Immunolocalization of Actin in Paramecium Cells
Department of Biology, University of Konstanz, Konstanz, Germany
Correspondence to: H. Plattner, Department of Biology, University of Konstanz, P.O. Box 5560, 78457 Konstanz, Germany. E-mail: helmut.plattner{at}uni-konstanz.de
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Summary |
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Key Words: actin cilia immunolocalization microfilaments vesicle traffic Paramecium
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Introduction |
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Different actin isoforms occurring in many organisms may serve specific functions in the respective cells (Pollard et al. 2000; Wagner et al. 2002
). For localization, antibodies (ABs) may be used at the light microscope (LM) and electron microscope (EM) levels, as well as for Western blots. Bicyclic peptide toxins, phalloidin or jasplakinolide, can bind rather specifically to F-actin, thus allowing fluorescence labeling (Wieland and Faulstich 1978
; Bubb et al. 2000
). This or the alternative approach, F-actin disruption by toxins of the type cytochalasin B and D or latrunculin A, is also widely used for functional analyses also with ciliates (see below).
In previous times, mainly before molecular biology approaches could be undertaken, biochemical, functional, and immunolocalization studies were tried to probe the potential function of F-actin in ciliates such as Paramecium (Tiggemann and Plattner 1981; Cohen et al. 1984
; Fok et al. 1985
; Kersken et al. 1986a
,b
), Tetrahymena (Mitchell and Zimmerman 1985
; Hirono et al. 1987b
,1989
; Hoey and Gavin 1992
), Pseudomicrothorax (Hauser et al. 1980
), Histriculus (Pérez-Romero et al. 1999
), Climacostomum (Fahrni 1992
), and Spirostomum (Zackroff and Hufnagel 1998
). However, with ciliates, F-actindisrupting drugs frequently had to be used in conspicuously high concentrations to abolish, e.g., phagocytosis (Fok et al. 1987
; Zackroff and Hufnagel 1998
,2002
). With a variety of protozoa of the phylum Alveolata, actin genes or partial sequences of it have been cloned. This holds in particular for ciliates, such as Tetrahymena (Zimmerman et al. 1983
; Cupples and Pearlman 1986
; Hirono et al. 1987a
) and Paramecium (Díaz-Ramos et al. 1998
), but also for their pathogenic relatives of the group of Apicomplexa such as Toxoplasma (Delbac et al. 2001
).
Our present analysis also addresses some special subcellular structures in Paramecium cells that contain multiple filament systems (Allen 1971; Cohen et al. 1984
,1987
; Cohen and Beisson 1988
; Keryer et al. 1990a
,b
; Allen et al. 1998
; Beisson et al. 2001
; Clérot et al. 2001
). We focus on regions with dense-core secretory vesicles ("trichocysts"), cortical filament bundles ("infraciliary lattice," cf. Allen 1971
,1988
), the narrow space between the plasma membrane and tightly attached cortical Ca2+-stores ("alveolar sacs," see Plattner and Klauke 2001
), in addition to abundant vesicles of the phago-/lysosomal and recycling system (Fok and Allen 1990
; Allen and Fok 2000
). Recent cloning of several actin genes of Paramecium tetraurelia in our laboratory opened up a new way to structural localization with this cell, whose regular "design" facilitates such studies. So far, studies on actin in Paramecium have not addressed all relevant aspects, and many aspects have remained controversial.
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Materials and Methods |
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Expression of Paramecium Actin-specific Peptides in Escherichia coli
For heterologous expression of actin-specific peptides we selected the amino acid sequence of actin1-1 (accession number AJ537442). After changing all deviant Paramecium glutamine codons (TAA and TAG) into universal glutamine codons (CAA and CAG) by PCR methods, the coding regions of either E57-P243 (N-terminal region) or L251-G366 (C-terminal region) of Paramecium actin1-1 were cloned into the XhoI/BamH1 restriction sites of pET 16b expression vector of the pET System from Novagen (Madison, WI) which employs a His10 tag for purification of the recombinant peptides.
Purification of Recombinant Actin1-1 Peptides
Recombinant actin1-1 peptides, actin1-1E57-P243 and actin1-1L251-G366 were purified by affinity chromatography on Ni2+-nitrilotriacetate agarose under native conditions, as recommended by the manufacturer (Novagen). The recombinant peptides were eluted with a step gradient, 100 to 1000 mM imidazole in 50 mM sodium phosphate (pH 6.0) with 300 mM NaCl added. The fractions collected were analyzed on SDS polyacrylamide gels, and those containing the recombinant peptides were pooled and dialyzed in phosphate-buffered saline (PBS).
