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Tracer and freeze-etching analysis of intra-cellular membrane-junctions in Paramecium

With a note on a new heme-nonapeptide tracer

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Summary

In paramecia the membranes of alveoli and trichocysts are permanently connected to the cell membrane by membrane-junctions, which consist of membrane-intercalated particles in a regular geometrical arrangement. Trichocysts contain secretory material discharged by exocytosis. In unfixed or fixed cells these two compartments were impermeable to the following tracers: To “microperoxidases”, i.e. a cytochrome c-derived heme-nonapeptide and a heme-undecapeptide (WM∼1650, 1900) applied in vivo, as well as to lanthanum and cytochrome c used during (La) or after (cytochrome c) fixation. The heme-nonapeptide was prepared by TPCK-trypsin digestion of cytochrome c and subsequent purification by Sephadex gel chromatography—a simple and inexpensive new procedure resulting in preparations of high yield and purity. Tracers entered alveoli only when the plasmalemma and the alveolar membranes ruptured upon glutardialdehyde fixation. In no case were transmembraneous channels detectable in regions containing membrane-intercalated particles; this holds true for all tracers used and for freeze-fracture replicas obtained by tantalum-tungsten evaporation. With regard to attachment sites over trichocysts our results do not support the assumptions by others according to which exocytosis would be driven by an osmotic shift via transmembraneous channels (which would be analogous to inter-cellular coupling phenomena mediated by gap-junctions), unless such channels would be assumed to operate as carriers rather than via diffusion. Tracers did not penetrate trichocysts before exocytosis occurred. The functional role of membrane-intercalated particles on trichocyst attachments remains nuclear. Despite some resemblance with gap-junctions all types of intra-cellular membrane-junctions investigated are functionally “tight” at the level of “resolution” obtained with tantalum-tungsten-shadowing and with the tracers used.

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References

  • Altman, P. L., Dittmer, D. S.: Biology Data Book. 2. Aufl., Bd. I. Bethesda (USA): Fedn. Amer. Soc. Exptl. Biol. (1972)

  • Bachmann, L., Abermann, R., Zingsheim, H. P.: Hochauflösende Gefrierätzung. Histochem. 20, 133–142 (1969)

    Google Scholar 

  • Bachmann, L., Schmitt, W. W., Plattner, H.: Improved cryofixation: Demonstrated on freeze-etched solutions, cell fractions and unicellular organisms. In: Proc. Fifth Europ. Congr. Electron Microscopy, (hrsg. von V. E. Cosslett) S. 244–245. London, Bristol: The Institute of Physics (1972)

    Google Scholar 

  • Bennett, M. V. L.: Function of electrotonic junctions in embryonic and adult tissues. Fed. Proc. 32, 65–75 (1973)

    Google Scholar 

  • Bennett, M. V. L., Spira, M.: Effects of fixatives for electrical coupling and tracer movements between embryonic cells. J. Cell Biol. 59, 22a (1973)

    Google Scholar 

  • Bennett, M. V. L., Spira, M., Pappas, G. D.: Effects of fixatives for electron microscopy on properties of electrotonic junctions between embryonic cells. J. Cell Biol. 55, 17a (1972a)

    Google Scholar 

  • Bennett, M. V. L., Spira, M., Pappas, G. D.: Properties of electrotonic junctions between embryonic cells of Fundulus. Develop. Biol. 29, 419–435 (1972b)

    Google Scholar 

  • Bone, Q., Denton, E. J.: The osmotic effects of electron microscope fixatives. J. Cell Biol. 49, 571–581 (1971)

    Google Scholar 

  • Dickerson, R. E., Takano, T., Eisenberg, D., Kallai, O. B., Samson, L., Cooper, A., Margoliash, E.: Ferricytochrome c. I. General features of the horse and bonito proteins at 2.8 Å resolution. J. biol. Chem. 246, 1511–1535 (1971)

    Google Scholar 

  • Elbers, P. F.: Ion permeability of the egg of Limnea stagnalis L. on fixation for electron microscopy. Biochim. biophys. Acta (Amst.) 112, 318–329 (1966)

    Google Scholar 

  • Fahimi, H. D., Drochmans, P.: Essais de standardisation de la fixation au glutaraldéhyde. I. Purification et détermination de la concentration du glutaraldéhyde. J. Microscopie 4, 725–736 (1965)

    Google Scholar 

  • Feder, N.: A heme-peptide as an ultrastructural tracer. J. Histochem. Cytochem. 18, 911–913 (1970)

    Google Scholar 

  • Feder, N.: Microperoxidase. An ultrastructural tracer of low molecular weight. J. Cell Biol. 51, 339–343 (1971)

