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Surface membrane fractions from Paramecium tetraureliacells contain a calmodulin-stimulated Ca 2+-ATPase responding to low levels of free Ca 2+and with features characteristic of a membrane-bound ATPase responding to low levels of free Ca 2+and with features characteristics of a membrane-bound ATPase. Among the different strains analyzed this enzyme was practically absent selectively from the ‘non-discharge” mutant nd9—28°C(from J. Beisson); if cultured at a permissive temperature (18°C), this strain showed identical values of calmodulin-stimulated Ca 2+-ATPase activity as wild-type cells ( 7S) or strains with mutations which do not affect exocytosis performance. We conclude that this calmodulin-stimulated Ca 2+-activated ATPase might be a prerequisite for membrane fusion in the course of exocytosis performance.

We cloned a protein phosphatase 2C gene from Paramecium (PtPP2C), which codes for one of the smallest PP2C isoforms (Klumpp, S., Hanke, C., Donella-Deana, A., Beyer, A., Kellner, R., Pinna, L. A., and Schultz, J. E. (1994) J. Biol. Chem. 269, 32774-32780). After mutation of 9 ciliate Q codons (TAA) to CAA PtPP2C was expressed as an active protein in Escherichia coli. The catalytic core region contains 284 amino acids as defined by C- and N-terminal deletions. The C terminus from amino acid 200-300 of PP2C isoforms has only about 20% similarity. To demonstrate that the carboxy end is in fact needed for activity, we generated an enzymatically active PtPP2C containing a C-terminally located tobacco etch virus-protease site. Upon proteolytic truncation enzyme activity was lost, i.e. the C terminus of PP2C is indispensable for enzyme activity. During these experiments isoleucine 214 was fortuitously identified to be essential for PP2C catalysis. Mutation of the hydrophobic amino acid to glycine in the ciliate or bovine isoforms resulted in inactive protein. Because Ile214 is in a loop region without defined secondary structure, our data clearly go beyond the x-ray structure. The functional equivalence of the 180 amino acid long C terminus from the bovine PP2C with the 100 amino acid long carboxy end of the PtPP2C was demonstrated by producing an active chimera, i.e. the PP2C from Paramecium has no obvious regions which may be specifically involved in subcellular localization or substrate recognition. Using antibodies against recombinant PtPP2C we localized the enzyme by immunogold labeling in the cytosol and nucleus and very distinctly on the ciliary microtubule/dynein complex. The data suggest a role for PtPP2C in the regulation of dyneins, i.e. in cellular cargo transport and ciliary motility.

Monospecific polyclonal antibodies against Paramecium tetraurelia calmodulin were prepared and labeled for calmodulin localization on different levels of resolution: by microinjection into living cells; with isolated cell surface complexes (cortices); on the ultrastructural level, using Lowicryl sections of non-permeabilized cells (with colloidal gold-protein A labeling of antibodies bound); or using permeabilized and gently fixed cells for incubation with peroxidase- or microperoxidase-tagged antibodies. Sites selectively labeled above cytoplasmic background largely coincided, irrespective of the method used, although sensitivity, resolution, and liability to redistribution of antigen were quite different. (The methodological diversification applied allowed for their mutual control.) Nonspecific binding can be largely excluded, since all these methods gave negative results with pre-immune sera. We reached the following conclusions on sites with selective calmodulin binding (above cytoplasmic background level) in P. tetraurelia cells. A pool of calmodulin co-localized with F-actin, not only in the cortex (including fibrous materials around ciliary basal bodies) but also around food vacuoles (phagosomes) and, to a lesser degree, around the buccal cavity. Trichocyst docking sites on the cell membrane, and coated pits also displayed calmodulin labeling, thus indicating the potential involvement of calmodulin in exo-endocytosis processes. Calmodulin was also enriched on membranes of compartments with presumable ion (possibly Ca2+) transport capacity, such as trichocysts and the osmoregulatory system. Not selectively labeled were nuclei, mitochondria, and some small lysosomal organelles (as identified in vivo by rhodamine 123 or acridine orange fluorescence, respectively).