Antibodies Used
ABs against the two recombinant actin peptides, actin1-1E57-P243 and actin1-1L251-G366, were raised either in rabbits or mice. After several boosts, positive sera were taken at day 60 and purified by two subsequent chromatography steps, a first step on a His-tag peptide column (24-amino acid peptide, to remove His tag-specific ABs), followed by an affinity step on the corresponding actin1-1 peptide. One of these ABs recognizes the N-terminal and the other the C-terminal region of actin1-1, yet results achieved in this study were indistinguishable with either type of ABs. Therefore, no further distinction is made, unless indicated. We used the sequence of Paramecium actin 1-1 because it is rather similar for numerous isoforms that we have cloned (R. Kissmehl, J. Mansfeld, E. Wagner, I. Sehring, H. Plattner, unpublished data) and thus should allow us to establish an overall distribution of actin, notably of F-actin, in Paramecium.
Mouse polyclonal ABs against Paramecium actin1-1 were selectively used for the colocalization at the LM level, in conjunction with an anti-centrin (Dictyostelium discoideum) polyclonal AB produced in rabbits (designation HisDdCentrin2 from R. Gräf, University of Munich) used to identify ciliary basal bodies (Daunderer et al. 2001).
Cell Fractionation
Cells were deciliated by a Mn2+-shock (for details, see below) and cilia were purified by differential centrifugation (Nelson 1995). Whole-cell homogenates were prepared in phase buffer (20 mM Tris-maleate, 20 mM NaOH, 20 mM NaCl, 250 mM sucrose, pH 7.0) by
100 hand strokes in a glass homogenizer equipped with a Teflon pestle. Soluble and particulate fractions were separated by centrifugation at 100,000 x g for 60 min at 4C. Cell surface complexes ("cortices") were prepared according to Lumpert et al. (1990)
, and trichocysts were isolated by the method of Glas-Albrecht and Plattner (1990)
. A protease-inhibitor cocktail containing 15 µM pepstatin A, 100 mU/ml aprotinin, 100 µM leupeptin, 0.26 mM TAME, 28 µM E64, and 0.2 mM Pefabloc SC was used throughout.
Electrophoretic Techniques and Western Blot Analysis
Protein samples were denatured by boiling for 3 min in sample buffer (0.4 M TrisHCl, 1% SDS, 0.5% DTT, 20% glycerol, pH 8.0) and subjected to electrophoresis on linear gradient (520%) SDS polyacrylamide gels using the discontinuous buffer system of Laemmli (1970). Before electrophoresis, samples were alkylated for 30 min at 20C by 2% iodoacetamide. Protein standards were used in accordance with manufacturer directions. Gels were either stained with Coomassie blue R250 or prepared for electrophoretic protein transfer onto nitrocellulose membranes. Protein blotting was performed at 2 mA/cm2 for 1 hr according to the technique of Kyhse-Andersen (1989)
using the semidry blotter from BioRad (Munich, Germany). ABs were diluted 1:1000 in 0.25% (w/v) non-fat dry milk and Tris-buffered saline, pH 7.5, and applied overnight at 4C. AB binding was visualized by a second AB coupled to alkaline phosphatase (Sigma: Taufkirchen, Germany) using 5-bromo-4-chloro-3-indolyl phosphate and Nitro Blue tetrazolium as substrates.
Immunofluorescence Labeling
Basic Procedure
Cells were washed twice in 5 mM Pipes buffer, pH 7.0, containing 1 mM KCl and 1 mM CaCl2. Cells were fixed in 4% (w/v) freshly depolymerized formaldehyde for 20 min at room temperature. Cells were then permeabilized and fixed in a mixture of 0.5% digitonin and 4% formaldehyde, dissolved in 5 mM Pipes buffer, pH 7.0, for 30 min. Cells were washed twice in PBS, 2 x 10 min in PBS with 50 mM glycine added and 30 min in this solution with 1% bovine serum albumin (BSA) added. The rabbit anti-actin AB was applied in a dilution of 1:50 in PBS (+1% BSA) for 90 min at room temperature. After 4 x 15 min washes in the same solvent, FITC-conjugated anti-rabbit ABs, diluted 1:50, were applied for 90 min, followed by 4 x 15 min washes in PBS. Samples were shaken gently during all incubation and washing steps.
