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The contractile vacuole complex (CVC) of freshwater protists sequesters the excess of water and ions (Ca(2+)) for exocytosis cycles at the pore. Sequestration is based on a chemiosmotic proton gradient produced by a V-type H(+)-ATPase. So far, many pieces of information available have not been combined to a comprehensive view on CVC biogenesis and function. One main function now appears as follows. Ca(2+)-release channels, type inositol 1,4,5-trisphosphate receptors (InsP3R), may serve for fine-tuning of local cytosolic Ca(2+) concentration and mediate numerous membrane-to-membrane interactions within the tubular spongiome meshwork. Such activity is suggested by the occurrence of organelle-specific soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) and Ras-related in brain (Rab) proteins, which may regulate functional requirements. For tubulation, F-Bin-amphiphysin-Rvs (F-BAR) proteins are available. In addition, there is indirect evidence for the occurrence of H(+)/Ca(2+) exchangers (to sequester Ca(2+)) and mechanosensitive Ca(2+)-channels (for signaling the filling sate). The periodic activity of the CVC may be regulated by the mechanosensitive Ca(2+)-channels. Such channels are known to colocalize with and to be functionally supported by stomatins, which were recently detected in the CVC. A Kif18-related kinesin motor protein might control the length of radial arms. Two additional InsP3-related channels and several SNAREs are associated with the pore. De novo organelle biogenesis occurs under epigenetic control during mitotic activity and may involve the assembly of γ-tubulin, centrin, calmodulin and a never in mitosis A-type (NIMA) kinase - components also engaged in mitotic processes.

We analysed in Paramecium tetraurelia cells the role of the infraciliary lattice, a cytoskeletal network containing numerous centrin isoforms tightly bound to large binding proteins, in the re-establishment of Ca2+ homeostasis following exocytosis stimulation. The wild type strain d4-2 has been compared with the mutant cell line Delta-PtCenBP1 which is devoid of the infraciliary lattice ("Delta-PtCenBP1" cells). Exocytosis is known to involve the mobilization of cortical Ca2+-stores and a superimposed Ca2+-influx and was analysed using Fura Red ratio imaging. No difference in the initial signal generation was found between wild type and Delta-PtCenBP1 cells. In contrast, decay time was greatly increased in Delta-PtCenBP1 cells particularly when stimulated, e.g., in presence of 1mM extracellular Ca2+, [Ca2+]o. Apparent halftimes of f/f0 decrease were 8.5 s in wild type and approximately 125 s in Delta-PtCenBP1 cells, requiring approximately 30 s and approximately 180 s, respectively, to re-establish intracellular [Ca2+] homeostasis. Lowering [Ca2+]o to 0.1 and 0.01 mM caused an acceleration of intracellular [Ca2+] decay to t(1/2)=33 s and 28 s, respectively, in Delta-PtCenBP1 cells as compared to 8.1 and 5.6, respectively, for wild type cells. We conclude that, in Paramecium cells, the infraciliary lattice is the most efficient endogenous Ca2+ buffering system allowing the rapid downregulation of Ca2+ signals after exocytosis stimulation.

Ca2+ signalling governs stimulated exocytosis and exocytosis-coupled endocytosis also in Paramecium cells. Upon stimulation, the < or =10(3) dense-core exocytotic organelles (trichocysts) can be synchronously (80 ms) released, followed by endocytotic membrane resealing (350 ms) and retrieval. Paramecium is the most synchronous dense-core exocytotic system known, allowing to dissect rapidly reversible Ca2+-dependent phenomena. This holds for the reversible de-/re-phosphorylation cycle of a 63 kD phosphoprotein, pp63/parafusin (pf), which we have cloned, immuno-localised, and characterised as phosphoglucomutase, the enzyme funneling glucose into the glycolytic pathway. It was isolated ex vivo, followed by MALDI analysis, while X-ray structure analysis was performed after heterologous expression. We found multiple phosphorylation of superficial Ser/Thr residues. Although present also in exo(-) mutants, pp63/pf is selectively de-phosphorylated only in exo(+) strains during synchronous exocytosis (80 ms) and re-phosphorylated within approximately 20 s, i.e., the time required to re-establish [Ca2+] homeostasis. We have isolated relevant protein phosphatases and kinases and probed their activity on pp63/pf in vitro. We consider Ca2+/calmodulin-activated PP2B (calcineurin, whose subunits have been cloned) relevant for de-phosphorylation. Re-phosphorylation can be achieved by two protein kinases that also have been cloned. One is activated by cGMP (PKG) which in turn is formed by Ca2+-activated guanylate cyclase. Another kinase, casein kinase 2, is inhibited by Ca2+ and, hence, activated with some delay in parallel to decreasing [Ca2+] after exocytosis. In total, several Ca2+-sensitive cycles cooperate whose protein components have been localised to the cell cortex. Regulation of the phosphorylation degree of pp63/pf may affect structure binding on a microscale and/or its enzymatic activity. All this may serve fueling substrate into glycolysis with increased ATP re-formation (compromised in exo(-) mutants) and NADH formation, with effects on Ca2+ signalling including mobilisation from cortical stores (alveolar sacs) and overall effects on ATP and Ca2+ dynamics during synchronous exo- and endocytosis.

Work with Paramecium has contributed to the actual understanding of certain aspects of exocytosis regulation, including membrane fusion. The system is faster and more synchronous than any other dense-core vesicle system described and its highly regular design facilitates correlation of functional and ultrastructural (freeze-fracture) features. From early times on, several crucial aspects of exocytosis regulation have been found in Paramecium cells, e.g. genetically controlled microdomains (with distinct ultrastructure) for organelle docking and membrane fusion, involvement of calmodulin in establishing such microdomains, priming by ATP, occurrence of focal fusion with active participation of integral and peripheral proteins, decay of a population of integral proteins ("rosettes", mandatory for fusion capacity) into subunits and their lateral dispersal during fusion, etc. The size of rosette particles and their dispersal upon focal fusion would be directly compatible with proteolipid V(0) subunits of a V-ATPase, much better than the size predicted for oligomeric SNARE pins (SCAMPs are unknown from Paramecium at this time). However, there are some restrictions for a straightforward interpretation of ultrastructural results. The rather pointed, nipple-like tip of the trichocyst membrane could accommodate only one (or very few) potential V(0) counterpart(s), while the overlaying domain of the cell membrane contains numerous rosette particles. Particle size is compatible with V(0), but larger than that assumed for the SNARE complexes. When membrane fusion is induced in the presence of antibodies against cell surface components, focal fusion is seen to occur with dispersing rosette particles but without dispersal of their subunits and without pore expansion. Clearly, this is required for completing fusion and pore expansion. After cloning SNARE and V(0) components in Paramecium (with increasing details becoming rapidly available), we may soon be able to address the question more directly, whether any of these components or some new ones to be detected, serve exocytotic and/or any other membrane fusions in Paramecium.

