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Lectin binding sites inParamecium tetraurelia cells

I. Labeling analysis predominantly of secretory components

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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.

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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

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Author notes

  1. N. Lüthe

    Present address: Abteilung für Experimentelle Kardiologie, Max Planck Institut für Physiologische und Klinische Forschung, D-6350, Bad Nauheim, Federal Republic of Germany

Authors and Affiliations

  1. Faculty of Biology, University of Konstanz, P.O.B. 5560, D-7750, Konstanz, Federal Republic of Germany

    N. Lüthe, H. Plattner & B. Haacke

  2. Department of Cell Biology and Electron Microscopy, ETH Zürich, CH-8092, Zürich, Switzerland

    P. Walther & M. Müller

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  1. N. Lüthe
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  5. M. Müller
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Lüthe, N., Plattner, H., Haacke, B. et al. Lectin binding sites inParamecium tetraurelia cells. Histochemistry 85, 365–376 (1986). https://doi.org/10.1007/BF00982666

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