    Google Scholar 

  • Gilula, N. B.: Isolation of rat liver gap junctions and characterization of the polypeptides. J. Cell Biol. 63, 111a (1974)

    Google Scholar 

  • Gilula, N. B., Branton, D., Satir, P.: The septate junction: A structural basis for intercellular coupling. Proc. nat. Acad. Sci. (Wash.) 67, 213–220 (1970)

    Google Scholar 

  • Gilula, N. B., Reeves, O. R., Steinbach, A.: Metabolic coupling, ionic coupling and cell contacts. Nature (Lond.) 235, 262–265 (1972)

    Google Scholar 

  • Graham, R. C., Karnovsky, M. J.: The early stages of absorption of injected horseradish peroxidase in the proximal tubules of mouse kidney: Ultrastructural cytochemistry by a new technique. J. Histochem. Cytochem. 14, 291–302 (1966)

    Google Scholar 

  • Habeeb, A. F. S. A., Hiramoto, R.: Reaction of proteins with glutaraldehyde. Arch. Biochem. Biophys. 126, 16–26 (1968)

    Google Scholar 

  • Hopwood, D.: A comparison of the crosslinking abilities of glutaraldehyde, formaldehyde and α-hydroxyadipaldehyde with bovine serum albumin and casein. Histochem. 17, 151–161 (1969)

    Google Scholar 

  • Karnovsky, M. J., Rice, D. F.: Exogenous cytochrome c as an ultrastructural tracer. J. Histochem. Cytochem. 17, 751–753 (1969)

    Google Scholar 

  • Kirshner, N., Viveros, O. H.: The secretory cycle in the adrenal medulla. Pharmacol. Rev. 24, 385–398 (1972)

    Google Scholar 

  • Kraehenbuhl, J. P., Galardy, R. E., Jamieson, J. D.: Preparation and characterization of an immunoelectron microscope tracer consisting of a heme-octopeptide coupled to Fab. J. exp. Med. 139, 208–223 (1974)

    Google Scholar 

  • Krejci, K., Machleidt, W.: Verbesserte Methodik der Aminosäurenanalyse im Nanomol-Bereich. Z. Physiol. Chem. 350, 981–993 (1969)

    Google Scholar 

  • Loewenstein, W. R.: Permeability of membrane junctions. Ann. N.Y. Acad. Sci. 137, 441–447 (1966)

    Google Scholar 

  • Loewenstein, W. R.: Membrane junctions in growth and differentiation. Fed. Proc. 32, 60–64 (1973)

    Google Scholar 

  • Margoliash, E., Smith, E. L., Kreil, G., Tuppy, H.: Amino-acid sequence of horse heart cytochrome c. The complete amino-acid sequence. Nature (Lond.) 192, 1125–1127 (1961)

    Google Scholar 

  • McNutt, N. S., Weinstein, R. S.: Membrane ultrastructure at mammalian intercellular junctions. Progr. Biophys. Molec. Biol. 26, 47–101 (1973)

    Google Scholar 

  • Mercer, E. H., Birbeck, M. S. C.: Electron Microscopy. A Handbook for Biologists. Oxford: Blackwell Scientific Publ. 1966

    Google Scholar 

  • Morel, F. M. M., Baker, R. F., Wayland, H.: Quantitation of human red blood cell fixation by glutaraldehyde. J. Cell Biol. 48, 91–100 (1971)

    Google Scholar 

  • Nickel, E., Grieshaber, E.: Elektronenmikroskopische Darstellung der Muskelkapillaren im Gefrierätzbild. Z. Zellforsch. 95, 445–461 (1969)

    Google Scholar 

  • Orwin, D. F. G., Thomson, R. W., Flower, N. E.: Plasma membrane differentiation of keratinizing cells of the wool follicle. J. Ultrastruct. Res. 45, 1–14 (1973)

    Google Scholar 

  • Penttila, A., Kalimo, H., Trump, B. F.: Influence of glutaraldehyde and/or osmium tetroxide on cell volume, ion content, mechanical stability, and membrane permeability of Ehrlich ascites tumor cells. J. Cell Biol. 63, 197–214 (1974)

    Google Scholar 

  • Peracchia, C.: Low resistance junctions in crayfish. I. Two arrays of globules in junctional membranes. J. Cell Biol. 57, 54–65 (1973a)

    Google Scholar 

  • Peracchia, C.: Low resistance junctions in crayfish. II. Structural details and further evidence for intercellular channels by freeze-fracture and negative staining. J. Cell Biol. 57, 66–76 (1973b)

