Exocytosis induction in Paramecium tetraurelia cells by exogenous phosphoprotein phosphatase in vivo and in vitro: possible involvement of calcineurin in exocytotic membrane fusion.

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.

Immunolabeling analysis of biosynthetic and degradative pathways of cell surface components (glycocalyx) in Paramecium cells.

Biosynthetic and degradative pathways of glycocalyx components are largely unknown in Paramecium and in some related parasitic protozoa. We isolated cell surface (glyco-)proteins, i.e., surface antigens (SAg) and used them in the native (nSAg) or denatured (dSAg) state to produce antibodies (AB) for immunolocalization by confocal imaging and by quantitative immunogold EM-labeling of ultrathin sections or of freeze-fracture replicas. Antibodies against nSAg or dSAg, respectively, yield different labeling densities over individual structures, thus indicating biosynthetic or degradative pathways, respectively. We derive the following biosynthetic way: ER --> Golgi apparatus --> non-regulated/non-dense core vesicle transport --> diffusional spread over non-ciliary (somatic) and ciliary cell membrane. For degradation we show the following pathways: Concentration of nSAg in the cytostome --> nascent digestive vacuole --> mature vacuoles --> release of dSAg at cytoproct, with partial retrieval by "discoidal vesicles". A second internalization pathway proceeds via coated pits ("parasomal sacs") --> early endosomes ("terminal cisternae") --> digestive vacuoles. Dense packing of SAg in the glycocalyx may drive them into the endo-/phagocytic pathway. Still more intriguing is the site of nSAg integration into the cell membrane by unstimulated exocytosis. We consider unconspicuous clear vesicles relevant for nSAg export, probably via sites which most of the time are occupied by coated pits. This could compensate for membrane retrieval by coated pits, while scarcity of smooth profiles at these sites may be explained by the much longer time period required for coated pit formation as compared to exocytosis.

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

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

Quantitative immunogold localization of protein phosphatase 2B (calcineurin) in Paramecium cells.

For immunogold EM labeling analysis, we fixed Paramecium cells in 4% formaldehyde and 0.125% glutaraldehyde, followed by low-temperature embedding in unicryl and UV polymerization. We first quantified some obvious but thus far neglected side effects of section staining on immunogold labeling, using mono- or polyclonal antibodies (Abs) against defined secretory and cell surface components, followed by F(ab)(2)- or protein A-gold conjugates. Use of alkaline lead staining resulted in considerable rearrangement and loss of label unless sections were postfixed by glutaraldehyde after gold labeling. This artifact is specific for section staining with lead. It can be avoided by staining sections with aqueous uranyl acetate only to achieve high-resolution immunogold localization of a protein phosphatase on unicryl sections. In general, phosphatases are assumed to be closely, although loosely, associated with their targets. Because the occurrence of protein phosphatase 2B (calcineurin) in Paramecium has been previously established by biochemical and immunological work, as well as by molecular biology, we have used Abs against mammalian CaN or its subunits, CaN-A and CaN-B, for antigen mapping in these cells by quantitative immunogold labeling analysis. Using ABs against whole CaN, four structures are selectively labeled (with slightly decreasing intensity), i.e., infraciliary lattice (centrin-containing contractile cortical filament network), parasomal sacs (coated pits), and outlines of alveolar sacs (subplasmalemmal calcium stores, tightly attached to the cell membrane), as well as rims of chromatin-containing nuclear domains. In other subcellular regions, gold granules reached densities three to four times above background outside the cell but there was no selective enrichment, e.g., in cilia, ciliary basal bodies, cytosol, mitochondria, trichocysts (dense-core secretory organelles), and non-chromatin nuclear domains. Their labeling density was 4- to 8.5-fold (average 6.5-fold) less than that on selectively labeled structures. Labeling tendency was about the same with Abs against either subunit. Our findings may facilitate the examination of molecular targets contained in the selectively labeled structures. (J Histochem Cytochem 48:1269-1281, 2000)

Filamentous actin in paramecium cells: functional and structural changes correlated with phalloidin affinity labeling in vivo.

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.

Filamentous actin in Paramecium cells: mapping by phalloidin affinity labeling in vivo and in vitro.

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.

Calmodulin in Paramecium tetraurelia: localization from the in vivo to the ultrastructural level.

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).

Ultrastructural and antigenic preservation of a delicate structure by cryopreparation: identification and immunogold localization during biogenesis of a secretory component (membrane-matrix connection) in Paramecium trichocysts.

Ultrastructure and antigenicity of the "mesh-like sheath" (MLS), a very delicate structure connecting the membrane and the paracrystalline matrix of Paramecium trichocysts, are well preserved after cryofixation (rapid freezing followed by freeze-substitution in methanol and embedding in Lowicryl K11M at 213K). The MLS is labeled by colloidal gold-bound antibodies (Ab-Au10nm) with primary antibody (Ab) against trichocyst components obtained by recloning hybridoma cells twice. We prepared Western blots from reduced gels obtained from subfractionated trichocysts. Trichocyst membranes displayed reactive bands of 68-70, 63-66, 43, 40 (strongest), and 57 and 54 KD, with a weak band of 38 KD. One of the most abundant protein bands of soluble secretory components (56-57 KD) was also strongly stained on blots. On ultra-thin sections pre-trichocysts display Ab-reactive material concentrated below the trichocyst membrane before the MLS can be recognized as a structural entity. Quantitative evaluation of Ab-Au10-labeled ultrathin sections also revealed passage of MLS materials through the very inconspicuous Golgi apparatus. This was substantiated by Ab-peroxidase labeling. We conclude that MLS components (whose ultrastructure is difficult to preserve) are largely membrane-associated, partly soluble proteins. They form a connection (released during exocytosis) between the abundant paracrystalline matrix components and the organelle membrane. MLS might thus maintain a peripheral aqueous space of functional importance.

