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 structure

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-[125I]-iodonaphthalene-1-azide (INA) labeling which reportedly sees integral proteins and cytoplasmic cell membrane-associated proteins. With INA (+DTT), bands of ≤ 55 kDa were similar as with hydrophilic iodogen (+DTT), but instead of large size bands including i-AGs, a group of 122, 104 and 94 kDa appeared. Several bands of the non i-AG type are compatible with integral (possibly oligomeric) or associated proteins of the cell membrane of established molecular identity, as we discuss. In summary, we can discriminate between i-AGs and some functionally important minor cell membrane components. Our methodical approach might be relevant also for an analysis of some related protozoan parasites

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

NSF regulates membrane traffic along multiple pathways in Paramecium.

N-ethylmaleimide (NEM)-sensitive factor (NSF), a regulator of soluble NSF attachment protein receptors (SNAREs), is required for vesicular transport in many eukaryotic cells. In the ciliated protozoon Paramecium, complex but well-defined transport routes exist, constitutive and regulated exocytosis, endocytosis, phagocytosis and a fluid excretory pathway through contractile vacuoles, that can all be studied independently at the whole cell level. To unravel the role of NSF and of the SNARE machinery in this complex traffic, we looked for NSF genes in Paramecium, starting from a partial sequence found in a pilot random sequencing project. We found two very similar genes, PtNSF1 and PtNSF2, which both seem to be expressed. Peptide-specific antibodies (Abs) recognize PtNSF as a 84 kDa band. PtNSF gene silencing results in decreasing phagocytotic activity, while stimulated exocytosis of dense core-vesicles (trichocysts), once firmly attached at the cell membrane, persists. Ultrastructural analysis of silenced cells shows deformation or disappearance of structures involved in membrane traffic. Aggregates of numerous small, smooth vesicles intermingled with branches of ER occur in the cytoplasm and are most intensely labeled with anti-NSF Ab-gold. Furthermore, elongated vesicles of approximately 30 nm diameter can be seen attached at cortical calcium storage compartments, the alveolar sacs, whose unknown biogenesis may thus be revealed. Involvement of PtNSF in some low frequency fusion events was visualized in non-silenced cells by immuno-fluorescence, after cautious permeabilization in the presence of ATP-gamma-S and NEM. Our data document that PtNSF is involved in distinct pathways of vesicle traffic in Paramecium and that actual sensitivity to silencing is widely different, apparently dependent on the turnover of membrane-to-membrane attachment formation

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°C to 33°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 156Dspecific 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 1old 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°C to 23°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 mdium. Altogether these combined processes may facilitate the installation of the new vsAg type

NSF regulates membrane traffic along multiple pathways in Paramecium

N-ethylmaleimide (NEM)-sensitive factor (NSF), a regulator of soluble NSF attachment protein receptors (SNAREs), is required for vesicular transport in many eukaryotic cells. In the ciliated protozoon Paramecium, complex but well-defined transport routes exist, constitutive and regulated exocytosis, endocytosis, phagocytosis and a fluid excretory pathway through contractile vacuoles, that can all be studied independently at the whole cell level. To unravel the role of NSF and of the SNARE machinery in this complex traffic, we looked for NSF genes in Paramecium, starting from a partial sequence found in a pilot random sequencing project. We found two very similar genes, PtNSF1 and PtNSF2, which both seem to be expressed. Peptide-specific antibodies (Abs) recognize PtNSF as a 84 kDa band. PtNSF gene silencing results in decreasing phagocytotic activity, while stimulated exocytosis of dense core-vesicles (trichocysts), once firmly attached at the cell membrane, persists. Ultrastructural analysis of silenced cells shows deformation or disappearance of structures involved in membrane traffic. Aggregates of numerous small, smooth vesicles intermingled with branches of ER occur in the cytoplasm and are most intensely labeled with anti-NSF Ab-gold. Furthermore, elongated vesicles of ~30 nm diameter can be seen attached at cortical calcium storage compartments, the alveolar sacs, whose unknown biogenesis may thus be revealed. Involvement of PtNSF in some low frequency fusion events was visualized in non-silenced cells by immuno-fluorescence, after cautious permeabilization in the presence of ATP-γ-S and NEM. Our data document that PtNSF is involved in distinct pathways of vesicle traffic in Paramecium and that actual sensitivity to silencing is widely different, apparently dependent on the turnover of membrane-to-membrane attachment formation

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 postembedding 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 Cand 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

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-fusiondeficient mutant strain (nd9-28°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

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 ~8-nm large IMP subunits, suggesting formation of SERCA aggregates (as known from sarcoplasmic reticulum). We found not only monomers of typical molecular size (~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