Deciliated Cells
Cells were washed twice in 5 mM Pipes buffer, pH 7.0, each containing 1 mM KCl and CaCl2, at room temperature and suspended in 50 mM MnCl2 solution in 10 mM Tris-HCl, pH 7.2. After 2 min at 4C, cells were removed by centrifugation and resuspended in the same solution. After 10 min of gentle shaking, 9095% of cells were deciliated. Deciliated cells were removed by centrifugation and washed twice in Pipes buffer before further use.
Deciliated cells were fixed in 8% (w/v) freshly depolymerized formaldehyde with 0.5% digitonin, 1 mM ATP, 10 mM MgCl2, and 10 mM KCl added, for 20 min on ice in Pipes buffer, pH 7.0. After fixation, cells were washed twice in PBS, 2 x 10 min in PBS with 50 mM glycine added and 30 min in this solution with 1% BSA added. The mouse anti-actin AB was applied in a dilution of 1:50 in PBS (+1% BSA) for 90 min at room temperature. After 4 x 15 min washes in the same solvent, FITC-conjugated anti-mouse ABs, diluted 1:50, were applied for 90 min, followed by 4 x 15 min washes in PBS. A second labeling with anti-centrin ABs from rabbits was performed as described above, using Texas Redconjugated anti-rabbit ABs. Anti-rabbit and anti-mouse fluorescent AB conjugates were from Sigma-Aldrich (St Louis, MO) and Serva (Heidelberg), respectively.
Light Microscopy
Cells were mounted with Mowiol supplemented with n-propylgallate to reduce fading. To analyze fluorescence staining, we used a conventional LM, type Axiovert 100TV (Zeiss; Oberkochen, Germany), or a confocal laser scanning microsope (CLSM) type LSM 510 (Zeiss) equipped with a Plan-Apochromat x63 oil immersion objective (numeric aperture 1.4). Images acquired with the LSM 510 software were processed with Photoshop software (Adobe Systems, San Jose, CA).
Fixation and Embedding for Postembedding EM Analysis
Using a quenched-flow apparatus (Knoll et al. 1991), Paramecium cells were rapidly injected into 8% formaldehyde plus 0.1% glutaraldehyde dissolved in Pipes buffer, pH 7.2 (0C), with 1 mM KCl and CaCl2 each added, further fixed for 60 min at 4C, washed in PBS (pH 7.4) + 50 mM glycine (2 x 10 min), dehydrated by increasing ethanol concentrations (30%, 50%, 70%, 90%, 96%, 2 x 15 min each, and 2 x 100%, 30 min each), and impregnated with LR Gold resin (London Resin, London, UK) at 0C, with two changes in 2-hr intervals each and then overnight, followed by UV-light polymerization at 35C for 72 hr.
Immunogold Labeling and EM Analysis
Postembedding Method
Ultrathin sections mounted on formvar-coated Ni grids were pretreated (2 x 10 min) with 20 µl of PBS, then for 10 min with PBS with 50 mM glycine added, and finally immersed in PBS supplemented with 0.5% BSA and 0.5% goat serum (2 x 10 min, room temperature), to eliminate nonspecific gold adsorption. Grids were then incubated with rabbit AB, diluted 1:20 in PBS supplemented with 0.3% BSA-c (BioTrend, Köln, Germany), pH 7.4, 1 hr at room temperature. BSA-c as an acetylated form reduces nonspecific adsorption of gold conjugates due to increased net charge.
Samples were washed in PBS/BSA-c (0.3%) three times, 10 min each, and treated for 1 hr with gold conjugates. We used goat anti-rabbit IgGs coupled to gold of 5 nm (Au5) provided by Sigma, diluted 1:30.
Preembedding Labeling
Without exception, cells were fixed with 8% formaldehyde + 0.1% glutaraldehyde and simultaneously treated with digitonin (Sigma) and the other additives, as described above for LM analysis of deciliated cells, incubated with primary rabbit ABs against Paramecium actin1-1, followed by Au5-conjugated second ABs, with the aim to make the narrow subplasmalemmal space accessible. After embedding in LR Gold (London Resin), sections were additionally subjected to the postembedding labeling procedure with the same primary and secondary ABs, respectively.
