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R Glas-Albrecht

Publications and source records attributed to R Glas-Albrecht.

5 recordsLinked to original sources
PubMed1992

Isolation of the membranes from secretory organelles (trichocysts) of Paramecium tetraurelia.

We present for the first time a method for isolation of the membranes of extrusive organelles (trichocysts) from sterile culture of different strains of Paramecium tetraurelia. First, trichocysts are isolated according to a new method (Glas-Albrecht, R. and Plattner, H. (1990) Eur. J. Cell Biol. 53, 164-172) with high purity and yield. Then the organelles are subjected to osmotic swelling. Since trichocysts then easily 'decondense' and entangle membranes, these cannot be isolated directly by centrifugation, but only by passage through a filter and subsequent centrifugation. Purity of membrane fractions is analysed by electron microscopy and SDS-PAGE, combined with silver staining or, after biotinylation, by avidin-peroxidase labelling. Molecular masses resolved in our gels are in a range from less than or equal to 15 to greater than or equal to 105 kDa. Main bands obtained with nd9-28 degrees C trichocyst membranes (most bands also being common to wild type trichocysts) are of about 16.5, 19-21, 27-29, 33-34, 44-45 (strong), 47-48 (strong), 57, 61, 65 (strong), 68-71, 75, 81, 94-95 (strong), 104 and greater than or equal to 110 kDa, from a total of approx. 23 bands resolved. There is no remarkable occurrence of dominant protein bands from trichocyst contents ('trichynins'), though these might represent up to 10(3)-times more of the total trichocyst proteins. The ratio of phospholipid/protein is approx. 0.2 mg/mg. The methodology developed might also be valuable for the isolation of extrusome membranes from some other protozoan species.

Animals↗
PubMed1992

Secretory organelles of Paramecium cells (trichocysts) are not remarkably acidic compartments.

Acridine orange (AO) trapping in conjunction with fluorescence microscopy was applied to Paramecium cells. Trichocysts were not labeled when analyzed with an image intensification system (as opposed to a lysosomal population). Only with increasing intensity of ultraviolet light (UV) did trichocysts (and to some extent the cytosol) exhibit orange fluorescence, both effects being paralleled by increasing cell damage. Therefore, in comparison with the reported cytosolic pH (6.8), trichocysts cannot be considered as essentially acidic compartments. This is supported by experiments in vitro, using isolated cortex fragments or isolated fractions of membrane-bounded trichocysts (greater than or equal to 90% non-leaky). Again, during UV illumination orange fluorescence was observed even in the absence of ATP and Mg2+. Furthermore, this AO fluorescence and the condensation state of trichocyst contents were not affected by NH3 or by any of the widely differing ion- and H(+)-exchange inhibitors or ionophores tested. Decondensation of trichocyst contents occurred only when Ca2+ ionophore A23187 or X537A was incorporated into trichocyst membranes and when Ca2+ was then added. In this case all trichocysts partially decondensed within their intact membranes. We conclude that AO might be trapped in trichocysts by the abundant acidic secretory components during observation with UV light, rather than by acidic luminal pH.

Acridine Orange↗
PubMed1990

High yield isolation procedure for intact secretory organelles (trichocysts) from different Paramecium tetraurelia strains.

The secretory organelles (trichocysts) of Paramecium tetraurelia, wild type (7S) and some mutant cells (non-discharge strains, nd), were isolated with their intact membranes using a self-generating Percoll gradient. This has become possible by detaching trichocysts before (nd) or during (7S) homogenization from the cell membrane, where they are normally firmly attached. Membrane integrity was tested in several ways (acridine orange trapping, Ca2+ insensitivity). Measurement of several marker enzymes, DNA and tubulin content showed considerable enrichment of trichocysts over other subcellular constituents. Calculation of the yield showed that more than 50% of the whole amount of trichocysts per cell can be isolated. However, with the method described trichocysts can be isolated in an intact state from a variety of Paramecium tetraurelia strains so that they now have become amenable to further analyses.

Animals↗
PubMed1987

Involvement of a 65 kDa phosphoprotein in the regulation of membrane fusion during exocytosis in Paramecium cells.

Antisera were raised against a phosphoprotein of 65 kDa (PP65) from Paramecium cells (shown before to be selectively dephosphorylated during synchronous exocytosis) and specified by immunoblotting. By immunofluorescence PP65 has been localized within the cortex, beneath the cell membrane. This corresponds to data obtained by cell fractionation, applying SDS-PAGE autoradiography to cortices prepared from 32P-prelabeled cells. Antisera against PP65 inhibit exocytosis in vivo (microinjection). Applying anti-PP65 antisera in vitro to cortices we could demonstrate inhibition not only of exocytosis, but also of PP65 dephosphorylation. We conclude that PP65 is involved in the regulation of membrane fusion during exocytosis.

Animals↗