A Paramecium cell has a stereotypically patterned surface, with regularly arranged cilia, dense-core secretory vesicles and subplasmalemmal calcium stores. Less strikingly, there is also a patterning of molecules; for instance, some ion channels are restricted to certain regions of the cell surface. This design may explain very effective and selective responses, such as that to Ca(2+) upon stimulation. It enables the cell to respond to a Ca(2+) signal precisely secretion (exocytosis) or by changing its ciliary activity. These responses depend on the location and/or type of signal, even though these two target structures co-exist side-by-side, and normally only limited overlap occurs between the different functions. Furthermore, the patterning of exocytotic sites and the possibility of synchronous exocytosis induction in the sub-second time range have considerably facilitated analyses, and thus led to new concepts of exocytotic membrane fusion. It has been possible to dissect complicated events like overlapping Ca(2+) fluxes produced from external sources and from internal stores. Since molecular genetic approaches have become available for Paramecium, many different gene products have been identified only some of which are known from "higher" eukaryotes. Although a variety of basic cellular functions are briefly addressed to demonstrate the uniqueness of this unicellular organism, this article focuses on exocytosis regulation., (Copyright 2002 Wiley Periodicals, Inc.)

In exocytosis, secretory granules contact plasma membrane at sites where microdomains can be observed, which are sometimes marked by intramembranous particle arrays. Such arrays are particularly obvious when membrane fusion is frozen at a subterminal stage, e.g., in neuromuscular junctions and ciliate exocytotic sites. In Paramecium, a genetic approach has shown that the "rosettes" of intramembranous particles are essential for stimulated exocytosis of secretory granules, the trichocysts. The identification of two genes encoding the N-ethylmaleimide-sensitive factor (NSF), a chaperone ATPase involved in organelle docking, prompted us to analyze its potential role in trichocyst exocytosis using a gene-silencing strategy. Here we show that NSF deprivation strongly interferes with rosette assembly but does not disturb the functioning of exocytotic sites already formed. We conclude that rosette organization involves ubiquitous partners of the fusion machinery and discuss where NSF could intervene in this mechanism.

A non-discharge mutant of Paramecium tetraurelia (nd12-35 degrees C, lacking exocytotic response upon stimulation with the nonpermeable polycationic secretagogue aminoethyldextran, AED), in the pawnA genetic context (d4-500r, lacking ciliary voltage-dependent Ca2+ influx), was shown to lack (45)Ca2+ entry from outside upon AED stimulation. In contrast, cells grown at 25 degrees C behave like the wildtype. To check the functional properties in more detail, fluorochrome-loaded 35 degrees C cells were stimulated, not only with AED (EC(100) = 10(-6) M in wildtype cells), but also with 4-chloro-meta-cresol, (4CmC, 0.5 mM), a permeable activator of ryanodine receptor-type Ca2+ release channels, usually at extracellular [Ca2+] of 50 microM, and eventually with a Ca2+ chelator added. We confirm that pwA-nd12(35 degrees C) cells lack any Ca2+ influx and any exocytosis of trichocysts in response to any stimulus. As we determined by x-ray microanalysis, total calcium content in alveolar sacs (subplasmalemmal stores) known to be mobilized upon exocytosis stimulation in wild-type cells, contain about the same total calcium in 35 degrees C as in 25 degrees C cells, and Ca2+ mobilization from alveoli by AED or 4CmC is also nearly the same. Due to the absence of any AED-induced Ca2+ influx in 35 degrees C cells and normal Ca2+ release from stores found by x-ray microanalysis one can exclude a "CICR"-type mechanism (Ca2+-induced Ca2+ release) and imply that normally a store-operated Ca2+ ("SOC") influx would occur (as in 25 degrees C cells). Furthermore, 35 degrees C cells display a significantly lower basal intracellular [Ca2+], so that any increase upon stimulation may be less expressed or even remain undetected. Under these conditions, any mobilization of Ca2+ from stores cannot compensate for the lack of Ca2+ influx, particularly since normally both components have to cooperate to achieve full exocytotic response. Also striking is our finding that 35 degrees C cells are unable to perform membrane fusion, as analyzed with the Ca2+ ionophore, A23187. These findings were corroborated by cryofixation and freeze-fracture analysis of trichocyst docking sites after AED or 4CmC stimulation, which also revealed no membrane fusion. In sum, in nd12 cells increased culture temperature entails multiple defects, notably insensitivity to any Ca2+ signal, which, moreover, cannot develop properly due to a lower basal [Ca2+] level and the lack of Ca2+ influx, despite normal store activation.

During exocytosis of dense-core secretory vesicles (trichocysts) in Paramecium, the protein pp63/parafusin (pp63/pf) is transiently dephosphorylated. We report here the structures of two crystal forms of one isoform of this protein which has a high degree of homology with rabbit phosphoglucomutase, whose structure has been reported. As expected, both proteins possess highly similar structures, showing the same four domains forming two lobes with an active-site crevice in between. The two X-ray structures that we report here were determined after crystallization in the presence of sulfate and tartrate, and show the lobes arranged as a closed and an open conformation, respectively. While both conformations possess a bound divalent cation, only the closed (sulfate-bound) conformation shows bound sulfate ions in the "phosphate-transfer site" near the catalytic serine residue and in the "phosphate-binding site". Comparison with the open form shows that the latter dianion is placed in the centre of three arginine residues, one contributed by subunit II and two by subunit IV, suggesting that it causes a contraction of the arginine triangle, which establishes the observed conformational closure of the lobes. It is therefore likely that the closed conformation forms only when a phosphoryl group is bound to the phosphate-binding site. The previously published structure of rabbit phosphoglucomutase is intermediate between these two conformers. Several of the known reversible phosphorylation sites of pp63/pf-1 are at positions critical for transition between the conformations and for binding of the ligands and thus give hints as to possible roles of pp63/pf-1 in the course of exocytosis., (Copyright 2002 Academic Press.)