We have tried to specify a widespread hypothesis on the requirement of ATP for exocytosis (membrane fusion). With Paramecium tetraurelia cells, synchronously (approximately 1 s) exocytosing trichocysts, ATP pools have been measured in different strains, including wild type cells, "non-discharge" (nd), "trichless" (tl), and other mutations. The occurrence of a considerable and rapid ATP consumption also in nd and tl mutations as well as its time course (with a maximum 3-5 s after exocytosis) in exocytosis-competent strains does not match the actual extent of exocytosis performance. However, from in vivo as well as from in vitro experiments, we came to the conclusion that ATP might be required to keep the system in a primed state and its removal might facilitate membrane fusion. (For the study of exocytosis in vitro we have developed a new system, consisting of isolated cortices). In vivo as well as in vitro exocytosis is inhibited by increased levels of ATP or by a nonhydrolyzable ATP analogue. In vitro exocytosis is facilitated in ATP-free media. In vivo-microinjected ATP retards exocytosis in response to chemical triggers, whereas microinjected apyrase triggers exocytosis without exogenous trigger. Experiments with this system also largely exclude any overlaps with other processes that normally accompany exocytosis. Our data also explain why it was frequently assumed that ATP would be required for exocytosis. We conclude that membrane fusion during exocytosis does not require the presence of ATP; the occurrence of membrane fusion might involve the elimination of ATP from primed fusogenic sites; most of the ATP consumption measured in the course of exocytosis may be due to other effects, probably to recovery phenomena.

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.

After the synchronous induction of exocytosis of secretory organelles (trichocysts) in Paramecium tetraurelia cells the process of membrane resealing and retrieval could be followed under synchronous conditions. The characteristic aggregates of membrane intercalated particles (MIPs) contained within the freeze-fractured cell membrane (rings and rosettes) and trichocyst membranes (annulus MIPs), in addition to collar striations on the top of trichocyst membranes, served as endogenous ultrastructural markers. This allowed us to follow the re-arrangement of membrane constituents during and after exocytosis with high temporal and spatial precision. Membrane specificity is maintained to a considerable extent (∼ 99·5%), as judged from the rare occurrence of aberrant resealing (according to freeze-fracture data) and from the rather minute shift of glycocalyx components (according to electron staining experiments) during normal membrane resealing. Coated pits are not involved in membrane retrieval (155 ghosts analysed); since the membrane regions involved in exocytotic fusion are backed by apposed materials, probably proteins, this may restrain membrane constituents from intermixing. Another factor for maintaining membrane specificity is the fact that resealing of the exocytotic opening occurs much more rapidly than in most other systems. The retrieval operates with a half-life of 3 (strain 7S) to 9 min (K401), the involvement of cortical microtubules in the retrieval can be largely excluded, since only two microtubules (of unidentified origin) were seen to approach ghost structures in 4074 cases analysed during this period of intense ghost retrieval. Phalloidin microinjected at a dose that blocked all cytoplasmic streaming (before synchronous exocytosis was induced) did not abolish membrane resealing and retrieval, which, therefore, may be passive processes.

A variety of trigger procedures were applied to analyse the exocytotic capability of different Paramecium tetraurelia strains. 7,S K 401, kin 241, nd g (18 °C) arecapable of exocytosis (permissive strains), in contrast to nd 6, nd 7, nd g (27 °C), tarn 38 and ftb A, although all procedures used enhance [Ca3+] in the cytoplasm of all strains tested and although strains nd 6, nd 7 and nd g (27 °C) contain a full set of morphologically normal trichocysts attached to the cell membrane. The results show that only those strains are permissive which were shown previously to contain a rosette of membrane-integrated particles and a Ca2+-ATPase activity in the cell membrane over the trichocyst attachment (exocytosis) sites. The results from trigger experiments with permissive and non-permissive strains would be compatible with a dual function of rosette particles as Ca2+ pumps and Ca2+ channels. Nevertheless, the latter aspect remains uncertain since we show that experiments along these lines published by others (introducing a Ca,+ ionophore from the outside) involve a solvent-induced artifact (pseudoexocytosis: matrix stretching in the absence of membrane fusion). In all strains, except for tarn 38 and ftb A (which have abnormal trichocysts incapable of being attached to the cell membrane), the isolated trichocyst matrix can be transferred from the contracted to the expanded state in vitro with certain trigger procedures. Our data clearly show that an increase of [Ca3+]t in the cytoplasm is not sufficient for exocytosis to occur and that non-permissivity is somehow due to an inability to perform membrane fusion. It remains open whether the lack of rosettes and Ca2+-ATPase activity at trichocyst attachment sites are the primary cause of non-permissivity’.

