ARTICLE |
Correspondence to: Hideho Ueda, Dept. of Anatomy, Yamanashi Medical University, Yamanashi 409-3898, Japan. E-mail: hueda@res.yamanashi-med.ac.jp
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Summary |
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Sarcoglycans are transmembrane proteins that are members of the dystrophin complex. Sarcoglycans cluster together to form a complex, which is localized in the cell membrane of skeletal, cardiac, and smooth muscle fibers. However, it is still unclear whether or not sarcoglycans are restricted to the sarcolemma. To address this issue, we examined -, ß-,
-, and
-sarcoglycan expression in femoral skeletal muscle from control and dystrophin-deficient mice and rats using confocal microscopy and immunoelectron microscopy. Confocal microscopy of the tissues in cross-section showed that all sarcoglycans were detected under the sarcolemma in rats and control mice.
- and
-sarcoglycan labeling demonstrated striations in the longitudinal section, suggesting that the proteins were expressed in the sarcoplasmic reticulum (SR) or transverse tubules (T-tubules). Moreover, such striations of both sarcoglycans were recognized in the dystrophin-deficient mouse skeletal muscle. Double labeling with phalloidin or
-actinin and
- or
-sarcoglycan showed different labeling patterns, indicating that
-sarcoglycan localization was distinct from that of
-sarcoglycan. Immunoelectron microscopy clarified that
-sarcoglycan was localized in the terminal cisternae of the SR, while
-sarcoglycan was found in the terminal cisternae and longitudinal SR over I-bands but not over A-bands. These data demonstrate that
- and
-sarcoglycans are components of the SR in skeletal muscle, suggesting that both sarcoglycans function independent of the dystrophin complex in the SR.
(J Histochem Cytochem 49:529537, 2001)
Key Words: sarcoglycan, sarcoplasmic reticulum, terminal cisternae, skeletal muscle, dystrophin complex
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Introduction |
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The clinical features of limbgirdle muscular dystrophy (LGMD) are quite similar to those of Duchenne muscular dystrophy (DMD) except for a lack of cognitive impairment. LGMD is caused by mutation of sarcoglycan, which consists of subunits a, b, d, and g. Mutations in a-, b-, d-, and g-sarcoglycans cause autosomal recessive LGMD types 2D, 2E, 2F, and 2C, respectively (-, ß-,
-,
-, and
-sarcoglycans are all transmembrane proteins, and their respective molecular weights are about 50 kD, 43 kD, 35 kD, 35 kD, and 50 kD (
-, ß-,
-, and
-sarcoglycans, while smooth muscle includes ß-,
-, and
-sarcoglycans (
-, and
-sarcoglycans are all Type II transmembrane proteins (amino terminus on the intracellular side), while
- and
-sarcoglycans belong to the Type I family (amino terminus on the extracellular side). ß-,
-, and
-sarcoglycans have a cluster of cysteine residues at the carboxyl terminus, similar to receptor molecules such as epidermal growth factor (EGF) receptors and laminin (
-, and
-sarcoglycans are tightly associated with one another to function as a unit, especially ß- and
-sarcoglycan (
-sarcoglycan can be dissociated from the complex under relatively mild conditions (
-, and
-sarcoglycans also reveals that they share significant homology with each other, particularly
- and
-sarcoglycans (
Previous reports have focused only on the sarcolemma, so little is known about whether sarcoglycans are localized in other membrane organelles such as sarcoplasmic reticulum (SR) or transverse tubules (T-tubules). - and ß-sarcoglycans are restricted to the sarcolemma but that
- and
-sarcoglycans are also localized in the SR.
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Materials and Methods |
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Animals and Tissue Preparations
All animals used in this study were cared for and handled in compliance with the Yamanashi Medical University Guidelines for the Use of Animals. We used adult female SpragueDawley rats, dystrophin-deficient mdx mice, and mouse controls. They were anesthetized with diethyl ether and sodium pentobarbital and perfused via the heart with 4% paraformaldehyde in 0.1 M phosphate buffer (PB, pH 7.4). The femoral muscle was removed and immersed in the fixative overnight at 4C. After rinsing in PBS, the muscle was immersed in 30% sucrose overnight at 4C. Then the muscle was embedded in OCT compound and sectioned at 10 µm thickness in a cryostat for immunohistochemistry.