Though only actual local free Ca2+ concentrations, [Ca2+], rather than total Ca concentrations, [Ca], govern cellular responses, analysis of total calcium fluxes would be important to fully understand the very complex Ca2+ dynamics during cell stimulation. Using Paramecium cells we analyzed Ca2+ mobilization from cortical stores during synchronous (< or = 80 ms) exocytosis stimulation, by quenched-flow/cryofixation, freeze-substitution (modified for Ca retention) and X-ray microanalysis which registers total calcium concentrations, [Ca]. When the extracellular free calcium concentration, [Ca2+]e, is adjusted to approximately 30 nM, i.e. slightly below the normal free intracellular calcium concentration, [Ca2+]i = 65 nM, exocytosis stimulation causes release of 52% of calcium from stores within 80 ms. At higher extracellular calcium concentration, [Ca2+]e = 500 microM, Ca2+ release is counterbalanced by influx into stores within the first 80 ms, followed by decline of total calcium, [Ca], in stores to 21% of basal values within 1 s. This includes the time required for endocytosis coupling (350 ms), another Ca2+-dependent process. To confirm that Ca2+ mobilization from stores is superimposed by rapid Ca2+ influx and/or uptake into stores, we substituted Sr2+ for Ca2+ in the medium for 500 ms, followed by 80 ms stimulation. This reveals reduced Ca signals, but strong Sr signals in stores. During stimulation, Ca2+ is spilled over preformed exocytosis sites, particularly with increasing extracellular free calcium, [Ca2+]e. Cortically enriched mitochondria rapidly gain Ca signals during stimulation. Balance calculations indicate that total Ca2+ flux largely exceeds values of intracellular free calcium concentrations locally required for exocytosis (as determined previously). Our approach and some of our findings appear relevant also for some other secretory systems.

The lipophilic fluorescent dye, FM1-43, as now frequently used to stain cell membranes and to monitor exo-endocytosis and membrane recycling, induces a cortical [Ca(2+)](i) transient and exocytosis of dense core vesicles ("trichocysts") in Paramecium cells, when applied at usual concentrations (

PP63 (parafusin) is a 63 kDa phosphoprotein, which exists in at least two different isoforms. It is very rapidly (80 ms) dephosphorylated during triggered trichocyst exocytosis. This occurs selectively in exocytosis-competent Paramecium tetraurelia strains. At least two protein kinases isolated from Paramecium, casein kinase type II kinase and cGMP-dependent kinase, are able to phosphorylate the two recombinant PP63/parafusin isoforms, both with phosphoglucomutase activity, in vitro. By performing mass spectrometric peptide mapping, we have investigated in vitro phosphorylation of recombinant PP63/parafusin by these kinases in comparison to in vivo phosphorylation of native PP63/parafusin isolated from Paramecium homogenates. Low picomolar quantities of proteolytic digests of recombinant and native PP63/parafusin, prior to and following alkaline phosphatase treatment, were directly analyzed by MALDI mass spectrometry. In native PP63-1/parafusin-1, six of 64 serine and threonine residues (S-196, T-205, T-280, T-371, T-373, and T-469) were found definitely, 27 were found possibly phosphorylated, 28 were identified as nonphosphorylated, and three were not covered by mapping. Three of the six certainly phosphorylated amino acids represent consensus phosphorylation sites for casein kinase II or cGMP-dependent protein kinase. In vitro phosphorylation studies of recombinant PP63/parafusin confirm that some of the sites found were used in vivo; however, also significant differences with respect to in vivo phosphorylation of native PP63/parafusin were observed. The two Paramecium protein kinases that were used do not preferably phosphorylate expected consensus sites in vitro. Homology structure modeling of PP63/parafusin with rabbit phosphoglucomutase revealed that the majority of residues found phosphorylated is located on the surface of the molecule.

We analyzed [Ca2+]i transients in Paramecium cells in response to veratridine for which we had previously established an agonist effect for trichocyst exocytosis (Erxleben & Plattner, 1994. J. Cell Biol. 127:935-945; Plattner et al., 1994. J. Membrane Biol. 158:197-208). Wild-type cells (7S), nondischarge strain nd9-28 degrees C and trichocyst-free strain "trichless" (tl), respectively, displayed similar, though somewhat diverging time course and plateau values of [Ca2+]i transients with moderate [Ca2+]o in the culture/assay fluid (50 microM or 1 mm). In 7S cells which are representative for a normal reaction, at [Ca2+]o = 30 nm (c.f. [Ca2+]resti = approximately 50 to 100 nm), veratridine produced only a small cortical [Ca2+]i transient. This increased in size and spatial distribution at [Ca2+]o = 50 microM of 1 mm. Interestingly with unusually high yet nontoxic [Ca2+]o = 10 mm, [Ca2+]i transients were much delayed and also reduced, as is trichocyst exocytosis. We interpret our results as follows. (i) With [Ca2+]o = 30 nm, the restricted residual response observed is due to Ca2+ mobilization from subplasmalemmal stores. (ii) With moderate [Ca2+]o = 50 microM to 1 mm, the established membrane labilizing effect of veratridine may activate not only subplasmalemmal stores but also Ca2+o influx from the medium via so far unidentified (anteriorly enriched) channels. Visibility of these phenomena is best in tl cells, where free docking sites allow for rapid Ca2+ spread, and least in 7S cells, whose perfectly assembled docking sites may "consume" a large part of the [Ca2+]i increase. (iii) With unusually high [Ca2+]o, mobilization of cortical stores and/or Ca2+o influx may be impeded by the known membrane stabilizing effect of Ca2+o counteracting the labilizing/channel activating effect of veratridine. (iv) We show these effects to be reversible, and, hence, not to be toxic side-effects, as confirmed by retention of injected calcein. (v) Finally, Mn2+ entry during veratridine stimulation, documented by Fura-2 fluorescence quenching, may indicate activation of unspecific Me2+ channels by veratridine. Our data have some bearing on analysis of other cells, notably neurons, whose response to veratridine is of particular and continuous interest.

We have localized a structure-bound fraction of the exocytosis-sensitive phosphoprotein, PP63/parafusin (PP63/pf), in Paramecium cells by widely different methods. We combined cell fractionation, western blots, as well as light and electron microscopy (pre- and post-embedding immunolabeling), applying antibodies against the recombinant protein. PP63/pf is considerably enriched in certain cortical structures, notably the outlines of regular surface fields (kinetids), docking sites of secretory organelles (trichocysts) and the membranes of subplasmalemmal Ca2+-stores (alveolar sacs). From our localization studies we tentatively derive several potential functions for PP63/pf, including cell surface structuring, assembly of exocytosis sites, and/or Ca2+ homeostasis.

