ARTICLE |
Correspondence to: Eeva-Liisa Punnonen, Inst. of Biotechnology, PO Box 56, FIN-00014 University of Helsinki, Finland. E-mail: Eeva-LiisaPunnonen@Helsinki.Fi.
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Summary |
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We describe a nonradioactive preembedding in situ hybridization protocol using digoxigenin-labeled RNA probes and tyramide signal amplification to increase the sensitivity of detection. The protocol is sensitive enough for electron microscopic localization of endogenous messenger RNAs encoding ß-actin and amphoterin. Three visualization methods were compared: diaminobenzidine enhanced by nickel, Nanogold enhanced by silver and gold toning, and fluorescently labeled tyramides. Diaminobenzidine and Nanogold can be used in both light and electron microscopy. The nickel-enhanced diaminobenzidine was the most sensitive visualization method. It is easy to accomplish but a drawback is poor spatial resolution, which restricts its use at high magnifications. Nanogold visualization has considerably better spatial resolution and is therefore recommended for electron microscopy. Fluorescent tyramides, especially TRITCtyramide, offer a good detection method for fluorescence and confocal microscopy. The methods were used to localize amphoterin and ß-actin mRNAs in motile cells. Both mRNAs were found in the soma and cell processes. In double labeling experiments, ß-actin mRNA localized to filamentous structures that also contained ribosomal proteins. Especially in the cortical cytoplasm, ß-actin mRNA was associated with actin filaments. Direct localization to microtubules was only rarely seen. (J Histochem Cytochem 47:99112, 1999)
Key Words: in situ hybridization, electron microscopy, tyramide signal amplification, mRNA localization, ß-actin, amphoterin
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Introduction |
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Subcellular compartmentalization of mRNA has been recognized as a mechanism for regulation of gene expression and protein sorting (
These recent findings have greatly added to the need for sophisticated electron microscopic localization methods for mRNA, which have not been widely available thus far. The ultrastructural localization of mRNA has mainly been limited by the low sensitivity of the methods described for in situ hybridization at the electron microscopic level. The specificity of mRNA localization is based on specific hybridization between the target and the complementary probe molecule (
Here we describe a sensitive preembedding in situ hybridization protocol for ß-actin and amphoterin mRNAs, both encoding cytoplasmic proteins that are synthesized outside the endoplasmic reticulum. ß-Actin mRNA is abundant and expressed in practically all cell types and is widely used as a control in Northern blot hybridization. Amphoterin mRNA is mainly expressed in the developing nervous system and motile cells in culture (
Tyramide signal amplification (
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Materials and Methods |
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Cell Culture
Rat C6 glioma cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calf serum at 37C in 5% CO2. For the experiments, the cells were plated on glass (light microscopy) or plastic (electron microscopy) Lab-Tek chamber slides (Naperville, IL) or on plastic Lab-Tek coverslips coated with laminin (Sigma; St Louis, MO) (10 µg/ml in PBS, overnight at 4C). The cells were plated in DMEM containing 1% bovine serum albumin 34 hours before fixation. These conditions were used to produce well-spread, motile cells having many processes. In some experiments, the cells were extracted with 0.1% Triton X-100 in extraction buffer (10 mM Pipes, pH 6.9, 0.3 M sucrose, 0.1 M NaCl, 3 mM MgCl2, 1 mM EGTA) for 60 sec at room temperature (RT) before fixation. This protocol removes the plasma membrane and most of the soluble cytoplasm, leaving the cytoskeleton intact, including the material that is associated with it (
Probes
Linearized Bluescript plasmid containing the full-length coding region (650 BP) of rat amphoterin (
In Situ Hybridization
The cell monolayers were fixed in 4% paraformaldehyde and 0.05% glutaraldehyde in 0.1 M Hepes buffer, pH 7.4, for 30 min at RT. In situ hybridization protocol was modified from
Detection Methods
Direct Visualization of Peroxidase with Diaminobenzidine.
DAB (Sigma) was diluted to 150 µg/ml in 50 mM Tris, pH 7.5. Nickel ions were added to the solution as diammonium nickel(II)sulfate 6-hydrate (BDH; Poole, UK) at 6 mg/ml to increase sensitivity (
Tyramide Signal Amplification.
