Histochemistry

, Volume 85, Issue 5, pp 365–376 | Cite as

Lectin binding sites inParamecium tetraurelia cells

I. Labeling analysis predominantly of secretory components
  • N. Lüthe
  • H. Plattner
  • B. Haacke
  • P. Walther
  • M. Müller
Article

Summary

Though all three lectins tested (ConA, RCA II, WGA) bound to the entire cell membrane, none bound selectively to the docking site of secretory organelles (trichocysts); the same results were achieved with FITC-conjugates, or, on the EM level, with peroxidase- or gold-labeling. Only WGA triggered the release of trichocysts and none of the lectins tested inhibited AED-induced synchronous exocytosis.

When exocytosis was triggered synchronously in the presence of any of these three lectins (FITC-conjugates), the resulting ghosts trapped the FITC-lectins and the cell surface was immediately afterwards studded with regularly spaced dots (corresponding to the ghosts located on the regularly spaced exocytosis sites). These disappeared within about 10 min from the cell surface (thus reflecting ghost internalization with a half life of 3 min) and fluorescent label was then found in ∼6–10 vacuoles, which are several μm in diameter, stain for acid phosphatase and, on the EM level, contain numerous membrane fragments (other-wise not found in this form in digesting vacuoles). We conclude that synchronous massive exocytosis involves lysosomal breakdown rather than reutilization of internalized trichocyst membranes and that these contain lectin binding sites (given the fact free fluorescent probes did not efficiently stain ghosts).

Trichocyst contents were analyzed for their lectin binding capacity in situ and on polyacrylamide gels. RCA II yielded intense staining (particularly of “tips”), while ConA (fluorescence concentrated over “bodies”) and WGA yielded less staining of trichocyst contents on the light and electron microscopic level. Only ConA- and WGA-staining was inhibitable by an excess of specific sugars, while RCA II binding was not. ConA binding was also confirmed on polyacrylamide gels which also allowed us to assess the rather low degree of glycosylation (∼1% by comparison with known glycoprotein standards) of the main trichocyst proteins contained in their expandable “matrix”.

Since RCA II binding could be due to its own glycosylation residues we looked for an endogenous lectin. The conjecture was substantiated by the binding of FITC-lactose-albumin (inhibitable by a mixture of glucose-galactose). This preliminary new finding may be important for the elucidation of trichocyst function.