Specificity of Immunogold Labeling and Further Processing
This was verified by the significant reduction of the number of Au5 particles on sections incubated with ABs that had been preadsorbed with the original antigen.
Further Processing and Quantitative Evaluation
After labeling, sections were rinsed with distilled water, fixed for 5 min with 2% glutaraldehyde, and routinely stained for 3 min with 2% aqueous uranyl acetate (unbuffered, pH 4.5). EM micrographs were taken at defined magnifications and enlarged to x77,000. Au5 grains were counted and referred to area size determined by superposition of square lattices with 5, 10.0 and 20.0 mm spacing, respectively, depending on the size of the structure to be analyzed (Plattner and Zingsheim 1983
). The actual area sizes to which the numbers of gold grains were referred were determined from the number of hit points.
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Results |
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We used conventional LM analysis to analyze immuno-FITC labeling of cilia with anti-actin ABs (Figure 4), thus taking advantage of a thicker optical section layer. While intracellular details are largely blurred, ciliary basal bodies and cilia on the outer cell surface are clearly labeled. This may also apply to cilia in the oral cavity, although this is not resolved in Figure 4.
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Figure 9 represents experiments with digitonin-permeabilized cells, showing AB-gold labeling in the narrow subplasmalemmal space between the plasmalemma and the outer side of alveolar sacs, while there is only spurious label occasionally seen after mere section labeling (Figures 5 and 7). Apart from this aspect, little label only is seen in the cell cortex with permeabilized cells (Figure 9). While digitonin permeabilization may be more appropriate than section labeling to show the presence of some actin in the very narrow outermost cytosolic space, particularly when enhanced by additional postembedding labeling (Figure 9), it may cause a serious overall loss of antigen. The abundance of cortical label after postembedding labeling justifies reliance in this study mainly on the postembedding procedure for further evaluation. Concomitantly, all figures presented with the exception of Figure 9 were obtained by this method.
Specification of Results Obtained with Postembedding Labeling
Beyond the general labeling of the cytosolic compartment of the cell cortex (Figures 5 to 8), we recognize that gold granules are enriched to a variable extent in a variety of structures.
The cytoplasm of cell surface ridges, typical of ciliated protozoa, are labeled (Figures 5 and 6). This also holds for the cytoplasm surrounding the tips of the elongate trichocyst organelles, as shown in cross-section (Figures 5 and 6) and in longitudinal section (Figure 7). The gold label associated with cortical basal bodies is somewhat variable and may in part sit inside this structure, as shown particularly in Figure 8B, where it shows up below the basal plate (Figure 8A). Gold label also occurs adjacent to cortical basal bodies, e.g., in the filamentous mass in Figure 6. This material is associated with the origin of a kinodesmal fiber emanating from a basal body from where the infraciliary lattice also emanates. From there, these filament bundles pass near adjacent trichocyst tips (Figure 5), as established by Allen (1971)(1988
). Although the bulk of the latter filament system is made of centrin (Beisson et al. 2001
), some actin clearly appears to be associated with it. Gold label also surrounds ghosts from discharged trichocysts (Figure 6).
Table 1 summarizes labeling densities on a quantitative level (gold grains per µm2). These are, in decreasing magnitude, as follows: 301.0 Au/µm2 for cytoplasmic regions around oral cavity and around food vacuoles, 141.5 for cell surface ridges, 111.9 for immediate surroundings of trichocysts, between 89.5 and 95.6 for infraciliary lattice, ciliary basal bodies, and cilia, followed by cortical cytoplasm (37.8) and the complex formed by the plasma membrane and the outer alveolar sacs membrane (25.9 Au/µm2). For statistics, see Table 1.
While the abundant filament bundles located in the cytoplasm around the oral cavity are made of materials other than actin (see "Discussion"), the distinct labeling in between such bundles (Figure 10A) again indicates association with actin. As in the cell cortex, some label may be associated with ciliary basal bodies around the oral cavity. Furthermore, we find intense labeling of cytosolic regions enriched in vesicles accumulated near the cytopharynx (Figure 10B). Many are oblong and thus represent discoidal vesicles known to serve membrane recycling from the cytoproct, i.e., formation of new phagocytic vacuoles (Fok and Allen 1988; Allen and Fok 2000
). In these domains of the cell, less labeling is seen immediately below the cell membrane than between the adjacent round and discoidal vesicles.
