ARTICLE |
Correspondence to: Michael J. Cullen, Dept. of Neurobiology, University Medical School, Framlington Place, Newcastle upon Tyne NE2 4HH, UK. m.j.cullen@ncl.ac.uk.
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Summary |
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An absence of dystrophin causes Duchenne muscular dystrophy, but the precise mechanism underlying necrosis of the muscle cells is still unclear. Dystrophin and ß-dystroglycan are components of a complex of at least nine proteins, the dystrophinglycoprotein complex (DGC), that links the membrane cytoskeleton to extracellular elements in skeletal and cardiac muscle. Biochemical studies indicate that dystrophin is bound to other components of the DGC via ß-dystroglycan, which suggests that the distribution of these two proteins should be almost identical. In this study, therefore, we examined the spatial relationship between dystrophin and ß-dystroglycan with a range of different imaging techniques to investigate the extent of the predicted co-localization. We used (a) double immunogold fracture-label, a freeze-fracture cytochemical technique that allows high-resolution face-on views of labeled membrane components in thin sections and in platinumcarbon replicas, (b) double immunogold labeling of cryosections and (c) confocal microscopy. Both dystrophin and ß-dystroglycan were found over the entire fiber surface and, when labeled singly, the nearest neighbor spacing of labeling sites for the two proteins was indistinguishable. With double labeling, very close co-localization could be demonstrated. The results support the conclusion that dystrophin and ß-dystroglycan directly interact at the muscle plasma membrane. (J Histochem Cytochem 46:945953, 1998)
Key Words: dystrophin, dystroglycan, immunogold, freeze-fracture
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Introduction |
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Dystrophin forms part of a large, tightly associated oligomeric complex of proteins, the dystrophinglycoprotein complex (DGC), located at the plasma membrane (-dystroglycan which, in turn, binds to merosin in the basal lamina (
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Since the first descriptions of the DGC based on biochemical data (
We have taken a different approach to obtaining a face-on view of the DGC by using immunogold labeling in combination with the freeze-fracture cytochemical technique, fracture-label (
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Materials and Methods |
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Tissue
Adult female rats (160200 g) were stunned and sacrificed by cervical dislocation. The external digitorum longus (EDL) and soleus muscles were quickly removed and immersed in fixative while pinned, slightly stretched, to dental wax. The primary fixative was 2% paraformaldehyde plus 0.01% glutaraldehyde in PBS at pH 7.4. For both fracture-label procedures and immunolabeling after cryosectioning, the muscles were cut into blocks approximately 1 x 1 x 1.5 mm after 1 hr and fixed for a further hour. For fracture-label procedures, the blocks were cryoprotected with 30% glycerol (in PBS buffer) for 2 hr before freezing. For immunolabeling after cryosectioning, the blocks were cryoprotected with 2.3 M sucrose for 2 hr or overnight before freezing.
Antibodies
A polyclonal antibody, P1583, to the last 17 amino acids of dystrophin was a gift from Dr. Henry Klamut (Ontario Cancer Institute, Toronto) and was raised by synthesizing the peptide (SSRGRNTPGPMREDIM) and conjugating this to BSA or keyhole limpet hemocyanin. The antibody against the peptide was raised in rabbit and purified using affinity chromatography on CNBrSepharose 4B columns as previously described (
Immunolabeling for Fracture-label
The glycerinated blocks of muscle were rapidly frozen by plunging them individually into liquid nitrogen slush (liquid nitrogen cooled to its melting point) and were then crushed under liquid nitrogen with a brass rod rotated in a copper well. Freeze-fracturing in this way generates fracture planes through the sample that preferentially follow the membrane planes, as in conventional freeze-fracture electron microscopy (
For single labeling, the muscle fragments were incubated overnight in the primary antibody followed, after washing, by the appropriate secondary goat anti-rabbit or anti-mouse antibody conjugated with 10-nm colloidal gold for 1 hr. The anti-dystrophin antibody was used at a dilution of 1:1000 and the anti-ß-dystroglycan at 1:50. After further washing, the specimens were postfixed in 2% paraformaldehyde plus 0.01% glutaraldehyde for 30 min. The larger fragments of the fractured muscle were then separated from the smaller pieces and prepared for platinumcarbon replication by partial dehydration (to 70% ethanol), air-drying, and mounting on the stage of a Balzers BAF 400T unit. Replicas were made by evaporation of platinum and carbon as in standard freeze-fracture, but at ambient temperature (
The remaining smaller fragments of fractured muscle were prepared for sectioning. They were postfixed in 2% osmium tetroxide, dehydrated through a graded series of alcohols, and embedded in Araldite. Semithin and ultrathin sections were cut at right angles to the fracture plane using a Reichert Ultracut E microtome.