Keywords

Acid Phosphatase Docking Site Lectin Binding Intense Staining Membrane Fragment 

Abbreviations

AED

aminoethyldextran

BSE

backscatter electrons

ConA

Concanavalin A

DAB

3,3′-diaminobenzidine

EM

electron microscope

FITC

fluorescein-isothiocyanate

kD

kiloDalton

ME

mercaptoethanol

MIP

membrane-intercalate particle

Mr

apparent molecular weight

PAGE

polyacrylamide-gel-electrophoresis

PAS

periodic acid Schiff

pI

isoelectric point

POX

peroxidase

RCA II

Ricinus communis agglutinin II

SDS

sodium dodecylsulphate

SEM

scanning electron microscope

WGA

wheat germ agglutinin

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Adoutte A, Garreau De Loubresse N, Beisson J (1984) Proteolytic cleavage and maturation of the crystalline secretion products ofParamecium. J Mol Biol 180:1065–1081Google Scholar
  2. Allen RD, Fok AK (1980) Membrane recycling and endocytosis inParamecium confirmed by horseradish peroxidase pulsechase studies. J Cell Sci 45:131–145Google Scholar
  3. Allen RD, Fok AK (1983) Phagosome fusion vesicles of Paramecium. I. Thin-section morphology. Eur J Cell Biol 29:150–158Google Scholar
  4. Allen RD, Fok AK (1984) Stages of digestive vacuoles in Paramecium: membrane surface differences and location. Eur J Cell Biol 35:149–155Google Scholar
  5. Allen RC, Spicer SS, Zehr D (1976) Concanavalin A-horseradish peroxidase bridge staining of α-1 glycoproteins separated by isoelectric focusing on polyacrylamide gel. J Histochem Cytochem 24:908–914Google Scholar
  6. Antrata R, Schauer P, Kvapil J, Kvapil J (1984) Single-crystal electron detectors, in Csanády A, Röhlich P, Szabó D (eds) Proc 8th Eur Congr Electron Microscopy Vol 1, Programme Committee, Budapest, pp 617–625Google Scholar
  7. Barondes SH (1981) Lectins: Their multiple endogenous cellular functions. Annu Rev Biochem 50:207–231Google Scholar
  8. Barondes SH (1984) Soluble lectins: a new class of extracellular proteins. Science 223:1259–1264Google Scholar
  9. Barondes SH, Cerra RF, Cooper DNW, Haywood-Reid PL, Roberson MM (1984) Localization of soluble endogenous lectins and their ligands at specific extracellular sites. Biol Cell 51:165–172Google Scholar
  10. Bernhard W, Avrameas S (1971) Ultrastructural visualization of cellular carbohydrate components by means of concanavalin A. Exp Cell Res 64:232–236Google Scholar
  11. Beyer EC, Tokuyasu KT, Barondes SH (1979) Localization of an endogenous lectin in chicken liver, intestines, and pancreas. J Cell Biol 82:565–571Google Scholar
  12. Bhavanandan VP, Katlic AW (1979) The interaction of wheat germ agglutinin with sialoglycoproteins. J Biol Chem 254:4000–4008Google Scholar
  13. Bilinski M, Plattner H, Tiggemann R (1981) Isolation of surface membranes from normal and exocytotic mutant strains of Paramecium tetraurelia. Ultrastructural and biochemical characterization. Eur J Cell Biol 24:108–115Google Scholar
  14. Brown WJ, Hunt RC (1978) Lectins. Int Rev Cytol 52:277–349Google Scholar
  15. Chailley B, N'Diaye A, Biosieux-Ulrich E, Sandoz D (1981) Comparative study of the distribution of fuzzy coat, lectin receptors, and intramembrane particles of the ciliary membrane. Eur J Cell Biol 25:300–307Google Scholar
  16. Chesnel A, Lerivray H, Jego P (1984) Isolement et charactérization partielle d'une lectine dans les sécretions de l'oviducte dePleurodeles waltl. Biol Cell 51:46aGoogle Scholar
  17. Daly JW, Witkop B (1971) Chemistry and pharmacology of frog venoms. In: Bücherl W, Buckley E (eds) Venomenous animals and their venoms, vol 2. Academic Press, New York, pp 497–519Google Scholar
  18. Enders GC, Werb Z, Friend DS (1983) Lectin binding to guineapig sperm zipper particles. J Cell Sci 60:303–329Google Scholar
  19. Estève JC (1972) L'appareil de Golgi des ciliés. Ultrastructure, particulièrement chezParamecium. J Protozool 19:609–618Google Scholar