Deeper inside the cell, small vesicles of different diameters are embedded in considerably labeled cytosol, frequently in close association with a large vacuole (Figures 11A and 11B). This arrangement suggests their identity either as lysosomes or as acidosomes in typical arrangement with phagosomes. These interpretations are suggested by the work of Allen and Fok (2000); e.g., considering the flat shape of the large vacuole indicating an early biogenetic stage of a food vacuole. Figure 11B shows association of actin label with parallel microtubular aggregates, the gold label unilaterally concentrated at sites where microtubules enter the section plane. Also in Figure 11B, a heavily labeled "trail" is in direct extension of the adjacent microtubular bundle. This indicates involvement of actin in phago-lysosomal vesicle trafficking, although after the preparation protocol required for immunogold analysis, distinct filaments are difficult to recognize. However, some of these gold aggregates may be the equivalent of the fluorescent strands visualized by anti-actin ABs in Figure 3.
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Discussion |
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Previous attempts to localize actin in Paramecium have led to controversies. One discrepancy concerned the composition of cortical filament bundles, notably of the infraciliary lattice emanating from ciliary basal bodies. While the bulk of this filament system has been established as centrin (Beisson et al. 2001), this does not necessarily preclude association of centrin filaments with actin, as we can show. Recall that widely different affinity stains for actin, including heavy meromyosin, have resulted in cortical labeling in Paramecium (Tiggemann and Plattner 1981
; Kersken et al. 1986a
,b
), as well as in Tetrahymena (Méténier 1984
). Theoretically, previous LM and preembedding-EM localization studies could have faced the problem of soluble antigen relocation and even loss during permeabilization. This would not easily be possible with the postembedding immuno-EM labeling procedure used now. Another hint to real cortical F-actin localization in Paramecium came from the in vivo labeling by injection of fluorescent phalloidin (Kersken et al. 1986a
,b
), resulting first in cortical labeling and, over longer time periods, in disappearance from the cortex and re-assembly as thick trans-cellular filament bundles of a type not previously seen. Conversely, aberrant phalloidin binding by F-actin formed by some isoforms may preclude labeling (Hirono et al. 1989
), while such forms may bind actin-specific ABs.
Additional Functional Aspects Derived from This Study
Cortical F-actin is generally required for cyclosisan actomyosin-based process (Shimmen and Yokota 2004). This is a permanent ongoing process also in Paramecium (Sikora et al. 1979
), where it serves the delivery of trichocysts to the cell cortex (Aufderheide 1977
) and the cycling of phago-lysosomal elements through the cell body (Fok and Allen 1988
,1990
; Allen and Fok 2000
). Myosins occur in Paramecium (Cohen et al. 1987
), just as in other protists (Gavin 2001
).
Our present EM analysis verifies that in the Paramecium cell cortex, actin is enriched at ciliary basal bodies, as discussed above on the LM level. From there it emanates to the infraciliary lattice and around trichocyst docking sites. The association of actin with ciliary basal bodies has led to the description of the "basal body cage," particularly in Tetrahymena (Hoey and Gavin 1992), where association with myosin has been demonstrated (Garcés et al. 1995
). The loose arrangement of gold label within and around basal bodies, as we see it here, suggests that during permeabilization for LM analysis, F-actin emanating from basal bodies may collapse to a compact arrangement. In sum, a more loosely arranged cortical F-actin in conjunction with myosin may underlie cytoplasmic streaming and possibly trichocyst docking. Concomitantly, inhibition of trichocyst docking by cytochalasin B (Beisson and Rossignol 1975
) would be compatible with both actin-based transport by cyclosis and enrichment of actin around trichocyst tips (this study).
