ARTICLE |
Correspondence to: H. Plattner, Faculty of Biology, University of Konstanz, 78434 Konstanz, Germany.
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Summary |
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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 Agold 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 48:12691281, 2000)
Key Words: calcineurin, ciliates, immunolocalization, Paramecium, phosphatase 2B, protozoa
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Introduction |
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A VARIETY OF CELLULAR PROCESSES are regulated by protein kinases and phosphatases (
CaN is widely distributed throughout eukaryotic cells, from the ciliated protozoan Paramecium (
In the ciliated protozoan Paramecium tetraurelia, immunological evidence first suggested the occurrence of CaN and a role in exocytosis regulation (
We applied low-temperature embedding to account for the fact that, in general, ~50% of CaN may be cytosolic, the other 50% being membrane bound (
This is the first ultrastructural immuno-EM (electron microscopic) localization of CaN. Among the intensely labeled structures in Paramecium, there are some targets to be expected from biochemical analyses but also some additional unexpected ones, thus providing important suggestions for future work. In detail, we find intensely labeled "infraciliary lattice" (cortical centrin-containing contractile filament system), outlines of "alveolar sacs" [suplasmalemmal calcium stores (
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Materials and Methods |
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Cell Cultures
Paramecium tetraurelia wild-type cells (strain 7S) were cultivated as described (
Fixation and Embedding
Cells were fixed for 30 min at 4C in 4% formaldehyde plus 0.125% glutaraldehyde in PBS, pH 7.2, washed three times for 10 min each in PBS containing 50 mM glycine, and dehydrated in increasing ethanol concentrations (30, 50, 70, 90, and 2 times 100%, 5 min each). This was followed by impregnation with unicryl resin (British BioCell; London, UK) at 0C, with two changes at 2-hr intervals and then overnight, followed by UV polymerization at -25C for 24 hr.
Antibody Labeling
Ultrathin sections collected on formvar-coated nickel grids were pretreated twice for 10 min with 20 µl of PBS + glycine (50 mM) and then immersed in PBS supplemented with 0.5% bovine serum albumin (BSA) and 0.5% goat serum for 10 min at room temperature (RT), to eliminate nonspecific gold adsorption. Then the grids were incubated with primary Abs diluted in PBS supplemented with 0.2% BSA-c (BioTrend; Köln, Germany) pH 7.4, for 1 hr at RT (or for 24 hr at 4C). Ab dilutions were 1:30. Note that BSA-c is an acetylated form which, due to increased net charge, reduces nonspecific adsorption of gold conjugates.
The primary Abs used were anti-CaN (subunit A+B) Abs (designated R299) as specified previously (
Samples were washed in PBS/BSA-c, three times for 10 min each, and treated for 1 hr with gold conjugates. We used either goat anti-rabbit (GR) IgGs, F(ab)2 fragments derived from these G
R IgGs, or protein A (pA), coupled to 5- or 10-nm gold (Au5, Au10) as indicated. Gold conjugates of IgGs and of pA were from BioTrend and from the University of Utrecht (Department of Cell Biology, School of Medicine, Utrecht, The Netherlands), respectively. Dilution was 1:20 for Abgold conjugates and 1:40 for pAgold conjugates.
The specificity of immunogold labeling was verified by a significant reduction in the number of gold particles on sections incubated with Abs preadsorbed with the original antigen, i.e., Abs against CaN from bovine brain (50 µg/ml of diluted Ab solution).
After labeling, sections were rinsed with distilled water, fixed for 5 min with 2% glutaraldehyde, and routinely stained for 5 min with 2% aqueous uranyl acetate only (unbuffered, pH 4.5). In experiments on the effects of section staining, these parameters were varied as follows.