Caffeine causes a [Ca2+]i increase in the cortex of Paramecium cells, followed by spillover with considerable attenuation, into central cell regions. From [Ca2+]resti approximately 50 to 80 nm, [Ca2+]acti rises within /=2 sec. Chelation of Ca2+o considerably attenuated [Ca2+]i increase. Therefore, caffeine may primarily mobilize cortical Ca2+ pools, superimposed by Ca2+ influx and spillover (particularly in tl cells with empty trichocyst docking sites). In nd cells, caffeine caused trichocyst contents to decondense internally (Ca2+-dependent stretching, normally occurring only after membrane fusion). With 7S cells this usually occurred only to a small extent, but with increasing frequency as [Ca2+]i signals were reduced by [Ca2+]o chelation. In this case, quenched-flow and ultrathin section or freeze-fracture analysis revealed dispersal of membrane components (without fusion) subsequent to internal contents decondensation, opposite to normal membrane fusion when a full [Ca2+]i signal was generated by caffeine stimulation (with Ca2+i and Ca2+o available). We conclude the following. (i) Caffeine can mobilize Ca2+ from cortical stores independent of the presence of Ca2+o. (ii) To yield adequate signals for normal exocytosis, Ca2+ release and Ca2+ influx both have to occur during caffeine stimulation. (iii) Insufficient [Ca2+]i increase entails caffeine-mediated access of Ca2+ to the secretory contents, thus causing their decondensation before membrane fusion can occur. (iv) Trichocyst decondensation in turn gives a signal for an unusual dissociation of docking/fusion components at the cell membrane. These observations imply different threshold [Ca2+]i-values for membrane fusion and contents discharge.

We have analyzed ultrathin sections from isolated bovine chromaffin cells grown on plastic support, after fast freezing, by quantitative electron microscopy. We determined the size and intracellular distribution of dense core vesicles (DVs or chromaffin granules) and of clear vesicles (CVs). The average diameter of DVs is 356 nm, and that of CVs varies between 35-195 nm (average 90 nm). DVs appear randomly packed inside cells. When the distance of the center of DVs to the cell membrane (CM) is analyzed, DV density is found to decrease as the CM is approached. According to Monte Carlo simulations performed on the basis of the measured size distribution of DVs, this decay can be assigned to a "wall effect." Any cortical barrier, regardless of its function, seems to not impose a restriction to a random cortical DV packing pattern. The number of DVs closely approaching the CM (docked DVs) is estimated to be between 364 and 629 (average 496), i.e., 0.45 to 0.78 DVs/micron2 CM. Deprivation of Ca2+, priming by increasing [Ca2+]i, or depolarization by high [K+]e for 10 s (the effect of which was controlled electrophysiologically and predicted to change the number of readily releasable granules [RRGs]) does not significantly change the number of peripheral DVs. The reason may be that (a) structural docking implies only in part functional docking (capability of immediate release), and (b) exocytosis is rapidly followed by endocytosis and replenishment of the pool of docked DVs. Whereas the potential contribution of DVs to CM area increase by immediate release can be estimated at 19-33%, that of CVs is expected to be in the range of 5.6-8.0%.

We had previously shown that an influx of extracellular Ca2+ (Ca2+e), though it occurs, is not strictly required for aminoethyldextran (AED)-triggered exocytotic membrane fusion in Paramecium. We now analyze, by quenched-flow/freeze-fracture, to what extent Ca2+e contributes to exocytotic and exocytosis-coupled endocytotic membrane fusion, as well as to detachment of "ghosts"-a process difficult to analyze by any other method or in any other system. Maximal exocytotic membrane fusion (analyzed within 80 msec) occurs readily in the presence of [Ca2+]e > or = 5 x 10(-6) M, while normally a [Ca2+]e = 0.5 mM is in the medium. A new finding is that exocytosis and endocytosis is significantly stimulated by increasing [Ca2+]e even beyond levels usually available to cells. Quenching of [Ca2+]e by EGTA application to levels of resting [Ca2+]i or slightly below does reduce (by approximately 50%) but not block AED-triggered exocytosis (again tested with 80 msec AED application). This effect can be overridden either by increasing stimulation time or by readdition of an excess of Ca2+e. Our data are compatible with the assumption that normally exocytotic membrane fusion will include a step of rapid Ca(2+)-mobilization from subplasmalemmal pools ("alveolar sacs") and, as a superimposed step, a Ca(2+)-influx, since exocytotic membrane fusion can occur at [Ca2+]e even slightly below resting [Ca2+]i. The other important conclusion is that increasing [Ca2+]e facilitates exocytotic and endocytotic membrane fusion, i.e., membrane resealing. In addition, we show for the first time that increasing [Ca2+]e also drives detachment of "ghosts"-a novel aspect not analyzed so far in any other system. According to our pilot calculations, a flush of Ca2+, orders of magnitude larger than stationary values assumed to drive membrane dynamics, from internal and external sources, drives the different steps of the exo-endocytosis cycle.

In Paramecium tetraurelia, polyamine-triggered exocytosis is accompanied by the activation of Ca2+-activated currents across the cell membrane (Erxleben. C., and H. Plattner. 1994. J. Cell Biol. 127:935-945). We now show by voltage clamp and extracellular recordings that the product of current x time (As) closely parallels the number of exocytotic events. We suggest that Ca2+ mobilization from subplasmalemmal storage compartments, covering almost the entire cell surface, is a key event. In fact, after local stimulation, Ca2+ imaging with high time resolution reveals rapid, transient, local signals even when extracellular Ca2+ is quenched to or below resting intracellular Ca2+ concentration ([Ca2+]e, < or = [Ca2+]i). Under these conditions, quenched-flow/freeze-fracture analysis shows that membrane fusion is only partially inhibited. Increasing [Ca2+], alone, i.e., without secretagogue, causes rapid, strong cortical increase of [Ca2+]i but no exocytosis. In various cells, the ratio of maximal vs. minimal currents registered during maximal stimulation or single exocytotic events, respectively, correlate nicely with the number of Ca stores available. Since no quantal current steps could be observed, this is again compatible with the combined occurrence of Ca2+ mobilization from stores (providing close to threshold Ca2+ levels) and Ca2+ influx from the medium (which per se does not cause exocytosis). This implies that only the combination of Ca2+ flushes, primarily from internal and secondarily from external sources, can produce a signal triggering rapid, local exocytotic responses, as requested for Paramecium defense.