In Paramecium nine rectangular aggregates of membrane-intercalated particles surround the freeze-cleaved membrane of the ciliary base. These ‘ciliary granule plaques’ occur independently of the ‘ciliary necklace’ which is observed in a more basal position on some cilia. In each individual plaque the ∼ 10 nm large granules are arranged in a square grid pattern: the granules of one plaque invariably form 3 vertical rows (number of horizontally arranged particles per row: Nh = 3) and 3–6 horizontal rows (mean value for Nv = 4 ·8); the centre-to-centre spacing of granules is dh = 22 nm in the horizontal and dv = 24 in the vertical direction. The ciliary granule plaques coincide with electron-dense deposits which can be produced in a position adjacent to the inner side of the basal ciliary shaft membrane by 3 procedures: (a) when cells are fixed in glutaraldehyde after incubation with Ca2+ concentrations (up to 25 mM) which are higher than normally present in the medium (about 0·5 HIM) but which do not immobilize the animals prior to fixation in glutaraldehyde; (b) when Ca2+ (up to 75 mM) was added to glutaraldehyde; (c) when cells were incubated with Ca2+’ as in (a) and then fixed in OsO4 solutions to which oxalate ions were added in concentrations equivalent to Ca2+. Comparable deposits were observed also with Ca2+ concentrations normally present in the medium, but they occurred much more rarely. The occurrence of electron-dense deposits was not enhanced by addition of Na+, Mg2+ or La3+ instead of Ca2+. In cross-sectioned ciliary bases the electron-dense Ca2+-dependent deposits face the 9 doublet microtubules. Occasionally the substructure of Ca2+-dependent deposits is resolved in median sections; their periodicity in the vertical direction (dv = 25nm; Nv = 5 ·2) corresponds to the periodicity of membrane-intercalated particles of freeze-cleaved plaques. Other parameters determined for Ca2+-dependent electron-dense deposits coincide also with those determined for freeze-cleaved ciliary granule plaques (values in parentheses are for cells with Ca2+ added before fixation with glutaraldehyde and ultrathin sectioning): The height of plaques (or deposits) is 84(115) nm; the distance from their lower end to the ciliary base is 138 (121) nm. Plaques (or deposits) are 45 (44) nm broad and the distance separating adjacent plaques (deposits) is 48 (50) nm. Plaques (deposits) are arranged with a horizontal periodicity of mean 93–102 (92) nm. It is concluded that the ciliary granule plaques are identical to or associated with Ca2+-binding sites involved in ciliary activity or other functions.

The contents of secretory vesicles ("trichocysts") were isolated in the condensed state from Paramecium cells. It is well known that the majority portion of trichocysts perform a rapid decondensation process during exocytosis, which is visible in the light microscope. We have analyzed this condensed leads to decondensed transition in vitro and determined some relevant parameters. In the condensed state, free phosphate (and possibly magnesium) ions screen local surplus charges. This is supported by x-ray spectra recorded from individual trichocysts (prepared by physical methods) in a scanning transmission electron microscope. Calcium, as well as other ions that eliminate phosphate by precipitation, produces decondensation in vitro. Under in vivo conditions, Ca2+ enters the vesicle lumen from the outside medium, once an exocytic opening has been formed. Consequently, within the intact cell, membrane fusion and protein decondensation take place with optimal timing. Ca2+ might then trigger decondensation in the same way by precipitating phosphate ions (as it does in vitro) and, indeed, such precipitates (again yielding Ca and P signals in x-ray spectra) can be recognized in situ under trigger conditions. As decondensation is a unidirectional, rapid process in Paramecium cells, it would contribute to drive the discharge of the secretory contents to the outside. Further implications on the energetics of exocytosis are discussed.

Synchronous exocytosis in Paramecium cells involves the rapid (less than or equal to 1 s) dephosphorylation of a 65-kD phosphoprotein, which, after a lag phase of approximately 5 s, is reversed within approximately 20 s. Exocytosis inhibitors suppress this reaction; stimulatory and inhibitory effects are dose dependent. The dephosphorylation of the 65-kD phosphoprotein occurs only in exocytosis-competent strains, but not in mutant strains that cannot carry out membrane fusion, or that are devoid of secretory organelles or cannot transport them to the cell membrane. Since under all conditions analyzed the transient dephosphorylation of the 65-kD phosphoprotein strictly parallels the actual amount of exocytosed organelles, this process might be involved in exocytosis performance, perhaps in its initiation.

