ARTICLE |
Correspondence to: H. Plattner, Dept. of Biology, Univ. of Konstanz, D-78434 Konstanz, Germany.
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Summary |
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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 stimulussecretion coupling research. (J Histochem Cytochem 47:841853, 1999)
Key Words: ATPase, Ca2+, calcium, exocytosis, microdomains, Paramecium, secretion, SERCA
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Introduction |
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This work examined the possibility of microdomain regulation of cortical Ca2+ concentrations [Ca2+], a frequently debated issue in current literature on stimulussecretion coupling (see below). In many cells, the endoplasmic reticulum (ER) forms a vast Ca2+ store (
In the ciliated protozoan Paramecium tetraurelia, alveolar sacs underlie the somatic cell membrane (Figure 1), i.e., the nonciliary cell body, except for regions at which cilia emerge and dense-cored secretory organelles (trichocysts) are attached in a distinct pattern for rapid exocytosis (
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Alveolar sacs are periodically arranged as distinct structural entities. The area facing the outside, i.e., the cell membrane (outer membrane region of alveolar sacs; OM-AS) is connected to the cell membrane by protein links, maintaining a subplasmalemmal space of ~15 nm (
Interestingly, alveolar sacs closely resemble skeletal muscle SR in different ways (
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Materials and Methods |
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Cell Cultures
Paramecium tetraurelia wild-type cells, strain 7S, were cultivated as described (
Biochemical and Immunochemical Analyses
AS fractions were prepared according to -32P]-ATP (16 x 1010 Bq/mol) as described. Some of these analyses were preceded by protein crosslinking using alveolar sacs (2.4 mg protein/ml) in 30 mM imidazole-HCl buffer, pH 6.8, containing 100 mM KCl and protease inhibitors, i.e., 10 µM E64 (Biomol; Hamburg, Germany) and freshly dissolved 280 µM phenylmethylsulfonylfluoride (Sigma; Deisenhofen, Germany). To alveolar sac samples, a 10-fold larger volume of crosslinker (EGS; ethylene-glycol-bis-[succinimidylsuccinate], 100 µM; Pierce, Rockford, IL) in DMSO (1% final concentration) was added for 10 or 30 min at 4C. This allows 32P labeling of the phosphoenzyme intermediate of the SERCA pump under conditions specified above and in more detail by
1 day at -70C. More extensive radioactive labeling studies with Paramecium cell surface proteins, with or without EGS treatment, served as controls because they revealed crosslinking selectively of densely packed proteins, like surface variant antigens, whereas other membrane proteins were not affected (unpublished observations).
Immunolocalization by Confocal Laser Scanning Microscopy
Methods applied and antibodies (Abs) against the PtSERCA peptide used were precisely as specified by
Ultrathin Section Immunogold Labeling
Two widely different preparation protocols, using either low temperature Unicryl embedding or cryosections (
Fixation and Preparation of Sections. Cells were fixed for 1 hr at 20C in 4% formaldehyde in 10 mM Tris-maleate, pH 7.0, with 10 mM MgCl2 and 1 mM CaCl2 added, washed in PBS with 50 mM glycine added, processed by progressive lowering of the temperature, embedded in Unicryl (British BioCell; London, UK), and UV polymerization at -20C according to the manufacturer's advice. Sections were washed with 0.2% BSA-c (BioTrend Chemikalien; Köln, Germany) in PBS before incubation with Abs (see below) and staining with 2% aqueous uranyl acetate. Alternatively, cells were suspended in 4% gelatin, fixed in 8% formaldehyde in 100 mM Pipes-HCl, pH 7.2, (48 hr, 4C), frozen, and used for ultrathin cryosectioning, Ab incubation (see below), and staining with 3% uranyl acetate + 2% type M-6385 methylcellulose from Sigma (mixture of 9:1 parts).
EM Immunocytochemistry on Sections.
The following Abs were used. Established rabbit Abs (diluted 1:15 in PBS) against the C-terminal region (amino acid residues 987999) of rat SERCA Type 3 (
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For visualization of Ab binding sites on sections we coupled protein A (Sigma) to 6-nm gold particles (pA-Au6, diluted 1:20 in PBS) or used rabbit anti-chicken Abs coupled to 10-nm gold particles (RaC-Au10, diluted 1:10 in PBS) from BioTrend.