In Paramecium tetraurelia cells synchronous exocytosis induced by aminoethyldextran (AED) is accompanied by an equally rapid dephosphorylation of a 63 kDa phosphoprotein (PP63) within 80 ms. In vivo, rephosphorylation occurs within a few seconds after AED triggering. In homogenates (P)P63 can be solubilized in all three phosphorylation states (phosphorylated, dephosphorylated and rephosphorylated) and thus tested in vitro. By using chelators of different divalent cations, de- and rephosphorylation of PP63 and P63 respectively can be achieved by an endogenous protein phosphatase/kinase system. Dephosphorylation occurs in the presence of EDTA, whereas in the presence of EGTA this was concealed by phosphorylation by endogenous kinase(s), thus indicating that phosphorylation of P63 is calcium-independent. Results obtained with protein phosphatase inhibitors (okadaic acid, calyculin A) allowed us to exclude a protein serine/threonine phosphatase of type I (with selective sensitivity in Paramecium). Protein phosphatase 2C is also less likely to be a candidate because of its requirement for high Mg2+ concentrations. According to previous evidence a protein serine/threonine phosphatase of type 2B (calcineurin; CaN) is possibly involved. We have now found that bovine brain CaN dephosphorylates PP63 in vitro. Taking into account the specific requirements of this phosphatase in vitro, with p-nitrophenyl phosphate as a substrate, we have isolated a cytosolic phosphatase of similar characteristics by combined preparative gel electrophoresis and affinity-column chromatography. In Paramecium this phosphatase also dephosphorylates PP63 in vitro (after 32P labelling in vivo). Using various combinations of ion exchange, affinity and hydrophobic interaction chromatography we have also isolated three different protein kinases from the soluble fraction, i.e. a cAMP-dependent protein kinase (PKA), a cGMP-dependent protein kinase (PKG) and a casein kinase. Among the kinases tested, PKA cannot phosphorylate P63, whereas either PKG or the casein kinase phosphorylate P63 in vitro. On the basis of these findings we propose that a protein phosphatase/kinase system is involved in the regulation of exocytosis in P. tetraurelia cells.

We have enriched phosphoglucomutase (PGM; EC 5.4.2.2) approximately 20-fold from Paramecium tetraurelia cells by combined fractional precipitation with (NH4)2SO4, gel filtration and anion-exchange chromatography yielding two PGM peaks. Several parameters affecting PGM enzymic activity, molecular mass and pI were determined. Phosphorylation studies were done with isolated endogenous protein kinases. Like the 63 kDa phosphoprotein PP63, which is dephosphorylated within 80 ms during synchronous trichocyst exocytosis [Höhne-Zell, Knoll, Riedel-Gras, Hofer and Plattner (1992) Biochem. J. 286, 843-849], PGM has a molecular mass of 63 kDa and forms of identical pI. Since mammalian PGM activity depends on the presence of glucose 1,6-bisphosphate (Glc-1,6-P2) (which is lost during anion-exchange chromatography), we analysed this aspect with Paramecium PGM. In this case PGM activity was shown not to be lost, due to p-nitrophenyl phosphate-detectable phosphatase(s) (which we have separated from PGM), but also due to loss of Glc-1,6-P2. Like PGM from various vertebrate species, PGM activity from Paramecium can be fully re-established by addition of Glc-1,6-P2 at 10 nM, and it is also stimulated by bivalent cations and insensitive to chelating or thiol reagents. The PGM which we have isolated can be phosphorylated by endogenous cyclic-GMP-dependent protein kinase or by endogenous casein kinase. This results in three phosphorylated bands of identical molecular mass and pI values, as we have shown to occur with PP63 after phosphorylation in vivo (forms with pI 6.05, 5.95, 5.85). In ELISA, antibodies raised against PGM from rabbit skeletal muscle were reactive not only with original PGM but also with PGM fractions from Paramecium. Therefore, PGM and PP63 seem to be identical with regard to widely different parameters, i.e. co-elution by chromatography, molecular mass, phosphorylation by the two protein kinases tested, pI values of isoforms, and immuno-binding. Recent claims that PP63 ('parafusin') would not be identical with PGM specifically in Paramecium are critically evaluated. Since some glycolytic enzymes are discussed as being associated with the Ca(2+)-release channel in muscle sarcoplasmic reticulum, and since sub-plasmalemmal Ca2+ stores in Paramecium closely resemble sarcoplasmic reticulum, a possible function of PP63/PGM in exocytosis regulation is discussed, particularly since dephosphorylation strictly parallels exocytosis.

Isolated subplasmalemmal Ca2+ stores ('alveolar sacs') from Paramecium tetraurelia cells sequester 45Ca2+ depending on ATP concentration. 45Ca2+ uptake is sensitive to SERCA-type Ca(2+)-ATPase inhibitors. They cause a slow release of 45Ca2+, as does caffeine. Of some importance are also the negative results we obtained with ryanodine, inositol 1,4,5-trisphosphate (InsP3), cyclic adenosinediphosphoribose (cADPR), 3',5'-cyclic guanosine monophosphate (cGMP, +/- beta-nicotinamide-adenine dinucleotide) or with increased [Ca2+]. These data were corroborated by experiments in vivo, including microinjection studies. Again ryanodine, InsP3, cADPR or cGMP did not trigger exocytosis, the trigger effect of SERCA inhibitors was sluggish, whereas caffeine induced exocytosis in a dose-dependent fashion. We then tested 45Ca2+ release also with isolated cell cortices (cell fragments containing cell membranes with stores and secretory organelles still attached). Under conditions which initiate exocytosis in vitro (depending on [ATP], reduction of [Mg2+] in presence of Ca2+; c.f. Lumpert et al. 1990, Biochem. J. 269, 639) we observed significant 45Ca2+ release with cortices as with isolated alveolar sacs. Our interpretation is as follows. (a) Alveolar sacs have a SERCA-type Ca(2+)-pump. (b) They have some sensitivity to caffeine, but none to ryanodine, InsP3 or cADPR. (c) There might be a direct functional coupling of these subplasmalemmal Ca2+ stores to the plasmalemma to which they are connected via feet-like structures; also like the SR, activation of this store is modulated by Mg2+ and ATP.

Paramecium tetraurelia wild-type (7S) cells respond to 2.5 mM veratridine by immediate trichocyst exocytosis, provided [Ca2+]o (extracellular Ca2+ concentration) is between about 10(-4) to 10(-3) M as in the culture medium. Exocytosis was analyzed by light scattering, light and electron microscopy following quenched-flow/freeze-fracture analysis. Defined time-dependent stages occurred, i.e., from focal (10 nm) membrane fusion to resealing, all within 1 sec. Veratridine triggers exocytosis also with deciliated 7S cells and with pawn mutants (without functional ciliary Ca channels). Both chelation of Ca2+o or increasing [Ca2+]o to 10(-2) M inhibit exocytotic membrane fusion. Veratridine does not release Ca2+ from isolated storage compartments and it is inefficient when microinjected. Substitution of Na+o for N-methylglucamine does not inhibit the trigger effect of veratridine which also cannot be mimicked by aconitine or batrachotoxin. We conclude that, in Paramecium cells, veratridine activates Ca channels (sensitive to high [Ca2+]o) in the somatic, i.e., nonciliary cell membrane and that a Ca2+ influx triggers exocytotic membrane fusion. The type of Ca channels involved remains to be established.