Antibodies
Mouse monoclonal anti-dystrophin (NCL-DYS2), mouse monoclonal anti-ß-dystroglycan (NCL-43DAG), mouse monoclonal anti--sarcoglycan (NCL-a-SARC), mouse monoclonal anti-ß-sarcoglycan (NCL-b-SARC), mouse monoclonal anti-
-sarcoglycan (NCL-g-SARC), and mouse monoclonal anti-
-sarcoglycan (NCL-d-SARC) were purchased from Novocastra (Newcastle-Upon-Tyne, UK). Mouse monoclonal dystrobrevin antibody was purchased from Transduction Labs. (Lexington, KY) and goat polyclonal
-actinin antibody from Santa Cruz Labs (Santa Cruz, CA). Alexa488-conjugated phalloidin was purchased from Molecular Probes (Eugene, OR). All sarcoglycan antibodies from Novocastra Laboratories have been used in other studies and their specificity is established (
Confocal Laser Scanning Microscopy
Dystrophin, dystrobrevin, and -, ß-, and
-sarcoglycan antibodies were used at 1:10 dilution, ß-dystroglycan and
-sarcoglycan antibodies at 1:50 dilution, and
-actinin antibody at 1:100 dilution. Cryosections were treated with 1% Triton X-100 in PBS and 10% rabbit serum for 30 min each. The sections were then incubated with primary antibodies overnight at 4C, biotinylated anti-mouse Ig (Histofine ABC Kit; Nichirei, Tokyo, Japan) for 30 min at room temperature, Alexa488 coupled to streptavidin (Molecular Probes) at 1:500 dilution for 60 min, and mounted with Vectashield (Vector Labs; Burlingame, CA). For the control, PBS replaced primary antibodies. A Leica TCS-4D confocal laser scanning microscopy (Heidelberg, Germany) was used for observations with a x63 or x100 oil-immersion objective lens. For double labeling with phalloidin or
-actinin and
-or
-sarcoglycan, we used Alexa488-conjugated phalloidin at 1:200 dilution or donkey anti-goat serum (Jackson ImmunoResearch; West Grove, PA) at 1:200 dilution and biotinylated anti-mouse Ig and streptavidin-conjugated Alexa594 (Molecular Probes) at 1:500 dilution as the fluorescent agent.
Conventional Electron Microscopy
Conventional electron microscopic preparation was described previously (
Immunoelectron Microscopy
Cryosections at 10-µm thickness were treated with 1% Triton X-100 and 1% H2O2 in PBS for 30 min and with 10% rabbit serum for 30 min. Then the sections were incubated with primary antibodies (anti--sarcoglycan at 1:50 dilution or anti-
-sarcoglycan at 1:10 dilution) overnight at 4C. The sections were then incubated with an ABC kit (Nichirei) according to the manufacturer's protocol, treated with a metal-enhanced DAB kit (Amersham; Poole, UK) and 1% OsO4 in PB for 60 min. The sections were dehydrated in a series of graded ethanols and embedded in Epon by the inverted gelatin capsule method. Ultrathin sections at 70 nm were stained with only uranyl acetate and were observed in an electron microscope (Hitachi H-7500).
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Results |
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- or
-sarcoglycan Expression in a Striated Pattern in the Rat and Control Mouse Skeletal Muscle
It is well known that dystrophin and dystrophin-associated proteins are expressed under the sarcolemma of the skeletal, cardiac, and smooth muscles. Our data showed that dystrophin (Fig 1A), ß-dystroglycan (Fig 1B), dystrobrevin (Fig 1C), and -, ß-,
-, and
-sarcoglycans (Fig 1E1H) were expressed along the sarcolemma of the rat skeletal muscle. Whereas
-sarcoglycan (Fig 1E and Fig 2A) and ß-sarcoglycan (Fig 1F and Fig 2B) were found just beneath the sarcolemma,
-sarcoglycan was detected not only under the sarcolemma (Fig 1G) but also in the sarcoplasm, in a striated pattern (Fig 2C). In addition,
-sarcoglycan produced striations that appeared different from those of
-sarcoglycan (Fig 2D). The serum control showed no immunoreactivity at all (Fig 1D). In normal mouse skeletal muscle, sarcoglycan labeling patterns were consistent with those in rat skeletal muscle (Fig 3A3H). Highly magnified images showed that
-sarcoglycan-labeled bands were composed of two fine striations (Fig 3G, inset). In contrast,
-sarcoglycan showed a network-like pattern in the cross-section (Fig 3D), which appeared as thick striations in the oblique section (Fig 3H). These data suggest that
- and
-sarcoglycans are localized in the membrane organelles in addition to the sarcolemma in skeletal muscle.
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- and
-sarcoglycan Expression in the mdx Mouse Skeletal Muscle
Previous studies reported that a primary dystrophin mutation leads to a secondary reduction or mislocalization of a number of dystrophin-associated proteins, including the sarcoglycan subunits (-sarcoglycan labeling in the sarcoplasm showed a variable appearance. For example, Fig 4A shows an almost normal appearance, with regular striations like those seen in the normal mice. Perinuclear regions are strongly labeled. Fig 4B shows that regenerating muscle fibers with central nuclei (arrow) have longitudinal labeling patterns instead of striations, and some muscle fibers show a completely disorganized labeling pattern with anti-
-sarcoglycan (Fig 4C). A sarcoplasmic banding pattern of
-sarcoglycan is also recognized (not shown). These data suggest that sarcoglycan expression in the SR is only minimally affected by dystrophin mutations.