  20. Evered D, Collins GM (eds) (1982) Membrane recycling. Ciba Foundation Symposium 92, Ciba Foundation. The Pitman Books Ltd LondonGoogle Scholar
  21. Gartner TK, Ogilvie ML (1984) Isolation and characterization of three Ca2−-dependentβ-galactoside-specific lectins from snake venoms. Biochem J 224:301–307Google Scholar
  22. Glossmann H, Neville DM (1971) Glycoproteins of cell surfaces. A comparative study of three different cell surfaces of the rat. J Biol Chem 246:6339–6346Google Scholar
  23. Goldstein IJ, Hayes CE (1978) The lectins: carbohydrate-binding proteins of plants and animals. Adv Carbohyd Chem Biochem 35:127–340Google Scholar
  24. Gordon JA, Staehelin LA, Kuettner CA (1977) Lectin-mediated agglutination of erythrocyte ghost membranes following depletion of membrane protein and intramembrane particles. Exp Cell Res 110:439–448Google Scholar
  25. Gurd JW, Evans WH (1976) Identification of liver plasma membrane glycoproteins which bind to125I-labelled concanavalin. A following electrophoresis in sodium dodecyl sulfate. Can J Biochem 54:477–480Google Scholar
  26. Hand AR, Oliver C (1981) Methods in cell biology, vol 23 Basic mechanisms of cellular secretion. Academic Press, New YorkGoogle Scholar
  27. Hardham AR (1985) Studies on the cell surface of zoospores and cysts of the fungusPhytophthora cinnamomi: The influence of fixation on patterns of lectin labeling. J Histochem Cytochem 33:110–118Google Scholar
  28. Hausmann K (1985) Protozoologie. Georg Thieme, StuttgartGoogle Scholar
  29. Hirano H, Parkhouse B, Nicolson GL, Lennox ES, Singer SJ (1972) Distribution of saccharide residues on membrane fragments from a myeloma-cell homogenate: Its implications for membrane biogenesis. Proc Natl Acad Sci USA 69:2945–2949Google Scholar
  30. Kersken H, Tiggemann R, Westphal C, Plattner H (1984) The secretory contents ofParamecium tetraurelia trichocysts: Ultrastructural-cytochemical characterization. J Histochem Cytochem 32:179–192Google Scholar
  31. Kersken H, Momayezi M, Braun C, Plattner H (1986a) Filamentous actin inParamecium cells: Functional and structural changes correlated with phalloidin affinity labeling in vivo. J Histochem Cytochem 34:455–465Google Scholar
  32. Kersken H, Vilmart-Seuwen J, Momayezi M, Plattner H (1986b) Filamentous actin inParamecium cells: Mapping by phalloidin affinity labeling in vivo and in vitro. J Histochem Cytochem 34:443–454Google Scholar
  33. Lüthe N, Plattner H (1986) Lectin binding sites inParamecium tetraurelia cells. II. Labeling analysis predominantly of nonsecretory components. Histochemistry 85:377–388Google Scholar
  34. Martin TW, Lagunoff D (1984) Mast cell secretion. In: Cantin M (ed) Cell biology of the secretory process. S Karger, Basel München Paris London New York, pp. 481–516Google Scholar
  35. Matt H, Plattner H (1983) Decoupling of exocytotic membrane fusion from protein discharge inParamecium cells. Cell Biol Int Rep 7:1025–1031Google Scholar
  36. Maylié-Pfenninger MF, Jamieson JD (1979) Distribution of cell surface saccharides on pancreatic cells. II. Lectin-labeling patterns on mature guinea pig and rat pancreatic cells. J Cell Biol 80:77–95Google Scholar
  37. Monsigny M, Roche AC, Midoux P (1984) Uptake of neoglycoproteins via membrane lectin(s) of L1210 cells evidenced by quantitative flow cytofluorometry and drug targeting. Biol Cell 51:187–196Google Scholar
  38. Muresan V, Sarras MP, Jamieson JD (1982) Distribution of sialoglycoconjugates on acinar cells of the mammalian pancreas. J Histochem Cytochem 30:947–955Google Scholar
  39. Ogilvie ML, Gartner TK (1984) Identification of lectins in snake venoms. J Herpetol 18:285–290Google Scholar
  40. Orci L, Amherdt M, Roth J, Perrelet A (1979) Inhomogeneity of surface labelling of B-cells at prospective sites of exocytosis. Diabetologia 16:135–138Google Scholar