Since it had been previously shown that in Paramecium cells exocytosis involves the dephosphorylation of a 65-kD phosphoprotein (PP), we tried to induce exocytotic membrane fusion by exogenous phosphatases (alkaline phosphatase or calcineurin [CaN]). The occurrence of calmodulin (CaM) at preformed exocytosis sites (Momayezi, M., H. Kersken, U. Gras, J. Vilmart-Seuwen, and H. Plattner, 1986, J. Histochem. Cytochem., 34:1621-1638) and the current finding of the presence of the 65-kD PP and of a CaN-like protein in cell surface fragments ("cortices") isolated from Paramecium cells led us to also test the effect of antibodies (Ab) against CaM or CaN on exocytosis performance. Microinjected anti-CaN Ab strongly inhibit exocytosis. (Negative results with microinjected anti-CaM Ab can easily be explained by the abundance of CaM.) Alternatively, microinjection of a Ca2+-CaM-CaN complex triggers exocytosis. The same occurs with alkaline phosphatase. All these effects can also be mimicked in vitro with isolated cortices. In vitro exocytosis triggered by adding Ca2+-CaM-CaN or alkaline phosphatase is paralleled by dephosphorylation of the 65-kD PP. Exocytosis can also be inhibited in cortices by anti-CaM Ab or anti-CaN Ab. In wild-type cells, compounds that inhibit phosphatase activity, but none that inhibit kinases or proteases, are able to inhibit exocytosis. Exocytosis cannot be induced by phosphatase injection in a membrane-fusion-deficient mutant strain (nd9-28 degrees C) characterized by a defective organization of exocytosis sites (Beisson, J., M. Lefort-Tran, M. Pouphile, M. Rossignol, and B. Satir, 1976, J. Cell Biol., 69:126-143). We conclude that exocytotic membrane fusion requires an adequate assembly of molecular components to allow for the dephosphorylation of a 65-kD PP and that this step is crucial for the induction of exocytotic membrane fusion in Paramecium cells. In vivo this probably involves a Ca2+-CaM-stimulated CaN-like PP phosphatase.

In living Paramecium cells, microinjected rhodaminyl (R)-phalloidin rapidly labels a thin cortical layer. This can be more clearly resolved with microinjected and fixed cells (allowing for better resolution) as well as with isolated pellicles (surface membrane complexes with trichocysts, microfilaments, and mitochondria attached). Labeling of a longitudinal and perpendicular pattern, reflecting the relief of the cell surface, and labeling of ciliary basal bodies then becomes clearly visible. Other structures labeled by R-phalloidin are the surfaces of food vacuoles of different sizes and, although inconsistently, the borders of the buccal cavity. Small acidic compartments (as identified by acridine orange fluorescence vital staining), probably representing acidosomes and small lysosomes, were not labeled. F-actin on food vacuole surfaces may somehow be involved in intracellular transport or fusion processes. No labeling was observed in association with the osmoregulatory system (contractile vacuoles and their ampullae and radial canals). The specificity of in vivo labeling obtained was supported by the abolition of R-phalloidin labeling when isolated pellicles were pretreated with unlabeled phalloidin or with DNAse I. It was also possible to discriminate among different layers of R-phalloidin binding in the cortex by detaching different layers of the surface complex from each other. Since localization of F-actin in ciliates has raised a considerable amount of dispute in the past, we also repeated all these experiments with RITC-labeled HMM, but we obtained essentially the same labeling pattern as with R-phalloidin. Ciliary basal bodies therefore clearly contain some F-actin. Our data shed some light on aspects of surface structuring and motility in these cells.

Rhodaminylated (R)-phalloidin microinjected into Paramecium tetraurelia cells at a final concentration of greater than or equal to 20 micrograms/ml produces considerable functional and structural changes. F-actin bundles (with 20 micrograms/ml phalloidin within 15 min) are formed, which subsequently (greater than 30 min) are sequestered into autophagic vacuoles; simultaneously, the originally intense fluorescence of a narrow cortical layer becomes more and more diminished. When such microinjected cells are processed for electron microscopy, they display concomitant ultrastructural alterations, namely, the formation of transcellular bundles of 5-7 nm-thick filaments, which subsequently appear in autophagosomes, as well as a considerable reduction of filamentous materials in the cortex. This, in turn, entails a considerable restructuring of the cortex, enabling free access of various structural components to the cortex. Higher doses of R-phalloidin abolish cytoplasmic streaming (e.g., 50 micrograms/ml after 20-30 min); although the cells may survive, new secretory organelles (trichocysts) are no longer docked to the cell membrane. In contrast, exocytosis of docked trichocysts (as well as subsequent membrane resealing and retrieval) is not impaired under any conditions. Cortical F-actin may account for the cytoplasmic streaming that may normally guarantee the delivery of new trichocysts to free docking sites at the cell membrane. When docking is inhibited by high R-phalloidin doses, excess free trichocysts are sequestered into autophagosomes (crinophagy). One of the most sensitive cell functions is food vacuole formation (assayed by prelabeling with India ink), which correlates with the presence of R-phalloidin labeling in the cytostomal region and around food vacuoles. The main conclusions from this work are that filamentous actin may be involved in structuring of the cortex and in cytoplasmic streaming, and may therefore influence the formation, and possibly the transcellular transport (cyclosis), of food vacuoles, as well as the docking of trichocysts, whereas it does not play a role in exocytosis per se or in the steps immediately following.