The cells were incubated in biotinylated tyramide (NEN Life Science Products) diluted 1:50 in amplification diluent (NEN Life Science Products) at RT for 410 min. For fluorescence microscopy, tyramide coupled to tetramethylrhodamine (TRITC) or fluorescein (FITC) was used. The cells were then washed in TBSsaponin three times for 5 min. After biotinyltyramide, the biotin epitopes were detected with (a) streptavidinperoxidase (NEN Life Science Products) diluted 1:100, (b) streptavidinNanogold (1.4 nm gold) (Nanoprobes; New York, NY) diluted 1:3001:400, or (c) streptavidinfluorescein (NEN Life Science Products) diluted 1:400 in TBSsaponin containing 1% bovine serum albumin or 0.5% blocking reagent (NEN Life Science Products), at RT for 1 hr. The cells were then washed in TBS-saponin four times for 5 min. Peroxidase activity (a) was visualized with DAB as described above. After Nanogold incubation (b), the samples were fixed in 1% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4 for 10 min. Nanogold was silver-enhanced with the HQ Silver kit (Nanoprobes) for 59 min once or twice and then toned with gold chloride (
Immunolabeling of Tubulin, Actin, and Ribosomal 60S Subunit
For double localization, TRITCtyramide or Nanogold enhanced by silver and gold was first used for detection of mRNA. Cells extracted with Triton X-100 before fixation were used for electron microscopic experiments. In this case, the cells were fixed in 0.1% glutaraldehyde in 0.1 M phosphate buffer for 10 min before the silver enhancement step. After in situ hybridization, cytoskeletal filaments were labeled with monoclonal antibodies. Anti-tubulin (clone E7) was from Developmental Studies Hybridoma Bank (Iowa City, IA) and anti-actin was from Boehringer (clone C4) or Sigma (AC-15, anti-ß-actin). Proteins of the ribosomal 60S subunit were labeled with a rabbit antibody kindly provided by Dr. John Hesketh (
Epon Embedding
The cells were postfixed in 1% osmium tetroxide in 0.1 M phosphate buffer, pH 7.4, dehydrated in a graded series of ethanol, and flat-embedded in Epon. After dehydration, the cells were covered with a thin layer of Epon. The samples were then examined under an inverted microscope, and gelatin capsules filled with Epon were inverted to the monolayers on sites containing an adequate number of labeled cells. Thin sections were cut parallel to the culture substrate, mounted on single-slot copper grids, and stained with uranyl acetate and lead citrate.
Microscopy
The samples were examined and photographed under an Olympus AX-70 light microscope, a Zeiss Axiovert 135M inverted fluorescence microscope with BioRad MRC1024 confocal equipment, or a Jeol JEM 1200EX transmission electron microscope.
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Results |
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Evaluation of the Detection Methods with Light Microscopy
Detection with Diaminobenzidine.
ß-Actin mRNA levels in spreading C6 cells were high enough to be detected by direct labeling with anti-digoxigeninperoxidase followed by DAB incubation (Figure 1A). It was essential to use nickel ions in the DAB solution to enhance the signal (
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Tyramide Amplification Followed by StreptavidinPeroxidase and DAB or by StreptavidinGold.
Tyramide was used to amplify the signals after hybridization with digoxigenin-labeled probe and labeling with anti-digoxigeninperoxidase. Peroxidase activity causes covalent incorporation of tyramide (in this case biotinylated tyramide) molecules to the reaction site (
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Visualization with Nanogold and silver enhancement, after tyramide amplification for 10 min, was not as sensitive as DAB and nickel. ß-Actin mRNA was readily detectable (Figure 1D), but amphoterin mRNA was only weakly visible, even after repeated incubations with the silver-enhancement solutions (Figure 2C).
In conclusion, visualization with DAB and nickel can be recommended for light microscopic experiments. It is less expensive and simpler to perform than ultrasmall gold and silver enhancement. In addition, the development of color can be followed during DAB incubation and the reaction can therefore be stopped once the desired signal intensity has been reached or background starts to appear. In our experiments, the anti-digoxigeninperoxidase conjugate was the most obvious source of background. This was evident after longer tyramide amplification (Figure 2B), both in cells hybridized with the sense probe and in cells treated identically but without any probe. No background developed if anti-digoxigeninperoxidase was omitted.
Detection for Fluorescence Microscopy
Two protocols were tested: indirect labeling with biotinyltyramide followed by streptavidinFITC, and direct labeling with FITC or TRITCtyramide. Indirect labeling was the most sensitive method. Both ß-actin (Figure 1E) and amphoterin (Figure 2E) mRNA were readily visible with this protocol, without background in cells hybridized with the sense probes (Figure 1F and Figure 2F). However, a longer biotinyltyramide amplification was necessary to visualize amphoterin mRNA. In direct detection, only TRITCtyramide gave signal for amphoterin mRNA. TRITCtyramide is therefore more sensitive than FITCtyramide. In addition, TRITCtyramide gave a better spatial resolution. It produced well-structured granular label (Figure 5), whereas FITCtyramide gave a more diffuse signal (not shown).