Freeze-fracturing and Replica Labeling
Live cells were rapidly frozen by two different protocols. (a) Cells were either sandwiched between two thin copper sheets and vigorously injected into melting propane (~123K) according to
Replicas obtained by Method (a) served for freeze-fracture replica labeling according to
Electron Microscopy and Image Analysis
Ultrathin sections and freeze-fracture replicas were evaluated in a Zeiss EM10 or a Leo EM912 Omega instrument.
Electron micrographs suitable for analysis of the IM-AS were selected by optical diffractometry and digitized with a pixel size corresponding to 0.77 nm at the specimen level. Standard correlation averaging techniques (
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Results |
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Figure 1 illustrates the potential Ca2+ fluxes in Paramecium, as outlined in the Introduction, and it organizes the results that follow. The previously described SERCA form from Paramecium alveolar sacs, PtSERCA (
Western blot analysis of subcellular fractions showed localization of PtSERCA to subplasmalemmal compartments, and we now show here that these correspond to alveolar sacs. Although occurrence of oligomeric SERCA forms has been inferred from biochemical studies with muscle, we report here similar results for PtSERCA and we characterize in situ for the first time an ultrastructural equivalent by image analysis.
Biochemical and Immunochemical Studies
Figure 2 shows proteins from alveolar sac fractions on a Western blot using established anti-SERCA Abs described in Materials and Methods. Note the band equivalent to single SERCA proteins at 106 kD and also a much larger band equivalent to oligomers of the SERCA proteins. The exact molecular weight is difficult to establish. However, the high molecular weight band of 300 kD appears to be due to oligomers, because there is a precedent for finding oligomers of SERCA proteins even on denaturing gels with SDS and even under reducing conditions (
Figure 3 shows 32P autoradiographs of phosphoenzyme intermediates. As with Western blots, there are bands at approximately 106 kD and, under conditions of Ca2+ and Mg2+, there is a higher molecular weight band as well.
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Figure 4 also shows phosphoenzyme intermediates under crosslinking conditions. EGS was used as a crosslinker of similar size as used in most recent studies with SERCA molecules (400 kD do not penetrate into the gel). Some additional bands are due to the fact that we analyzed alveolar sac fractions rather than purified enzyme.
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The specificity of Ab binding on Western blots and of phosphoenzyme intermediate 32P labeling has been established previously (
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Immunocytochemistry
High-resolution confocal laser scanning microscopic analysis of permeabilized cells labeled with Abs against PtSERCA peptide showed almost exclusive localization of scale-like cortical structures equivalent to alveolar sacs (Figure 5). Results after omission of permeabilization or first Ab incubation or peptide pretreatment of Abs were negative (not shown).
For immuno-EM localization (Figure 6 and Figure 7), two widely different Abs and preparation protocols were used (see Materials and Methods). Both of these widely differing procedures revealed heavy labeling of alveolar sacs, selectively of their inner profiles (IM-AS). With anti-ratSerca 3 Abs, some label was found in the cytoplasm (Figure 6). This is compatible with their crossreactivity with a potential SERCA isoform in the ER, in agreement with the distribution of the original antigen in some other cell types (
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Freeze-fracture and Correlation Averaging Image Analysis
We analyzed ultrastructural details of the alveolar sac membranes by freeze-fracture replication after cryofixation without chemical pretreatment (Figure 8 and Figure 9). To orient the confusingly dense packing of the three membrane layers, we identified the plasmalemma by the freeze-fracture replica gold-labeling approach using Abs against cell surface components (see Materials and Methods). The survey in Figure 8 reveals alveolar sacs as distinct structural entities. Four alveolar sacs surround a trichocyst docking site, each site being in line with a row of cilia (
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A characteristic cobblestone pattern was observed sometimes selectively on the plasma fracture face (PF face) of the IM-AS regions (Figure 10). In general, the metal distribution on the shadowed replicas gave the impression of more or less densely packed annular depressions on the fractured membrane. The optical diffractograms of the micrographs indicate a periodicity of ~21 nm. For image analysis, a small fraction of the image (100 x 100 nm) was selected as reference and cross-correlated with the entire area of interest. Sites of maximal correlation of the motifs are interactively optimized, resulting in a peak list comprising ~1800 entities/µm2, which is ~90% of the total number (Figure 11). The average of these motifs looks very similar to the ones in Figure 12, their nearest neighbors, but are only vaguely indicated. When the distortions of the peak positions (which are mainly due to the curvature of the fractured membrane) were taken into account and the motifs were translationally and rotationally (± 5°) aligned, the hexagonal arrangement of the motifs became clearly visible (Figure 12). The shortest repeat distance of the motifs is 21 nm ± 5% according to the power spectra. The image obtained in Figure 12 suggests that the structure may be composed of subunits (~8-nm diameter), an observation that is supported by the analysis of several replicas. To check the results obtained by correlation averaging, single particle analysis using multivariate statistical analysis (MSA) was applied to areas with densely packed motifs and to less densely packed areas. The class average of the motifs thus obtained agreed well with the findings by correlation averaging (not shown).
