac­ flagellated protist Euglena gracilis (Euglenozoa) ity (Vannini et al., 1981; Bre et al., 1986; Strycek with its complex and unusual surface structure. et al., 1992). When the nutrition of Euglena gracilis This unicellular organism possesses a cell mem­ is varied drastically, a change in the cell envelope brane complex, the pellicle. It is composed of re­ with its glycoresidue-rich membrane can be de­ petitive membrane domains - the ridges and tected. It was shown by lectin binding studies that grooves - in an undulating manner, underlaid a lack of cobalamine (vitamine B12) leads to a loss with an epiplasmatic layer of fibrils and a regular of N-acetyl-galactosamine (GalNAc) residues on pattern of microtubules which are interpreted as the one hand (Lefort-Tran et al., 1980; Bre and the peripheral membrane skeleton (Dubreuil and Lefort-Tran, 1984; Bre et al., 1986) and to a muci­ Bouck, 1988). The intussusceptive surface growth laginous layer outside of the cell on the other hand takes place by inserting a new ridge between two (Bre and Lefort-Tran, 1978). old ones (Hofmann and Bouck, 1976; Nakano et Previous studies using a complement bio-assay al., 1987). (Ruppel and Benninghoff, 1983; Benninghoff The complexity of this structure is also reflected et al., 1986) showed that cultivation of Euglena in the chemical composition of the pellicle: the gracilis in magnesium (Mg2+) and potassium (K+) pellicle dry weight consists of about 70-80% pro­ deficient media leads to a change in the comple­ tein, between 6 and 17% carbohydrates and ment binding capacity. A possible explanation is that the sugar residues vary according to the starv­ ing conditions so that the proteins of the human complement system cannot be activated via the al­ ternative pathway, leading to complete inhibition Abbreviations: BS-I, Bandeiraea simplicifolia; ConA, of cytolysis by complement (Johnson, 1994). Canavalia ensiformis; HPA, Helix pomatia; LCH. Lens Deficiency of potassium as main intracellular culinaris; LPH, Limulus polyphemus; RCA, Ricinus cation shows a direct influence on cells. Beside in­ communis; TGA, Tetragonolobus purpureas; WGA, Trit- icum vulgaris; FITC, fluorescein-isothiocyanate hibition of cell proliferation, potassium starvation Reprint requests to Dr. A. Preisfeld. leads to a decrease of the deficient cation in the Fax: 0521-1066-6039. cell. Potassium effects through its important role 0939-5075/97/0100-0033 $ 06.00 © 1997 Verlag der Zeitschrift für Naturforschung. All rights reserved. D Dieses Werk wurde im Jahr 2013 vom Verlag Zeitschrift für Naturforschung This work has been digitalized and published in 2013 by Verlag Zeitschrift in Zusammenarbeit mit der Max-Planck-Gesellschaft zur Förderung der für Naturforschung in cooperation with the Max Planck Society for the Wissenschaften e.V. digitalisiert und unter folgender Lizenz veröffentlicht: Advancement of Science under a Creative Commons Attribution Creative Commons Namensnennung 4.0 Lizenz. 4.0 International License. 34 A. Preisfeld et al. ■ Euglena Mucilage as cofactor for a series of enzymatic reactions in KH2P04 was replaced in each buffer system by photophosphorylation on photosynthesis, in gly­ NaH2P04. Cells were then resuspended in PBS colysis on carbohydrate metabolism and in binding (phosphate buffer containing 140 mM NaCl). of mRNA to ribosomes on protein synthesis (Ltittge and Clarkson, 1989; for review see Bhan- Growth analysis del and Malik, 1988). Magnesium is cofactor for a lot of enzymatic reactions, too, structural compo­ Number of cells, protein content (Lowry et al., nent of ribosomes and nucleic acids and part of 1951), carbohydrate (Roe, 1955) and chlorophyll chlorophyll in photoautotrophic organisms (Hilt content (Schmid, 1971) were determined every et al., 1987). Deficiency leads to inhibition of cell 24h at the end of dark period. division and shows direct effects on the cellular level of potassium ions because of its influence on Lectin assay Na-K-Cl-cotransport (Smith and Maguire, 1993). For detection of carbohydrate residues on the Both cations are considered to influence the per­ pellicle of Euglena gracilis the following lectins meability of membranes (Ryan, 1993). (Table I) conjugated with fluoresceine isothiocya- In our subsequent investigations on surface