A correlated electrophysiological and light microscopic evaluation of trichocyst exocytosis was carried out the Paramecium cells which possess extensive cortical Ca stores with footlike links to the plasmalemma. We used not only intra- but also extracellular recordings to account for polar arrangement of ion channels (while trichocysts can be released from all over the cell surface). With three widely different secretagogues, aminoethyldextran (AED), veratridine and caffeine, similar anterior Nain and posterior Kout currents (both known to be Ca(2+)-dependent) were observed. Direct de- or hyperpolarization induced by current injection failed to trigger exocytosis. For both, exocytotic membrane fusion and secretagogue-induced membrane currents, sensitivity to or availability of Ca2+ appears to be different. Current responses to AED were blocked by W7 or trifluoperazine, while exocytosis remained unaffected. Reducing [Ca2+]o to < or = 0.16 microM (i.e., resting [Ca2+]i) suppressed electrical membrane responses triggered with AED, while we had previously documented normal exocytotic membrane fusion. From this we conclude that the primary effect of AED (as of caffeine) is the mobilization of Ca2+ from the subplasmalemmal pools which not only activates exocytosis (abolished by iontophoretic EGTA injection) but secondarily also spatially segregated plasmalemmal Ca(2+)-dependent ion channels (indicative of subplasmalemmal [Ca2+]i increase, but irrelevant for Ca2+ mobilization). The 45Ca2+ influx previously observed during AED triggering may serve to refill depleted stores. Apart from the insensitivity of our system to depolarization, the mode of direct Ca2+ mobilization from stores by mechanical coupling to the cell membrane (without previous Ca(2+)-influx from outside) closely resembles the model currently discussed for skeletal muscle triads.

Exo- and endocytotic processes induced by depolarization of isolated neurosecretory nerve terminals show a close temporal correlation, which suggests a short time of integration of the neurosecretory granule membrane with the plasma membrane. In order to determine minimal time requirements for exocytosis-coupled endocytosis to occur, we have analyzed by electron microscopy uptake of horseradish peroxidase (HRP) as a fluid phase marker at the onset of depolarization. We have applied rapid mixing and sampling (quenched flow) to assess events in sub-second time periods after stimulation. A significant number of labelled endocytotic vacuoles was observed during the first second of depolarization. This number then further increased by a factor of about 2 (within 5 s) and 4 (within 50 s). Thus, as for exocytosis, the rate of endocytosis decreased considerably during prolonged stimulation. These data indicate i) that a substantial proportion of secretory granules undergoes exocytosis very shortly after stimulation, and ii) that, following exocytosis, the minimal time required for consecutive membrane retrieval is in the sub-second range.

We had previously shown that a phosphoprotein of 63 kDa ('PP63') is rapidly and selectively dephosphorylated during synchronous (less than or equal to 1 s) trichocyst exocytosis in Paramecium cells and then rephosphorylated within less than or equal to 1 min [Zieseniss & Plattner (1985) J. Cell Biol. 101, 2028-2035]. Using a new quenched-flow device, we now find a strict correlation between PP63 dephosphorylation and the process of membrane fusion, both occurring within 80 ms. Uptake of 32P over 90 min, followed by exocytosis and rephosphorylation for 1 min, results in a rather selective phosphorylation of the dephosphorylated form, P63, to PP63. Solubilization by repeated freezing and thawing allows isolations of P63 and PP63. On isoelectric focusing autoradiograms they have pI values of 6.05, 5.95 (major spots), 5.85 and 5.75. All spots are sensitive to alkaline, but not to acidic, hydrolysis (except for the pI-6.05 spot). On two-dimensional-gel autoradiograms the most prominent spot, of pI 5.95, is most extensively de- and re-phosphorylated. This spot, from de- and re-phosphorylated samples, was used to produce monospecific antibodies. A cortical localization of PP63 was revealed by producing Western blots from isolated cell-surface fragments ('cortices') and by immunofluorescence labelling. We assume that both P63 and PP63 are attached to cortical structures, e.g. around trichocysts, though they are partly soluble. This localization and the strict correlation of PP63 dephosphorylation with exocytotic membrane fusion suggests a role in fusion regulation.

The synchrony of trichocyst exocytosis in Paramecium allows temporal correlation of associated events. Using quenched flow we observed a Ca2+ influx concurrent with exocytosis within 80 ms after stimulation with the secretagogue aminoethyldextran. Cyclic AMP did not change in depency of stimulation. Cyclic GMP transiently increased after 500 ms, culminating at 2 s, and thus considerably lags behind exocytosis induction and influx of Ca2+. Both Ca2+ influx and rise in cGMP are known to be induceable also by Ba2+ or veratridine, allegedly via the opening of ciliary Ca2+ channels. However, only veratridine stimulated exocytosis. We conclude that both aminoethyldextran and veratridine induce an exocytosis-associated Ca2+ influx, which is responsible for the rise in cGMP, through an as yet unknown pathway.

Synchronous exocytosis in Paramecium cells was analyzed on a subsecond time scale. For this purpose we developed a quenched flow device for rapid mixing and rapid freezing of cells without impairment (time resolution in the millisecond range, dead time approximately 30 ms). Cells frozen at defined times after stimulation with the noncytotoxic secretagogue aminoethyldextran were processed by freeze substitution for electron microscopic analysis. With ultrathin sections the time required for complete extrusion of secretory contents was determined to be less than 80 ms. Using freeze-fracture replicas the time required for resealing of the fused membranes was found to be less than 350 ms. During membrane fusion (visible 30 ms after stimulation) specific intramembranous particles in the cell membrane at the attachment sites of secretory organelles ("fusion rosette") disappear, possibly by dissociation of formerly oligomeric proteins. This hitherto unknown type of rapid change in membrane architecture may reflect molecular changes in protein-protein or protein-lipid interactions, presumably crucial for membrane fusion. By a modification of the quenched flow procedure extracellular [Ca++] during stimulation was adjusted to less than or equal to 3 x 10(-8) M, i.e., below intracellular [Ca++]. Only extrusion of the secretory contents, but not membrane fusion, was inhibited. Thus it was possible to separate both secretory events (membrane fusion from contents extrusion) and to discriminate their Ca++ requirements. We conclude that no Ca++ influx is necessary for induction of membrane fusion.