Double Labeling and Controls
In double labeling experiments the two proteins were labeled serially. A typical sequence would be as follows: (a) polyclonal antibody to dystrophin; (b) goat anti-rabbit conjugated to 10-nm gold; (c) MAb to ß-dystroglycan; (d) goat anti-mouse conjugated to 5-nm gold. To control against steric hindrance of one conjugate by the other, the labeling sequence was repeated with the monoclonal preceding the polyclonal, and to control against the positioning being affected by the size of the gold conjugate the sequence was repeated with the secondary antibodies conjugated to the alternative size of gold. In some experiments, instead of a secondary goat anti-mouse conjugate, a biotinylated anti-mouse and streptavidingold system was used. Control experiments were also carried out in which dystrophin and ß-dystroglycan were double labeled with an irrelevant non-co-localizing protein (myosin). Other controls run in parallel were (a) omission of primary antibodies and (b) a single primary with both secondaries. Cross-fractured cells in positively labeled samples served as internal controls.
Immunolabeling After Cryosectioning
Our methods of immunolabeling cryosections have been extensively documented in previous publications (
Confocal Laser Scanning Microscopy
For immunoconfocal microscopy, 10-µm cryosections were cut at -25C and thaw-mounted on poly-L-lysine-coated glass slides. The sections were treated with 0.3% Triton X-100 for 15 min to improve permeability to the reagents and blocked with 0.5% bovine serum albumin (BSA) at room temperature. For single labeling, the sections were then incubated overnight with primary antibody followed by the appropriate anti-mouse or anti-rabbit secondary antibody. The anti-ß-dystroglycan antibody was used at a concentration of 1:50 and the anti-dystrophin antibody at 1:1000. The mouse 43DAG/8D5 (anti-ß-dystroglycan) was followed by Cy3mouse (1:500) for 1 hr and the rabbit (anti-dystrophin) with anti-rabbit-FITC (1:20) for 1 hr. For double labeling, sections were exposed sequentially to (a) polyclonal anti-dystrophin overnight, (b) monoclonal anti-ß-dystroglycan for 4 hr, (c) anti-rabbitFITC for 1 hr, and (d) Cy3anti-mouse 1:500 for 1 hr. All sections were mounted using Citifluor mounting medium. The following controls were run in parallel: (a) omission of primary antibody; (b) switching of detection systems in single labeling experiments (e.g., using mouse monoclonal followed by anti-rabbit secondary); and (c) reversing the order of primary antibodies in the double labeling procedure.
The immunolabeled sections were examined by confocal laser scanning microscopy using a Leica TCS 4D, equipped with an argon/krypton laser and fitted with the appropriate filter blocks for detection of fluorescein and Cy3 fluorescence. Double labeled samples were imaged using simultaneous dual channel scanning. Both single optical sections and projection views from sets of 10 consecutive single optical sections taken at intervals between 0.6 and 1 µm were examined. All specimens were examined within 24 hr of immunolabeling.
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Results |
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Single Labeling
When fractured samples were labeled individually for either dystrophin or ß-dystroglycan, the label was seen on those parts of the replicas that covered the P half of the plasma membrane, i.e., the half membrane leaflet left adjacent to the sarcoplasm after fracturing (
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The distribution of label as seen on the platinumcarbon replicas was confirmed in sections of the resin-embedded samples of the same tissue, fractured and immunolabeled in the same experiments (Figure 2B and Figure 2C). Gold particles were positioned at the surface of the sectioned fibers, verifying that the protein molecules of the fractured surfaces were accessible for immunolabeling. In both transversely (Figure 2B) and longitudinally (Figure 2C) orientated fibers there appeared to be no preferential labeling of zones corresponding to specific segments of the underlying myofibrils.