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When these images are analyzed, it must be recalled, first, that they represent fracture faces in which the original structure of IMPs will be more or less deformed, thus limiting the feasibility of interpretation. Second, the images represent the distribution of a fairly thick Pt/C deposit (as obtained by the routine freeze-fracture method applied) which reflects more than the relief of the shadowed surface. Rather, the thickness of the deposit and, even more important, self-shadowing and decoration effects limit resolution of relief details to a degree that structural elements smaller than 10 nm may be only vaguely indicated (
Because no fixation or crosslinking was applied in these experiments before fast freezing, such oligomeric structures must occur in the IM-AS in the living cell. For the following reasons, we consider these IMP aggregates to be potential SERCA equivalents. (a) Such structures are realized exclusively in the IM-AS region, i.e., where immunogold labeling predominates after application of widely different methods. Direct identification by freeze-fracture replica labeling, although highly desirable, is not possible. (b) IMP size and arrangement are compatible with a SERCA-type molecule but not with any of the known Ca2+ release channels. (c) Aggregation of SERCA molecules notoriously occurs in the SR, by far the best known Ca2+ store. These arguments are discussed in more detail below.
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Discussion |
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To substantiate the partial aggregation and microzonal arrangement of SERCA molecules in alveolar sacs, we combined different criteria, using Western blots, phosphoenzyme intermediate analysis, IMP size, and IMP aggregation analysis (freeze-fracturing combined with MSA image analysis), as well as EM immuno-localization studies. With the Abs against a PtSERCA peptide previously characterized by Western blots (
What Are the Ultrastructural Characteristics of Alveolar Sacs?
Freeze-fracture replica labeling allows clear identification of freeze-fracture levels within the cell membrane/OM-AS/IM-AS complex. Therefore, we could ascertain that the OM-AS region is rather poor in IMPs which do not form any conspicuous aggregates. OM-AS fractures, however, enabled us to recognize lateral suture-like connections formed by double IMP rows, first described by
How Should We Address the Possible Identity of IMP Aggregates in the IM-AS Region?
Because Ca2+ stores must contain Ca2+ release channels, these must be excluded as equivalents of IMP aggregates in IM-AS. According to the [Ca2+]i dynamics determined during exocytosis stimulation, we presume mobilization of Ca2+ from alveolar sacs is the first step and that Ca2+ influx from the medium is a second step (
Could Enrichment of SERCA Molecules Account for the Freeze-fracture Appearance of the IM-AS Region?
The following arguments support this assumption. Whereas IMP density is low or moderate in the OM-AS region, it is very high in the IM-AS region, just as in the SR (
How does this compare with other systems? Analyses of vertebrate systems [
How Can This Arrangement of SERCA Molecules in Alveolar Sacs Account for Functional Requirements?
Our main conclusion is that each alveolar sac is a separate Ca2+ storage unit, with SERCA-type pumps enriched in the IM-AS region. Clearly, [Ca2+]i in the narrow 15-nm subplasmalemmal space would be regulated most efficiently by the plasmalemmal Ca2+ pump. Finally, on stimulation of exocytosis, a large flow of Ca2+, released from alveolar sacs and enforced by Ca2+ influx (
Are there still some other possible functions of alveolar sacs? On induction of ciliary beat reversal by cell membrane depolarization, Ca2+ enters the cell via voltage-dependent Ca2+ channels in the ciliary membrane (
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Acknowledgments |
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Supported by DFG grant SFB156/B4 and by the Forschergruppe "Cell surface structure and function."
We thank Ms C. Braun, S. Kolassa, and B. Kottwitz for excellent technical assistance, and J. Hentschel, S. Huber, and N. Klauke for probing some samples. We gratefully acknowledge the generous help of Dr F. Wuytack (University of Louvain, Belgium) with anti-SERCA 3 Abs.
Received for publication November 4, 1998; accepted February 23, 1999.
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