Cytoskeleton-secretory vesicle interactions during the docking of secretory vesicles at the cell membrane in Paramecium tetraurelia cells.

Stationary-phase cells of Paramecium tetraurelia have most of their many secretory vesicles ("trichocysts") attached to the cell surface. Log-phase cells contain numerous unoccupied potential docking sites for trichocysts and many free trichocysts in the cytoplasm. To study the possible involvement of cytoskeletal elements, notably of microtubules, in the process of positioning of trichocysts at the cell surface, we took advantage of these stages. Cells were stained with tannic acid and subsequently analyzed by electron microscopy. Semithin sections allowed the determination of structural connections over a range of up to 10 micrometer. Microtubules emanating from ciliary basal bodies are seen in contact with free trichocysts, which appear to be transported, with their tip first, to the cell surface. (This can account for the saltatory movement reported by others). It is noteworthy that the "rails" represented by the microtubules do not directly determine the final attachment site of a trichocyst. Unoccupied attachment sites are characterized by a "plug" of electron-dense material just below the plasma membrane; the "plug" seems to act as a recognition or anchoring site; this material is squeezed out all around the trichocyst attachment zone, once a trichocyst is inserted (Westphal and Plattner, in press. [53]). Slightly below this "plug" we observed fasciae of microfilaments (identified by immunocytochemistry using peroxidase labeled F(ab) fragments against P. tetraurelia actin). Their arrangement is not altered when a trichocyst is docked. These fasciae seem to form a loophole for the insertion of a trichocyst. Trichocyst remain attached to the microtubules originating from the ciliary basal bodies--at least for some time--even after they are firmly installed in the preformed attachment sites. Evidently, the regular arrangement of exocytotic organelles is controlled on three levels: one operating over a long distance from the exocytosis site proper (microtubules), one over a short distance (microfilament bundles), and one directly on the exocytosis site ("plug").

Localization of actin in the cortex of Paramecium tetraurelia cells by immuno- and affinity-fluorescence microscopy.

The cortex of the ciliate Paramecium tetraurelia has been examined both with whole cells and with isolated surface complexes (pellicles) for the presence and distribution of actin, using direct fluorescence labeling with antibodies monospecific for Paramecium actin, with DNase I or with heavy meromyosin, all tagged with FITC as a fluorochrome. The results obtained with the three different methods were similar. Actin is concentrated in the cilia and--clearly visible only after deciliation--in the basal bodies. The docking site of secretory vesicles (trichocysts) onto the cell membrane does not display any selective actin content. This does not preclude the interaction with actin at other sites of the trichocysts, since diffuse fluorescence labeling in deeper layers of the cell did not allow us yet to analyze the distribution of actin throughout the cell body. However, our data would preclude any direct involvement of actin at the site of exocytotic membrane fusion proper.

Isolation of surface membranes from normal and exocytotic mutant strains of Paramecium tetraurelia. Ultrastructural and biochemical characterization.

A density gradient centrifugation method for the isolation of the surface membrane complex from Paramecium tetraurelia cells is presented. The resulting "pellicles" consist predominantly of the somatic cell membrane and the underlying alveolar membranes. Marker enzyme activities for other cell components are low and SDS-polyacrylamid-gel electrophoreses indicate the presence of only minor amounts of ciliary and secretory proteins. Pellicles were prepared from different strains: (a) Exocytosis-capable strains with the normal set of exocytotic organelles ("trichocysts") docked to the cell membrane (strains 7S, K 401, and 9-18 degrees C), (b) exocytosis-uncapable strains (although with normal trichocyst attachment: nd 9-27 degrees C, nd 6, nd 7) and (c) strain from tam 38 with empty docking sites and rare, defective, free trichocysts. A Ca2+-stimulated ATPase was present in the pellicles from all strains with Km (CA2+) values between 0.19 to 0.88 mM Ca2+ and Vmax between 286 to 787 nMoles Pi/mg protein/min. Km and Vmax was identical for all strains of group (a). Vmax was significantly lower for all strains of group (b) and still lower for group (c). Similar group differences were found for Km (except for strain nd 6). Freeze-fracture analysis shows that the disruption of the membrane-to-membrane attachments during fractionation is paralleled by the disarrangment of the regular arrays ("rings", "rosettes") of membrane-integrated particles.