Histograms of the nearest neighbor distances of dystrophin and ß-dystroglycan labeling sites on the platinumcarbon replicas showed a very similar distribution with matching modes and means (mean ± SD 149.26 ± 59.57 and 147.24 ± 59.67, respectively) (Figure 3). The distributions of the nearest neighbor distances suggest that the positions of the sites are not completely random and that there is some regularity of structure. This was confirmed by comparing the means for the observed data with means calculated from 19 computer-simulated patterns of sites, each forming a completely random pattern and containing the same number of points as the number seen experimentally (technically a realization of a two-dimensional Poisson process). The mean nearest neighbor distances for all 19 simulated distributions were greater than the observed distances, providing strong evidence that there is an underlying systematic pattern to the positioning of the labeling sites.
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Double Labeling
When fractured samples were labeled consecutively for dystrophin and ß-dystroglycan, pairs of gold particles of different size could frequently be detected (Figure 4). The members of each pair were often in very close proximity to each other. As with single labeling, there was occasional clumping of the gold probe, again attributed to multiple labeling of the primary antibody by the secondary conjugate. This was more common with the 5-nm particles than the 10-nm particles, because the smaller markers are less subject to steric hindrance. Some 10-nm particles showed no indication of being associated with a 5-nm particle, but if the smaller particle lay directly above or below the larger one it would not be optically detectable in the electron beam. Therefore, the percentage of sites that in fact show double labeling may be greater than is immediately apparent. The very close proximity of the gold particles in each pair, along with the low percentage of single particles, shows clearly that the pairing is not occurring by random association.
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Close co-localization after double labeling of dystrophin and ß-dystroglycan was also demonstrated when the procedure was carried out on cryosections. Gold particles of both sizes were seen in close proximity to the plasma membrane (Figure 5). Further evidence for the co-localization of the C-termini of dystrophin and ß-dystroglycan was provided by confocal laser scanning microscopy through simultaneous visualisation of the fluorescent probes by dual-channel imaging (Figure 6). In the combined image, the two individual images coincide almost exactly. The overall labeling pattern was one of a continuous distribution at the fiber periphery with occasional repeating points at which it appeared to be more concentrated.
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Discussion |
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In this study we sought to establish the spatial relationship between the C-terminal domains of dystrophin and ß-dystroglycan at the plasma membrane of skeletal muscle by applying double immunogold electron microscopy with complementary dual-channel scanning immunoconfocal microscopy. To achieve this, we applied the fracture-label technique, which permits high-resolution visualization of labeled membrane components both in sections and in platinumcarbon replicas of the fractured tissue face (
In previous double labeling studies we have examined proteins that, by fluorescence imaging, may appear to co-localize but which, at the resolution of the electron microscope, can be identified as being situated in different cellular compartments (
The interest in the confocal microscopy is not so much that it shows the expected co-localization but that it provides a broader scan of the overall distribution of the proteins. Earlier immunofluorescence work has suggested that dystrophin is arranged in costameres, i.e., that it forms a band at the level of the Z-line or either side of the Z-line (
In conclusion, we have shown that the C-termini of dystrophin and ß-dystroglycan are extremely closely associated at the cytoplasmic surface of the plasma membrane. The separation between the two epitopes is on the same order as the size of the gold probes (510 nm) or less than this. This is consistent with the biochemical evidence that dystrophin is bound to ß-dystroglycan and is of interest because, whereas the binding site on ß-dystroglycan is known to be at the C-terminus (1- and ß1-syntrophin, which are cytoplasmic proteins of the glycoprotein complex (Figure 1) (
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Acknowledgments |
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MJC and JW were supported by The Wellcome Trust, grant number 046045/Z/95/Z; SS, SR, and NJS were supported by The British Heart Foundation, grant numbers FS/94044 and PG/93136.
We thank Dr Henry Klamut (Ontario Cancer Institute, Toronto) for the gift of the polyclonal antibody P1583, Dr Louise Anderson (Dept of Neurobiology, University of Newcastle upon Tyne) for the monoclonal antibody 43DAG/8D5, and Dr Trevor Cox (Dept of Mathematics and Statistics, University of Newcastle upon Tyne) for assistance with the spatial statistics.
Received for publication November 19, 1997; accepted March 25, 1998.
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