gly- nate (FITC, Sigma, Deisenhofen, Germany) have coconjugates of Euglena gracilis we examined by been used. lectin binding studies whether lack of magnesium 200 |_il cellsuspension (106 cells/ml PBS) were in­ and potassium initiated a change of the carbohy­ cubated with diluted FITC-lectin (final concentra­ drate residues. Electron microscopic investigations tion of lectin 100 [il/ml in PBS, except W GA and after treatment of cells with ruthenium red gave LPH: 200 (il/ml) for 30 min at 25 °C. After rinsing an insight into the mucilage deposition in the con­ three times with PBS cells were picked up in 200 fil trol and the “starved” pellicle. PBS and measured in a FacScan flowcytometer counting the fluorescence of 5.000 cells each run Materials and Methods (Becton Dickinson; Software Simulset, Hewlett- Cultivation o f Euglena gracilis Packard). An aliquot was examined with an Or­ thoplan / Leitz Fluorescence Microscope, BP 450- Euglena gracilis strain Z (Klebs 1224-5/25) ob­ 490 nm; LP 515 nm. Images were taken with a Ko­ tained from Algensammlung Göttingen was grown dak Ektachrom slide film, 400 ASA. For determin­ under axenic conditions with a light/dark change ing the specifity of lectins a control was performed of 14:10 h at 30 °C. Cell suspension was bubbled using the appropriate sugar(s) as inhibitors prior with air containing 3% C 02. Every three days at to lectin assay (lactose with RCA and BS-I; N- the end of a dark period, the culture was diluted: acetylgalactosaminewith HPA and BS-I; N-Acetyl- 10 ml (0.5 x 106 cells/ml) of the suspension were neuraminic acid with WGA and LPH; a-methyl- transferred to 100 ml fresh medium (Cramer and D-mannoside with ConA and LCH; glucose with Myers, 1952). The deficient media differed from ConA and fucose with TGA). the normal “control” media only in regard to the “starved” minerals: MgS04 x 7 H20 was substi­ Staining o f Euglena gracilis with alcian blue for tuted with K2S04 for the magnesium deficient me­ light microscopy dium; whereas KH2P 0 4 was replaced by NaH2P 04 for the potassium deficient medium. Higher con­ Anionic groups of mucus can be demonstrated centrations of ions needed for substitution (so­ in the pellicle of Euglena gracilis (Cogburn and dium and potassium) did not influence the cells in Schiff, 1984) by histochemical staining with alcian previous investigations (Scholten-Beck, 1996). blue (Sigma). Cells were harvested after three Control cells were rinsed and resuspended with days of cultivation in each media by centrifugation fresh deficient media. For further investigations of 10 ml cellsuspension (106 cells/ml) immediately cells were harvested after three days at the end of after the dark period. For the histochemical pro­ a dark period and rinsed three times with phos­ cedure cells were stained in a solution containing phate buffer (0.02 m KH2P 0 4-Na2H P 0 4, pH 7.2). 0.1% alcian blue in 0.5 m acetic acid (pH 2.5) at In experiments with potassium deficient cells room temperature for 15 min, rinsed three times A. Preisfeld et al. ■ Euglena Mucilage 35 with water and examined with an Olympus BH nol in distilled water). After infiltration, samples microscope. Images were taken with a 200 ASA were embedded in water-mixable Transmit resin Kodak Ektachrom film with brightfield and phase (TAAB), filled into BEEM capsules and left to contrast optics. polymerize for 24 hours at 70 °C. Ultrathin sections of about 60-70 nm were Staining o f Euglena gracilis with ruthenium red for made with a diamond knife (DuPont) on an Ultra­ electron microscopy cut (Reichert Ultracut E) and put onto 200 mesh Cells were harvested as described for alcian blue Cu-grids. Cells were counter-stained with 2% staining and prepared for electron microscopy as (w/v) aquaeous uranylacetate (Merck) and 2% (w/ follows: v) lead citrate (Merck). Ruthenium red (RR) powder (Sigma) was dis­ solved and a 0.5% (w/v) stock solution was pre­ Results pared. A pellet of 10 ml cellsuspension (106 cells/ Growth analysis ml) rinsed three times with EM buffer (0.05 m NaH2P04-Na2HP04, pH 7.0) was fixed with 2.5% Euglena gracilis grown in potassium and magne­ glutaraldehyde (w/v; Serva, Heidelberg, Germany) sium free media show rapid changes in physiologi­ and 0.1% ruthenium red (v/v) in 0.05 m EM buffer cal behaviour.