Effect of Section Staining on Ab Labeling
We analyzed the effect of alkaline lead citrate, frequently used as a standard stain, on immunogold labeling (Fig 1). We used monoclonal Abs (MAbs) against a well-defined secretory component, the "mesh-like sheath" (which in Paramecium links the contents of a trichocyst with its membrane) as previously specified (
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Then the gold-labeled sections were processed either with or without 5-min fixation with 2% glutaraldehyde. Sections were stained in different ways, either with 2% aqueous (acidic) uranyl acetate as above or with standard alkaline lead citrate (
Evaluation of Labeling Density
Gold grains were counted on distinct structures of randomly taken samples and referred to area size of the respective structure analyzed (
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Results |
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Effects of Section Staining with Lead
We show some extreme situations occurring after gold labeling followed by application of different section staining procedures. Two different labelings were analyzed: (a) with MAbs against a defined secretory component contained in trichocysts, followed by F(ab)2 goat anti-mouse IgGAu5 conjugates (Fig 1 and Fig 2A), and (b) with rabbit Abs against glycocalyx components, followed by pAAu10 (Fig 2B). Briefly, lead stain at pH 12.0, or just application of pH 12.0, caused dramatic redistribution and loss of gold label. This can be impeded by fixation of sections after immunogold labeling. This step is not required when uranyl only is applied. In this case, labeling density is approximately the same with or without section fixation.
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Quantitative evaluation (Fig 2) revealed approximately the same effects with both labeling procedures analyzed, using either F(ab)2 or pAgold conjugates. Therefore, our findings may be of general interest. Essentially, we established the following details highlighted in Fig 1 and summarized in Fig 2. (a) Aqueous uranyl acetate (pH 4.5), a standard electron "stain" used to enhance structural contrast, does not impair immunogold labeling. This includes two aspects, sensitivity and structurelabel correlation, i.e., the number of gold grains per area is large and grains are strictly asscociated with the respective structures ("mesh-like sheath" and glycocalyx components, respectively). Quantification substantiates that the situation does not improve when sections are fixed with glutaraldehyde before uranyl staining. The other extreme is staining with alkaline (pH 12.0) lead solution (Fig 1C) which, although frequently used to further enhance structural contrast even in immunocytochemical work, causes severe dislocation and even loss of gold grains (Fig 2A and Fig 2B). This artifact cannot be eliminated by uranyl application before lead staining (Fig 1D and Fig 2A, Fig B). Mimicing the high pH, essential for the lead stain to work (
In consequence, we recommend section staining with aqueous uranyl acetate only, section fixation after gold labeling not being mandatory. However, postfixation must be applied if structural contrast should be further increased by successive staining with uranyl and lead. In the analyses shown below, we applied only uranyl staining followed by fixation.
Localization of CaN Using Anti-CaN Abs (R299)
These Abs (designated R299) were prepared against whole bovine brain CaN and have been characterized on Western blots, not only with CaN subfractions obtained from Paramecium (
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Fig 3 demonstrates the occurrence of label on the cell surface membrane complex. Because this is formed by the cell membrane and the closely apposed flat alveolar sacs, specific assignment of label to the cell membrane and to inner and outer regions of alveolar sacs, is not always possible. Some situations in Fig 3 and in some subsequent images suggest that not only the inner side of alveolar sacs (or closely apposed materials) but also the much tighter complex formed by the outer part of alveolar sacs and the cell membrane is labeled. Fig 3 also shows significant labeling of a parasomal sac, with a few gold granules scattered in the cytosol.
Fig 4 is an example of significant labeling in the (macro-)nucleus. Label may be concentrated on regions with electron dense (hetero-)chromatin, or with their rims.
Because trichocyst docking sites are not frequently encountered, they are documented separately. In Fig 5, a docking site is cut tangentially. Therefore, much of its membrane-associated materials surrounding the upper two thirds of the narrow trichocyst tip are exposed. It is precisely this "collar" region (cf.
Fig 6 documents in more detail labeling of a parasomal sac or of its apical and basal regions. In this figure the sac can be easily identified by its association with a ciliary basal body, which is not labeled. Another structure not so commonly encountered is the infraciliary lattice, formed of filament bundles enriched in centrin. This is also regularly labeled.
Fig 7 and Fig 8, showing again a cortical cell region, demonstrate the specificity of the type R299 Ab. The inner part of alveolar sacs (their "inner membrane") and the contractile "infraciliary lattice" are clearly labeled in Fig 7, in contrast to Fig 8. This difference is due to the use of Abs before (Fig 7) and after (Fig 8) preadsorption with the original antigen, before section labeling with GRAu5.
In summary, after statistical evaluation of a great number of samples to take into account the wide variability of labeling densities, the four regularly labeled structures in Paramecium are the cell surface complex (alveolar sacs + cell membrane), the infraciliary lattice, parasomal sacs, and chromatin borders (Fig 9).