Immunolocalization of protein phosphatase type 1 in Paramecium cells using antibodies against recombinant protein and peptides.

We localized protein phosphatase Type 1 (PP1) in Paramecium cells using antibodies (specified on Western blots) against recombinant protein, amino- or carboxy-terminal peptides, or peptide segments containing both terminals and an intermediate segment. Cell fractionation and ELISA revealed high PP1 concentrations in cilia, corresponding to observations by immunofluorescence and immunogold labeling analyses. We compared ELISA results obtained with MnCl2- or detergent-mediated deciliation and immunolocalizations obtained with digitonin and saponin- or detergent-mediated permeabilization. We observed that detergents at too high concentrations can displace the antigen from its original position. Quantitative evaluation of immunogold labeling revealed a predominant localization of PP1 in cilia, notably in the narrow space between the membrane and the outer microtubule doublets, as ascertained by immunogold labeling of Lowicryl sections obtained after rapid freezing and freeze-substitution. This localization to the periphery of cilia is compatible with previous suggestions of PP1 involvement in ciliary beat regulation, notably of cilia on the free cell surface. Immunolabeling occurs along the entire length of surface cilia. Despite much higher PP1 concentrations in cilia, ELISA values for absolute PP1 content were considerably higher in deciliated cells. This may indicate still other functional aspects of PP1. Along these lines, we also discuss the differences observed when immunochemical and enzymatic data are compared.

Microdomain arrangement of the SERCA-type Ca2+ pump (Ca2+-ATPase) in subplasmalemmal calcium stores of paramecium cells.

We localized SERCA pumps to the inner region of alveolar sac membranes, facing the cell interior, by combining ultrastructural and biochemical methods. Immunogold labeling largely predominated in the inner alveolar sac region which displayed aggregates of intramembrane particles (IMPs). On image analysis, these represented oligomeric arrangements of approximately 8-nm large IMP subunits, suggesting formation of SERCA aggregates (as known from sarcoplasmic reticulum). We found not only monomers of typical molecular size ( approximately 106 kD) but also oligomeric forms on Western blots (using anti-SERCA antibodies, also against endogenous SERCA from alveolar sacs) and on electrophoresis gelautoradiographs of 32P-labeled phosphoenzyme intermediates. Selective enrichment of SERCA-pump molecules in the inner alveolar sac membrane region may eliminate Ca2+ after centripetal spread observed during exocytosis activation, while the plasmalemmal Ca2+ pump may maintain or reestablish [Ca2+] in the narrow subplasmalemmal space between the outer alveolar sac membrane region and the cell membrane. We show for the first time the microzonal arrangement of SERCA molecules in a Ca2+ store of a secretory system, an intensely discussed issue in stimulus-secretion coupling research.

Hydrophobic and hydrophilic radio-iodination, crosslinking, and differential extraction of cell surface proteins in Paramecium tetraurelia cells.

We combined widely different biochemical methods to analyze proteins of the cell surface of P. tetraurelia since so far one can isolate only a subfraction of cell membrane vesicles enriched in the GPI-anchored surface antigens ("immoblization" or "i-AGs"). We also found that i-AGs may undergo partial degradation by endogenous proteases. Genuine intrinsic membrane proteins were recognized particularly with lipophilic 5-[(125)I]-iodonaphthalene-1-azide (INA) labeling which reportedly "sees" integral proteins and cytoplasmic cell membrane-associated proteins. With INA (+DTT), bands of

Temperature-induced change of variant surface antigen expression in Paramecium involves antigen release into the culture medium with considerable delay between transcription and surface expression.

We analyzed temperature-induced changes of variant surface antigen (vsAg) expression in Paramecium primaurelia, using immuno-techniques and mRNA determinations. Upon a 23 degrees C to 33 degrees C shift, the old vsAg, type 156G, remains on the cell surface for a time, when already mRNA for the new form, 156D, is expressed. A considerable amount of 156D-specific mRNA is formed 45-48 h after the temperature shift, while 156D surface expression reaches maximal levels only after >72 h. A new aspect of these experiments is that, during this transition, the old vsAg is steadily released in high-molecular-weight form into the culture medium, as found by dot blot and Western blot analysis of concentrated culture medium. The new vsAg form is first inserted into the somatic cell membrane, before it spreads also into cilia. In the reverse transition, 33 degrees C to 23 degrees C, the adaptation on the level of transcription and surface expression is considerably faster. While we had previously shown, under steady-state conditions (constant temperature), the occurrence of a degradation pathway by endocytotic and phagocytotic ingestion of vsAg this may proceed in parallel to the steady release of old vsAg from the cell surface into the medium. Altogether these combined processes may facilitate the installation of the new vsAg type.

Immunolocalization of the exocytosis-sensitive phosphoprotein, PP63/parafusin, in Paramecium cells using antibodies against recombinant protein.

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.

Functional characterization and localization of protein phosphatase type 2C from Paramecium.

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.

Ir-gene control of antibody class production.

The antibody response in mice to DNP-insulin is under Ir-gene control. The Ir gene defects in two strains have been analyzed. In both cases the IgG immune response was impaired whereas the IgM response was not affected. One H-2 gene haplotype was characterized by lack of IgG response, independent of the immunization protocol. A second H-2 haplotype manifested a low response of IgG after immunization with Bordetella pertussis as an adjuvant but a high response after complete Freund's adjuvant. It is proposed that a low level of T cell help induces the production of IgM antibodies, intermediate levels allow few IgG clones to develop, and high levels induce a heterogeneous IgG response.