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Electron Microscopy
Visualization of the hybridized probes with DAB and nickel ions produced electron-dense precipitates that were visible as spots in the cytoplasm (Figure 3A). ß-Actin was readily detectable without tyramide amplification (not shown), whereas a 10-min biotinyltyramide amplification step, followed by incubation with streptavidinperoxidase, was necessary to reliably visualize amphoterin mRNA (Figure 3A). The ultrastructure of the cells, especially endoplasmic reticulum, endosomal vesicles, and the Golgi apparatus, was good (Figure 3A). Some of the mitochondria were partially swollen (not shown).
The use of ultrasmall gold (11.4 nm in diameter) is essential for penetration of the conjugates into cells if the use of drastic permeabilization methods is to be avoided to preserve the ultrastructure of membrane-bound organelles. Ultrasmall gold has the additional advantage of high sensitivity. Compared to 5- or 10-nm colloidal gold particles, it has been shown to markedly increase the sensitivity of labeling (
Silver enhancement is necessary for visualization of the ultrasmall gold in heavy metal-stained samples.
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In addition to producing larger gold clusters after gold toning, repeated silver enhancement also produced more intense labeling. This is probably due to the nonhomogeneous growth of ultrasmall gold particles in the silver enhancement step (
Subcellular Localization of ß-Actin and Amphoterin mRNAs
Both ß-actin and amphoterin mRNAs were found in the cell body and cell processes (Figure 1E, Figure 2E, Figure 3A, Figure 3B, and Figure 4AC). This is compatible with the role of both proteins in cell motility and process growth (
In the cell body, the mRNA was restricted to certain regions of cytoplasm. Typically, ß-actin mRNA was localized in the region rich in cytoplasm surrounding the nucleus, not reaching the thin peripheral area (Figure 5A and Figure 5C). However, the cytoplasm at one side of the nucleus, especially if the nucleus was asymmetrically located, was devoid of label, as was a small area in the perinuclear cytoplasm (Figure 5A and Figure 5C). Electron microscopic images suggested that the Golgi apparatus is located in the area devoid of ß-actin mRNA (Figure 3B).
Association of ß-Actin mRNA with the Cytoskeleton
To increase the contrast of the cytoskeletal filaments, we extracted the cells with Triton X-100 before fixation to wash out the plasma membrane and soluble cytoplasm. This also enabled the use of colloidal gold particles for double labeling of mRNA and proteins. Labeling for ß-actin mRNA was detected in the extracted cells (Figure 6AD), suggesting that the mRNA is associated with the insoluble cytoskeleton. The mRNA was associated with characteristic knobbly, filamentous structures that also showed labeling for the proteins of ribosomal 60S subunit (Figure 6A). Some of the ribosomes are known to be associated with the cytoskeleton (
Previous studies have shown that many mRNAs, including ß-actin mRNA, are associated with the cytoskeleton, especially with actin and/or tubulin filaments (
Overlap of fluorochromes at the light microscopic level can be caused either by localization in close proximity, or by real co-localization, of the two labels (
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Discussion |
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Electron microscopic in situ hybridization has earlier been used to localize abundant mRNAs, such as messages for cytoskeletal proteins (
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Amplification of the signal always decreases the spatial resolution of detection. We used tyramide signal amplification to enhance the sensitivity. In a quantitative study,
We observed that ß-actin mRNA was restricted to certain areas of the cytoplasm (Figure 3B, Figure 5A, and Figure 5B). The compartmentalization phenomenon was also seen in cells extracted with Triton X-100 before fixation, suggesting that it was not caused by hindered diffusion of the probe. This result may be an indication of compartmentalization of the cytoplasm into structural areas and nonstructural channels, which are used for intracellular transport and protein synthesis (
Both ß-actin and amphoterin mRNA localized as separate spots in the cytoplasm. Granular localization of mRNAs encoding proteins synthesized on cytoskeleton-bound polysomes has also been reported in earlier light microscopic studies. (
Most ß-actin mRNA labeling inside the cortical cytoplasm was associated to knobbly filamentous structures. We also detected immunolabeling for the ribosomal 60S subunit associated with these structures. This suggests that ß-actin mRNA and ribosomes are associated, either for translation or for transport.
Our recent experiments using drugs that disrupt microtubules or microfilaments showed that both ß-actin and amphoterin mRNAs are transported along and anchored to the actin microfilament network (not to microtubules) in neuroblastoma cells (
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Acknowledgments |
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We thank Mervi Lindman for excellent technical assistance and Dr John Hesketh for the ribosome antibody.
Received for publication June 3, 1998; accepted September 22, 1998.
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