Labeling with Abs Against Subunits of CaN
Examples of labeling with ABs against CaN-A are shown in Fig 10 and Fig 11. On Western blots, Abs against the A- and the B-subunit strongly and selectively react with the respective subunits (data not shown). Although the labeling intensity achieved on ultrathin sections may be somewhat less than with Abs against both subunits (R299), the tendencies are all the same, as shown below.
In Fig 10, the surface membrane complex is cut tangentially and therefore displays rather intense labeling. Fig 10 also contains two parasomal sacs in oblique (bottom left) or cross-section (middle right). In both situations they are clearly labeled, as is some basal body-associated material. Fig 11 shows labeling of an emerging trichocyst docking site, although in this particular case much less cytosolic labeling and labeling of the surface membrane complex is present.
Similar situations are shown in Fig 12 and Fig 13 for Abs against the B-subunit. Again, in Fig 12, the surface complex of cell membrane and alveolar sacs is labeled, as is a cross-cut parasomal sac. Fig 13 is a detail containing an emerging trichocyst whose docking site is again intensely labeled.
Fig 14 and Fig 15 document for Abs against the A- and B-subunit, respectively, that in the macronucleus heterochromatic areas are preferentially stained, notably at their periphery. These data correspond to those with Ab type R299.
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Discussion |
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Technical Note
CaN may only partially be present in a doublet, particularly when not associated with a membrane. Therefore, we tried to avoid loss or displacement of antigen, as occurs with too weak fixation (
We found that gold label is displaced and even removed from antigenic sites when a section is stained with alkaline lead citrate and that this is due to its obligatory pH of 12.0. We found this with gold conjugates of Abs and of pA applied to unicryl sections. Because unicryl is one of the methacrylate resins most widely used in immunogold labeling studies, the deleterious effect of section staining with alkaline lead solution that we detected may be of more general importance, although embedding media other than unicryl have not yet been analyzed in this regard. This deleterious effect was unexpected because the use of this stain, in addition to aqueous uranyl acetate, is quite common, as one can recognize in monographs on immunocytochemistry. If increased contrast is desired, uranyl staining may be prolonged or the EM set-up must to be selected accordingly. We do not know whether the AbAu complex, or the antigen, or the antigenAb complex is actually removed. Because after any application of pH 12.0 gold grains still may be present, but at a greater distance from the original localization of the antigen, the latter possibility also appears feasible.
Molecular Identity of CaN Substrates
Only a few substrates of CaN are well characterized, so that immunolocalization may yield new hints. Well-defined targets are the PP1 inhibitors DARPP-32 (
The Abs recognizing the entire CaN molecule (type R299), i.e., subunits A and B, may be useful to recognize CaN on sections, because they have been extensively probed on Western blots (
Although not yet established for CaN-B of Paramecium, association with membranes may be facilitated by myristoylation (
CaN at the Infraciliary Lattice
One of the main components of the dominant cortical filament system in Paramecium, the infraciliary lattice, is centrin (20 kD (
CaN at Outlines of Alveolar Sacs and at Trichocyst Docking Sites
Paramecium was the first system for which involvement of CaN in exocytosis regulation could be demonstrated. Microinjection of anti-CaN Abs inhibited trichocyst exocytosis (
In the Paramecium cell cortex, distribution of CaN coincides in part with that of the exocytosis-sensitive PP63/parafusin (
In summary, it remains to be analyzed in more detail whether in Paramecium CaN can act precisely at the exocytosis site and/or at the cortical calcium stores.
CaN at Parasomal Sacs
Detachment of clathrin-coated pits from the cell membrane during endocytotic vesicle formation depends on CaN-mediated dephosphorylation of the large monomeric GTP-binding protein dynamin (
CaN in the Nucleus
Thus far, CaN has been localized, at the light microscopic level only, in the nuclei of mouse spermatids (
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Acknowledgments |
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Supported by a grant from the Deutsche Forschungsgemeinschaft, Schwerpunkt "Struktur und Funktionssteuerung an Zelloberflächen."
We are grateful to Dr Karin Hauser for calculating sequence identities, to Sylvia Kolassa for skillful technical assistance, and to Anne Keller for revising the English text.
Received for publication December 2, 1999; accepted April 4, 2000.
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