Cortex preparations isolated from Paramecium tetraurelia cells consist of surface with secretory organelles (trichocysts) still attached. In the absence of nucleotides, in media with a pCa of 5-5.5 and a pH of greater than or equal to 6.5, maximal exocytosis occurred when the Mg2+ concentration was lowered from 10 to 0.5 mM. ATP, as well as its non-hydrolysable analogues adenosine 5'-[gamma-thio]triphosphate (ATP[S]) and adenosine 5'[beta gamma-imido]triphosphate (App[NH]p), inhibited exocytosis at a concentration equivalent to that occurring in vivo (as determined by h.p.l.c.), but preincubation with ATP augmented the exocytotic response. GTP and its analogues only slightly stimulated exocytosis in vitro, but sensitivity to Ca2+ was increased significantly, in particular with GTP. These effects of nucleotides were rapidly reversible. Intracellular GTP concentrations (0.35 mM) would suffice for full activation with the pCai values assumed to occur in these cells during activation. On microinjection, ATP inhibited the secretagogue response in intact cells. Whereas microinjected GTP stimulated exocytosis (membrane fusion) without a secretagogue added, Gpp[NH]p remained without any effect; GTP[S] permanently abolished any triggered secretory response. Concomitantly, h.p.l.c. analysis of triggered and untriggered cells showed that GTP hydrolysis occurs immediately after synchronous (1 s) exocytosis in vivo. The precise site(s) of action of GTP during signal transduction in Paramecium cells remain to be determined.

In Paramecium cells Ca++-stimulated triggering of the exocytosis of secretory vesicles ("trichocysts") was achieved by ionophores X-537 A or A 23187. Under triggering conditions electron dense deposits were present in some "resting" trichocysts and regularly in discharging trichocysts; upon subsequent fixation deposits occurred on the trichocyst membrane (on the inner side or within the membrane) and on the "inner lamellar sheath" from where deposits seemed to "radiate" into the secretory materials. Similar results were obtained with glutardialdehyde fixation alone which also triggers exocytosis but only at low concentrations. Element analysis by energy dispersive x-ray microanalysis ascertained the presence of Ca and P in deposits occurring in trichocysts. Those "resting" trichocysts which were devoid of deposits did not contain Ca or P enriched. Hence, an abrupt Ca++-influx into individual trichocysts just before exocytosis seems to be involved in the triggering mechanism, possible in combination with the sudden activation of an ATPase system localized at those sites of the trichocysts which primarily contain the deposits. When paramecia were treated only with Ca++ and then fixed with OsO4 plus oxalate or merely with glutardialdehyde, electron scattering deposits were formed also on the inner side of the cell membrane and within the ciliary shaft (but rarely in trichocysts). Deposits obtained on cilia (including "ciliary granule plaques") also contained Ca, P and S. Cells contain osmiophilic "calcium-storing vacuoles" which were selectively rich in Ca and S but devoid of P.

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").

Catecholamine-storing chromaffin cells, isolated from bovine adrenal medullae by collagenase digestion, were stimulated with carbachol at 37 degrees C; aliquots for controls were kept at 37 degrees C: Starting from this temperature, cells were ultrarapidly frozen with the use of a sandwich-propane-jet procedure and freeze-fractured. The replicas were analysed quantitatively for exocytotic activity: After stimulation the cell membrane displayed a significant increase of exo-endocytotic openings varying in size from 20 to 300 nm. The number of openings increased in parallel to the catecholamine output. At no stage could a clearing of membrane-intercalated particles (MIPs) be observed. Openings of all size classes were etchable. Results from the PF-face were comparable with those of the EF-face. We conclude that (i) exocytosis in isolated chromaffin cells starts as a focal event; the smallest possible stages are about 10 nm in size, (ii) fusion proceeds without previous rearrangement of MIPs, and (iii) the opening starts without formation of a diaphragm.

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.

We used aminoethyldextran (AED) as a secretagogue for the simultaneous exocytosis of a great proportion of secretory organelles (trichocysts) from Paramecium tetraurelia cells and we applied freeze-fracturing and ultrathin sectioning for a quantitative analysis of the re-arrangement of ultrastructural details within the cell membrane during re-insertion of new trichocysts. Characteristic arrangements of membrane-intercalated particles (MIPs) occur over trichocyst docking sites: 300 nm large double rings of MIPs and a MIP rosette in the middle. Empty sites, displaying a compressed ring ("parenthesis") without a rosette, are expanded to full size rings when a trichocyst is docked. We obtained a first hint on the possible existence of a short lived (approximately 5 min) adaptation stage, represented by rings without a rosette. This could mean that docking of a trichocyst would induce the assembly of rosette MIPs over a newly docked trichocyst. The reformation of rosettes is paralleled by an increasing number of extrudable trichocysts which underscores the causal role of rosette MIPs in exocytosis performance. New trichocysts are inserted at the old predetermined sites after removal of ghosts and formation of a "plug" as a receptor-type structure. The number of non-docked, free trichocysts in the cytoplasm is only slightly changed, indicating a continuous synthesis and docking rate (approximately 2-3 organelles per min). Since in strain 7S ghosts are removed within approximately 10 min and docking goes on over 9 h, there occur many empty docking sites in the time period in between, with a maximum of approximately 1 h after AED triggering, thus providing a unique situation for further experiments.

Aminoethyldextran (AED) was used to trigger the synchronous release of trichocysts from Paramecium tetraurelia cells (see [8]) by a mechanism involving exocytotic membrane fusion and resealing (see [5]). Ultrastructural changes were analyzed by quantitative evaluation of ultrathin sections. In resting cells the percentage of potential trichocyst-docking sites which are actually occupied by a trichocyst was 58%; 36% of potential docking sites contained ghosts and 6% a "plug" of electron-dense material. We derived from our data that paramecia would discharge permanently and spontaneously trichocysts (without AED) at a rate of 2-3 per min (which we then also verified by counting the spontaneous release rate) and that this value is equivalent to the docking rate. For the synchronous expulsion of trichocysts in response to AED we had determined that the degree of synchrony is more than a hundred times better than in most other systems (see [8]). We have determined the half-lives (HL) for different events involved in exocytosis and re-docking as follows: approximately 3 sec for trichocyst discharge, approximately 3 sec for the formation of ghosts, 8 min for the clearing of ghosts from the cell surface, 4 min for the formation of "plugs". Trichocysts are docked with a HL of 40 min and "plugs" (considered as receptor-type structures for trichocyst docking) disappear with a concomitant HL of 50 min. Evidently the clearing of ghosts allows for re-formation of "plugs" but the respective HL values signal that "plugs" may also be formed anew. The relatively slow decline of the percentage of "plugs" (after their azimuth 15 min after AED triggering) may also indicate the synthesis of new docking sites. After a period of over approximately 3 h following AED triggering, the original situation is roughly re-established and maintained over the whole period of population growth analyzed.