Assembly of F-actin around nascent phagosomes is well established, not only in mammalian cells but also in Paramecium cells (Allen and Fok 1983; Fok and Allen 1988
). In detail, fusion of acidosomes with the nascent food vacuole depends on F-actin (Fok et al. 1987
), as does maturation along the phago-lysosomal pathway, where multiple fusion/fission processes occur (Allen and Fok 1985
; Allen et al. 1995
). Interestingly, in our study, gold labeling immediately below the cytopharyngeal plasma membrane is less intense than between the closely packed globular and discoidal vesicles slightly below. This can be seen in line with the following reports. In Dictyostelium, F-actin prevents clustering of endosomal vacuoles (Drengk et al. 2003
). Alternatively, in yeast, actin is required for Ca2+-mediated vacuole interaction leading to fusion (Merz and Wickner 2004
). The final step of this cycle in Paramecium, exocytotic release of spent phago-lysosomes, can also be inhibited by cytochalasin B (Allen and Fok 1985
). In agreement with this previous work, the site of phagosome formation, vacuoles of different size, and the cytoproct are clearly labeled with anti-actin ABs in our CLSM and EM pictures. Therefore, the fine filaments described at the cytoproct by Cohen et al. (1984)
are, at least to some extent, F-actin. However, centrin also occurs at the cytoproct, according to the CLSM pictures presented in Figure 3.
At the EM level, we see that the cytosolic compartment around large and small vacuoles is frequently heavily labeled (even when filaments are difficult to discern due to faint contrast resulting from preparation for immuno-EM analysis). This holds, e.g., for domains with clearly visible microtubule bundles deep inside the cell and for regions with discoidal vesicles approaching the cytopharynx. The latter are delivered along microtubule rails, using dynein as a motor (Schroeder et al. 1990). Therefore, actin at these sites may serve not as a motor, but rather as a kind of scaffold. In sum, apart from association with non-actin filaments (see below), we see that actin is also associated with the second cytoskeletal element, the microtubules. This agrees with functional data obtained by combined drug application (Fok et al. 1985
).
Label also occurs around the oral cavity outside the site of phagosome formation in the cytopharynx. Such filaments are known not to represent actin, either in Paramecium (Clérot et al. 2001), or in other ciliates (Viguès et al. 1999
). In these regions, F-actin may again serve structuring of these firmly established subcellular domains and/or vesicle trafficking. Interestingly, co-assembly of polymerizing actin with other filament components from Tetrahymena can be produced in vitro (Mitchell and Zimmerman 1985
).
Vesicles deeper inside the cytoplasm, often close to a large phagosome, are also surrounded by gold label. All this reflects that actin is present throughout the cell in LM analyses, frequently as strands. Actin may thus participate directly or indirectly in vesicle trafficking, including cyclosis.
Not only ciliary basal bodies, but also the ciliary shaft, are labeled by anti-actin ABs. Labeling of cilia has been reported previously based on peroxidase-based preembedding immunostaining in Paramecium (Tiggemann and Plattner 1981) and in quail oviducts (Sandoz et al. 1982
). Because this method is subject to redistribution artifacts (Plattner and Zingsheim 1983
), we considered a re-analysis by Western blots and by the postembedding EM methodology to be necessary. It is known only from flagella of the green alga, Chlamydomonas (Mitchell 2000
; Hayashi et al. 2001
; Hirono et al. 2003
), that actin is mandatory for normal beat activity. This may apply also to cilia of Tetrahymena, whose 14S axonemal dynein binds actin (Muto et al. 1994
). More details on the role of actin in cilia remain to be elucidated.
Another poorly understood aspect concerns coupling of cortical calcium stores to the cell membrane. With mammalian cells, one of the molecules considered to establish such connections, particularly for store-operated Ca2+-influx, is actin (Patterson et al. 1999; Rosado and Sage 2000
; Kunzelmann-Marche et al. 2001
; Wang et al. 2002
). Interestingly, we find gold label that may be associated with the narrow subplasmalemmal space not only using a variation of the general labeling procedure that faciliates access of ABs (Figure 9), but also, though to a lesser extent, using postembedding labeling (Figures 5 and 7). This becomes evident particularly after statistical evaluation (Table 1). Although cytochalasin B application did not change concomittant Ca2+ signals (Mohamed et al. 2003
), we keep this question open because the different actin isoforms found in Paramecium (Kissmehl et al., in preparation) may have different drug sensitivities.
Our present immunogold EM analysis largely depends on the preparation schedule used, whereas we obtained no such clear-cut labeling pattern with other approaches (data not shown). The current approach implied rapid injection (spraying) of cells in 0C aldehyde fixative, containing high formaldehyde and very low glutaraldehyde concentrations, followed by low temperature embedding and UV polymerization at 35C. This can considerably restrict diffusion of macromolecules and, even more, of filamentous aggregates. Therefore, we consider the current approach, elaborated on a (semi-)quantitative basis, more reliable than some previous attempts to localize actin in such cells.
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Acknowledgments |
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Footnotes |
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