  41. Pape R, Plattner H (1985) Synchronous exocytosis in Paramecium cells. V. Ultrastructural adaptation phenomena during re-insertion of secretory organelles. Eur J Cell Biol 36:38–47Google Scholar
  42. Parish RW, Schmidlin S, Müller U (1977) The effects of proteases on proteins and glycoproteins ofDictyostelium discoideum plasma membranes. Exp Cell Res 110:267–276Google Scholar
  43. Pfenninger KH, Maylié-Pfenninger MF (1981) Lectin labeling of sprouting neurons. I. Regional distribution of surface glycoconjugates. J Cell Biol 89:536–546Google Scholar
  44. Plattner H, Miller F, Bachmann L (1973) Membrane specializations in the form of regular membrane-to-membrane attachment sites inParamecium. A correlated freeze-etching, and ultrathin-sectioning analysis. J Cell Sci 13:687–719Google Scholar
  45. Plattner H, Matt H, Kersken H, Haacke B, Stürzl R (1984) Synchronous exocytosis inParamecium cells. I. A novel approach. Exp Cell Res 151:6–13Google Scholar
  46. Plattner H, Pape R, Haacke B, Olbricht K, Westphal C, Kersken H (1985a) Synchronous exocytosis inParamecium cells. VI. Ultrastructural analysis of membrane resealing and retrieval. J Cell Sci 77:1–17Google Scholar
  47. Plattner H, Stürzl R, Matt H (1985b) Synchronous exocytosis in Paramecium cells. IV. Polyamino compounds as potent trigger agents for repeatable trigger-redocking cycles. Eur J Cell Biol 36:32–37Google Scholar
  48. Schnitzler S, Renner H, Furkert J (1982) The role of N-acetylneuraminic acid in the triggering of rat mast cells by polycationic molecules. Agents Actions 12:108–110Google Scholar
  49. Schrével J, Gros D, Monsigny M (1981) Cytochemistry of cell glycoconjugates. Prog Histochem. Cytochem 14:1–269Google Scholar
  50. Selman GG, Jurand A (1970) Trichocyst development during the fusion cycle ofParamecium. J Gen Microbiol 60:365–372Google Scholar
  51. Steers E, Beisson J, Marchesi VT (1969) A structural protein extracted from the trichocyst ofParamecium aurelia. Exp Cell Res 57:392–396Google Scholar
  52. Tiggemann R, Plattner H, Rasched I, Baeuerle P, Wachter E (1981) Quantitative data on peroxidatic markers for electron microscopy. With a note on actin identification inParamecium cells. J Histochem Cytochem 29:1387–1396Google Scholar
  53. Vilmart J, Plattner H (1983) Membrane integrated proteins at preformed exocytosis sites. J Histochem Cytochem 31:626–632Google Scholar
  54. Virtanen I, Miettinen A, Wartiovaara J (1978) Lectin-binding sites are found in rat liver cell plasma membrane only on its extracellular surface. J Cell Sci 29:287–296Google Scholar
  55. Wagh PV, Bahl OP (1981) Sugar residues on proteins. Crit Rev Biochem 10:307–377Google Scholar
  56. Walther P, Křîž S, Müller M, Ariano BH, Brodbeck V, Ott P, Schweingruber ME (1984) Detection of protein A gold 15 nm marked surface antigens by backscattered electrons. Scanning Electron Microscopy III:1257–1266Google Scholar
  57. Westphal C, Plattner H (1981) Ultrastructural analysis of the cell membrane-secretory organelle interaction zone inParamecium tetraurelia cells. I — In situ characterization by electron “staining” and enzymatic digestion. Biol Cell 42:125–140Google Scholar
  58. Wyroba E (1980) Release of Paramecium antigen to the non-nutrient medium. Cell Biol Int Rep 4:1–10Google Scholar
  59. Zingsheim HP, Plattner H (1976) Electron microscopic methods in membrane biology. In: Korn ED (ed) Methods in membrane biology. Plenum Press, New York, pp 1–146Google Scholar

Copyright information

© Springer-Verlag 1986

Authors and Affiliations

  • N. Lüthe
    • 1
  • H. Plattner
    • 1
  • B. Haacke
    • 1
  • P. Walther
    • 2
  • M. Müller
    • 2
  1. 1.Faculty of BiologyUniversity of KonstanzKonstanzFederal Republic of Germany
  2. 2.Department of Cell Biology and Electron MicroscopyETH ZürichZürichSwitzerland

Personalised recommendations