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.

Under certain conditions it is possible in Paramecium cells to induce selectively the fusion of the secretory organelle membrane with the cell membrane without the involvement of any further steps (release of secretory contents, etc.). A Ca2+-mobilizing fusogen was used in the presence of components which inhibit the discharge of the secretory contents (Mg2+ and EGTA, mainly). One can thus produce many exocytotic openings with the secretory contents (which are normally vigorously discharged) still retained.

Parameters tetraurelia cells were used for analyzing the crucial but also controversial question regarding the organization of those sites of the cell membrane that undergo fusion during exocytosis. Paramecia are unique in that they display a "rosette" of from eight to ten MIPs (Membrane-intercalated particles, as seen on freeze-fracture replicas) at each of their numerous preformed exocytosis sites. We analyzed these structures by exposing slightly fixed cells to various enzymes and by subsequent freeze fracturing. We show that "rosette" MIPs are selectively sensitive to proteolytic enzymes. Since "rosettes" are known to be necessary for membrane fusion, this cytochemical result would be in line with the assumption that membrane-integrated proteins may play an active role in regulating exocytotic membrane fusion.

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.

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.

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.

We found that different polyamino compounds trigger the instantaneous and simultaneous release of trichocysts from Paramecium tetraurelia cells (monoxenically bacterized cultures), provided amino groups are spaced in intervals of approximately 1.0 nm; in this case even diamines or oligopeptides exert some trigger effect. The most potent trigger agent found was aminoethyldextran (AED, MW = 40 kDa) which was used mainly as a derivative with 40 -NH2/molecule. A maximal response (strain K401) was achieved at 1.38 X 10(-6) M, a half maximal response at 1.08 X 10(-6) M. AED acts by a dual effect, i.e., not only by statistically properly spaced amino groups but also by some additional effect of the dextran molecule, since this greatly enhances the effect of oligoamines, although it exerts no trigger effect per se. From a total of approximately 1120 or approximately 1230 trichocysts per cell (strain K401 or 7S) approximately 95% are releasable by AED. In these strains the number of non-releasable trichocysts corresponds closely to the number of undocked trichocysts floating in the cytoplasm, so that practically all trichocysts which are docked to the cell membrane, can be released. (We also analysed different mutant strains for their response to AED.) Massive trichocyst release does not impair cell viability or culture growth, and multiple release-redocking cycles can be performed; up to 5 trigger-docking cycles were tested with individual cells in 12 h intervals. AED-triggered exocytosis requires a free extracellular [Ca2+] of greater than or equal to 10(-5) M; it is inhibited by EGTA (ethyleneglycol-bis(beta-aminoethyl ether)-N,N'-tetraacetate), by a short pH 5.5 shock or by neomycin at 10(-5) M concentration.(ABSTRACT TRUNCATED AT 250 WORDS)

From a total number of approximately 1100-1300 secretory organelles ("trichocysts") in a Paramecium tetraurelia cell, approximately 90% are docked to the cell membrane. Approximately 90% of this subpopulation can be discharged from the cells within seconds, when exposed to the novel trigger agent aminoethyldextran (AED) at a concentration of 10(-6) M. No deleterious side effects were recognized with this trigger agent even over long time periods. By application of AED close to cells with the use of a micropipette we found that triggering of trichocyst release by AED involves a local, non-propagated effect and that all regions of the cell body are equally reactive. It requires exogenous Ca2+. It is independent of ciliary Ca2+ channels, since deciliated cells or ciliary mutations with "Ca2+-tight" cilia respond to AED with normal exocytosis performance. The massive and rapid occurrence of trichocyst release in response to AED allowed for a freeze-fracture analysis of intramembraneous changes (see Olbricht et al., Exp cell res 151 (1984) 14 [23]) which also shows the involvement of exocytosis) as well as for a long-term study of the re-attachment of trichocysts (see Haacke & Plattner, Exp cell res 151 (1984) 21 [10]) under synchronous conditions.

Since Paramecium tetraurelia cells were found to discharge synchronously most of their secretory organelles ('trichocysts') when exposed to 10(-6) M aminoethyldextran (AED) [17], this was now used for a freeze-fracture and -etching analysis of intramembranous changes during exocytosis performance, in conjunction with a rapid freezing method. In controls the potential exocytosis sites of the cell membrane revealed a 'rosette' of approximately 8 membrane-intercalated particles (MIPs) within a 300 nm large double 'ring' of MIPs (see [18]). During exocytosis we found the following changes: (a) Membrane fusion starts as a focal event, the smallest recognizable openings measuring 20-30 nm in diameter. (b) The exocytotic opening always forms in the center of the rosette. (c) Rosette MIPs may stay very close to the exocytotic opening, or they may partly be dispersed as the exocytotic opening is formed. (d) No diaphragm is formed during exocytotic membrane fusion. (e) The exocytotic opening is increasing to a size where it fills the total fusogenic zone contained within a ring, but not any further. (f) Rosette MIPs become further dispersed through the rings. (g) Resealing involves the transformation of rings into a collapsed form ('parenthesis'). (h) A resealed exocytosis site contains no conspicuous MIP aggregates, such as rosettes or 'annulus' structures from the trichocyst membrane, indicating a clear separation of both components.

Microinjected alkaline phosphatase triggered exocytosis of trichocysts. Among a variety of inhibitors and stimulators interfering with fusogenic mechanism discussed in the literature, only phosphatase inhibitors inhibited exocytosis. Various other enzymes were also tested with a new in vitro system (Vilmart-Seuwen et al., 1986), but results were mostly negative. (The possible involvement of proteases remains questionable.) Positive results obtained with micro-injected alkaline phosphatase are in line with our previous results: (a) The occurrence of a cytochemical reaction for phosphatase activity precisely at fusion sites (Plattner et al., 1980); (b) the occurrence of protein dephosphorylation during exocytosis (Gilligan and Satir, 1982; Zieseniss and Plattner, 1985) and (c) the negative modulatory effect of ATP on exocytotic membrane fusion (Vilmart-Seuwen et al., 1986).

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.

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 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, Ca2+-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 greater than 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 degrees 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 X 10(6) ATP molecules.

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.

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.