    Google Scholar 

  • Plattner, H.: Ciliary granule plaques: Membrane-intercalated particle aggregates associated with Ca++-binding sites in Paramecium. J. Cell Sci. 18, 257–269 (1975)

    Google Scholar 

  • Plattner, H.: Intramembraneous changes upon cationophore-triggered exocytosis in Paramecium. Nature (Lond.) 252, 722–724 (1974)

    Google Scholar 

  • Plattner, H., Fuchs, S.: x-ray microanalysis of calcium binding sites in Paramecium. With special reference to exocytosis. Histochem. 45, 23–47 (1975)

    Google Scholar 

  • Plattner, H., Miller, F., Bachmann, L.: Membrane specializations in the form of regular membrane-to-membrane attachment sites in Paramecium. A correlated freeze-etching and ultrathin-sectioning analysis. J. Cell Sci. 13, 687–719 (1973)

    Google Scholar 

  • Raviola, E., Gilula, N. B.: Gap junctions between photoreceptor cells in the vertebrate retina. Proc. nat. Acad. Sci. (Wash.) 70, 1677–1681 (1973)

    Google Scholar 

  • Reese, T. S., Bennett, M. V. L., Feder, N.: Cell-to-cell movement of peroxidases injected into the septate axon of crayfish. Anat. Rec. 169, 409 (1971)

    Google Scholar 

  • Revel, J. P., Karnovsky, M. J.: Hexagonal array of subunits in intercellular junctions of the mouse heart and liver. J. Cell Biol. 33, C7-C12 (1967)

    Google Scholar 

  • Robinson, R. A., Stokes, R. H.: Electrolyte solutions. 2. Aufl. London: Butterworths 1959

    Google Scholar 

  • Satir, B., Schooley, C., Satir, P.: Membrane reorganization during secretion in Tetrahymena. Nature (Lond.) 235, 53–54 (1972)

    Google Scholar 

  • Satir, B., Schooley, C., Satir, P.: Membrane fusion in a model system. Mucocyst secretion in Tetrahymena. J. Cell Biol. 56, 153–176 (1973)

    Google Scholar 

  • Schatzki, P. F., Newsome, A.: Particle size of lanthanum salts used as ultrastructural tracers. J. Cell Biol. 59, 304a (1973)

    Google Scholar 

  • Schultz, S. G., Solomon, A. K.: Determination of effective hydrodynamic radii of small molecules by viscosimetry. J. gen. Physiol. 44, 1189–1199 (1961)

    Google Scholar 

  • Seligman, A. M., Karnovsky, M. J., Wasserkrug, H. L., Hanker, J. S.: Nondroplet ultrastructural demonstration of cytochrome oxidase activity with a polymerizing osmiophilic reagent, diaminobenzidine (DAB). J. Cell Biol. 38, 1–14 (1968)

    Google Scholar 

  • Simionescu, N., Simionescu, M., Palade, G. E.: Permeability of intestinal capillaries. Pathway followed by dextrans and glycogens. J. Cell Biol. 53, 365–392 (1972)

    Google Scholar 

  • Smith-Sonneborn, J., Klass, M., Cotton, D.: Parental age and life span versus progeny life span in Paramecium. J. Cell Sci. 14, 691–699 (1974)

    Google Scholar 

  • Vassar, P. S., Hards, J. M., Brooks, D. E., Hagenberger, B., Seaman, G. V. F.: Physicochemical effects of aldehydes on the human erythrocyte. J. Cell Biol. 53, 809–818 (1972)

    Google Scholar 

  • Staehelin, L. A.: Structure and function of intercellular junctions. Int. Rev. Cytol. 39, 191–283 (1974)

    Google Scholar 

  • Zingsheim, H. P., Plattner, H.: Electron microscopic methods in membrane biology. In: Methods in Membrane Biology, hrsg. E. D. Korn. New York: Plenum Press (in press)

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Authors and Affiliations

  1. Institut für Zellbiologie, Universität München, Fed. Rep. Germany

    Helmut Plattner & Doris Wolfram

  2. Institut für Technische Chemie, Technische Universität München, Fed. Rep. Germany

    Luis Bachmann

  3. Institut für Physiologische Chemie und Physikalische Biochemie, Universität München, Fed. Rep. Germany

    Elmar Wachter

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  1. Helmut Plattner
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  2. Doris Wolfram
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  3. Luis Bachmann
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  4. Elmar Wachter
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Plattner, H., Wolfram, D., Bachmann, L. et al. Tracer and freeze-etching analysis of intra-cellular membrane-junctions in Paramecium . Histochemistry 45, 1–21 (1975). https://doi.org/10.1007/BF00508048

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