Possible involvement of a calmodulin regulated Ca2+ -ATPase in exocytosis performance in Paramecium tetraurelia cells.

Surface membrane fractions from Paramecium tetraurelia cells contain a calmodulin-stimulated Ca2+-ATPase responding to low levels of free Ca2+ and with features characteristic of a membrane-bound ATPase. Among the different strains analyzed this enzyme was practically absent selectively from the 'non-discharge' mutant nd9-28 degrees C (from J. Beisson): if cultured at a permissive temperature (18 degrees C), this strain showed identical values of calmodulin-stimulated Ca2+-ATPase activity as wild-type cells (7S) or strains with mutations which do not affect exocytosis performance. We conclude that this calmodulin-stimulated Ca2+-activated ATPase might be a prerequisite for membrane fusion in the course of exocytosis performance.

The secretory contents of Paramecium tetraurelia trichocysts: ultrastructural--cytochemical characterization.

The secretory contents ("matrix") of Paramecium tetraurelia trichocysts expand by a factor of 4.5 when they undergo a Ca2+-mediated decondensation in the course of exocytosis. This is paralleled by a concomitant increase in the interval of the periodic banding of the matrix from 12 nm to 45-51 nm, which becomes visible with different electron stains for proteins and negatively charged groups. Recent reports of actin in secretory contents led us to investigate its redistribution and artifactual adsorption to the trichocyst contents upon their expansion. To visualize this effect we used peroxidase-labeled F(ab) fragments from an IgG directed against Paramecium actin, a DNAase I-gold complex, and the induction of F-actin polymerization. The trichocysts were analyzed in situ as well as after isolation by density-gradient centrifugation. Additionally, in response to current reports in the literature, we reanalyzed trichocyst contents for any possible presence of calmodulin. We applied three independent in situ methods for this: autofluorescence after trifluoperazine affinity labeling, calmodulin-fluorescence affinity labeling, and an electron microscopic immunocytochemical method. All three methods failed to reveal any significant labeling of structurally intact trichocysts in situ, although we also showed that discharged trichocysts avidly adsorb calmodulin from the culture medium. From the present data we conclude that the decondensation of trichocysts during exocytosis is mediated by a sudden conformational rearrangement of secretory proteins in the trichocyst contents, without the involvement of any other regulatory or contractile proteins, which occur only in the cytoplasm. Trichocyst contents are not significantly--if at all--glycosylated.

Quantitative data on peroxidatic markers for electron microscopy. With a note on actin identification in Paramecium cells.

Several important points of heme-peptide cytochemistry were quantitatively analyzed, with particular regard to their use in electron microscopic immunocytochemistry. A simple procedure is presented for the preparation of heme-octapeptide (H-8-P) microperoxidase. H-8-P, hemenonapeptide (H-9-P), and various horseradish peroxidase (HRP) isoenzymes were used for coupling with immunoglobulin (Ig)G or the papain-cleavage fragments from IgG (Fab) molecules. Ultracentrifugation and spectrophotometric analyses revealed the following characteristics of the conjugates: a) They are of a uniform size class; b) their diameters were calculated, and ranged from 5.6 (Fab-H-8-P; H-9-P) to 10.5 (IgG-HRP); c) the persistence of antigen binding capacity was ascertained; d) the deactivation of the marker peroxidase activity due to coupling was as low as 20-30%; e) optimal conditions for use of the electron microscopic (EM) with 3,3'-diaminobenzidine media were elaborated (with a pH optima somewhat different from some standard methods in current use); and f) on the basis of the quantitative data presented, an optimal compromise (either in favor of higher peroxidase activity with HRP conjugates or of smaller size with microperoxidase-Fab conjugates) can be achieved. Finally, the identification of isolated purified actin and of actin in cortical microfilament bundles and ciliary basal bodies of Paramecium cells served as a test object for the usefulness of conjugation products and optimized assay conditions for EM immunocytochemistry.