In different Paramecium tetraurelia strains the occurrence of a Ca2+-ATPase (or p-nitro-phenylphosphatase) activity at the preformed attachment and exocytosis sites of the secretory vesicles (trichocysts) was analysed by electron-microscopic cytochemistry and X-ray microanalysis. In conjunction with freeze-fracture studies it was found that only those strains, which contain rosette particles, display this Ca2+-ATPase activity (7S, K 40I, nd 9 (18 degrees C), while other strains (nd 6, nd 9 (27 degrees C), tam 38) are devoid of both these characteristics. The presence (absence) of rosette particles and of Ca2+-ATPase activity at the preformed exocytosis sites is correlated with the capability (incapability) of performing exocytosis in these strains. We discuss several possible interpretations of this structure-function correlation.

In Paramecium cells a synchronized discharge of trichocysts (which involves only the final exocytosis steps of membrane fusion, content discharge and membrane resealing) was achieved with ATPase-blockers, Ca3+-ionophores, lipid solvents (including lysolecithin), polyethyleneglycol, anaesthetics (Dibucain) and cationic detergents (cetyltrimethylammonium bromide (CTMAB) and cetylpyridinium chloride (CPC)). Only Dibucain - and to some extent cationic detergents -can trigger exocytosis independently of extracellular Ca2+, possibly by mobilizing intracellular Ca2+. The internal free [Ca2+] necessary for exocytosis can be estimated to be > 10−6 to 10−4 M. Membrane-free trichocyst contents were isolated by density gradient centrifugation; they are converted from the contracted to the expanded state by Dibucain, CTMAB and CPC, and also by exogenous ATPase (Apyrase). Thus, it is possible to de-couple the discharge (stretching) process from membrane-related phenomena. Since only the latter are inhibited by low temperature (0°C), membrane lipids probably have to be in a fluid state for exocytosis to occur. At least 2 steps appear to be involved: when membrane fusion is initiated, an independent matrix-bound system is activated for the synchronized stretching process. The energy requirement for one discharge event is estimated to be about 14 × 106 ATP molecules.

The outer membrane complex of Paramecium was investigated by ultrathin-sectioning techniques and by freeze-etching of unfixed cells without cryoprotectants. Granules were found in the freeze-etched cortical membrane complex in a highly ordered arrangement; morphometric analyses and partial disruption of this membrane complex showed that some of these granular membrane specializations (which we call types a, b, e, f, g) represent membrane-to-membrane attachment sites. Type a: granules arranged in rings (single or double), 300 nm in diameter, connect plasmalemma and alveolar membranes around trichocysts. Type b: within these a-type rings, a concentric zone (180 nm diameter) of granules connects plasma membrane and trichocyst membrane. Type c: the trichocyst membrane further contains a central ring or patch, 80 nm in diameter, of rather large granules where it is in contact with the crystalline trichocyst matrix. Type d: ‘ciliary necklaces’ are formed by groups of triple rows of 3–6, most frequently 5 granules, with 21-nm periodicity. Rows of granules also connect the alveolar membranes to the apical portion of the trichocyst membrane (type e) and the alveolar membrane to the plasmalemma around cilia (type f). Type g: the inner alveolar membrane displays an intense granularity and contains double rows of granules along alveolar septa at attachment sites between 2 alveolar membranes. Upon experimental discharge of the majority of trichocysts only the innermost concentric circles of membrane-bound granules of this region (b- and c-type) disappeared from the plasmalemma, while the outermost a-type rings of granules persisted for a longer time. Among other possible functions, these regular membrane-to-membrane attachments are likely to maintain the specific cellular shape.