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- and
-sarcoglycan Topology Revealed by Double Immunolabeling with Phalloidin or
-Actinin
To determine whether or not - and
-sarcoglycans are localized in the same region, we used double labeling with phalloidin or
-actinin. Phalloidin attaches to actin filaments to show I-bands, and
-actinin localizes in Z-bands in the striated muscle to anchor actin filaments (
-sarcoglycan was detected in the central parts of I-bands (Fig 5A5C) and along Z-bands (Fig 6A6C). On the other hand,
-sarcoglycan was found broadly over I-bands (Fig 5D5F), especially around Z-bands (Fig 6D6F), but was not recognized over A-bands (Fig 5D5F). These data demonstrate that
-sarcoglycan localization is different from that of
-sarcoglycan.
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- and
-sarcoglycan Localization in the SR
To clarify the ultrastructural localization of - and
-sarcoglycans, immunoelectron microscopic examinations were performed. Skeletal and cardiac muscles have special apparatuses for excitationcontraction coupling in the junctional SR (terminal cisternae) and T-tubules (Fig 7A).
-Sarcoglycan was detected along the cell membrane of myofibers (Fig 7B, upper left inset) and in the terminal cisternae of the SR (Fig 7B and upper right inset).
-Sarcoglycan was detected along the cell membrane (Fig 7C, upper left inset) and in the longitudinal SR over the I-band, including terminal cisternae (Fig 7C and upper right inset). These data demonstrate that
-sarcoglycan is a component of the terminal cisternae as well as the sarcolemma, whereas
-sarcoglycan is more broadly expressed in the SR. The schematic diagram in Fig 8 summarizes sarcoglycan localization.
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Discussion |
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In this study, we clearly demonstrated that -sarcoglycan was localized in the terminal cisternae of the SR, and that
-sarcoglycan was found in the SR over I-bands, including terminal cisternae. Moreover, their expression was not affected by the absence of dystrophin. These data give a clue to sarcoglycan function.
Recent developments in molecular genetics have allowed development of -, ß-,
-, and
-sarcoglycan knockout mice to analyze the biological roles of these proteins (
-Sarcoglycan-deficient mice show ongoing muscle necrosis with age, loss of sarcolemmal integrity, and changes of absolute force (
-sarcoglycan gene (
-sarcoglycan-deficient mice show muscular dystrophy and cardiomyopathy due to impairment of sarcolemmal integrity (
-sarcoglycan develop a phenotype that closely parallels the human LGMD, with marked cardiomyopathy (
-dystroglycan was enriched and fully glycosylated but was not tightly associated with membranes in
-sarcoglycan-deficient mice. In addition,
-sarcoglycan deficiency in mice produced an almost complete secondary reduction of both ß- and
-sarcoglycans but that their mRNA was normally produced. They also found that staining for dystrophin, ß-dystroglycan, and laminin was intact, suggesting that the mechanical dystrophindystroglycanlaminin link was unaffected by
-sarcoglycan deficiency (
- and
-sarcoglycan expression in the SR appears to be independent of dystrophin deficiency.
Terminal cisternae are important structures for calcium releasecalcium uptake mechanisms through triad configuration with a T-tubule. It is assumed that calcium influx through dihydropiridine receptors on the T-tubule induces calcium release from the terminal cisternae via ryanodine receptors, resulting in muscle contraction. The SR contains proteins that are characteristically associated with calcium regulation, such as ryanodine receptors, calsequestrin, sarcoplasmic or endoplasmic reticulum calcium ATPase (SERCA), phospholamban, triadin, junctin, and myotonic dystrophy protein kinase (-, and
-sarcoglycans contain cysteine residues at the carboxyl terminus (extracellular part;
-, and
-subunits also function as receptors for a yet unidentified ligand (
-sarcoglycan deficiency. Taken together with our present data demonstrating
-sarcoglycan localization in the junctional SR, we suggest that
-sarcoglycan may be associated with calcium regulation. On the other hand, the
-sarcoglycan localization is quite unusual. To our knowledge, no protein is reported to be localized in the SR over I-bands. Two possible functions of
-sarcoglycan are hypothesized. First,
-sarcoglycan may be related to calcium metabolism. The cytoplasmic domain of
-sarcoglycan has five tyrosine residues, and recent studies imply that bidirectional signaling with integrins may involve the sarcoglycan subunits (
-sarcoglycan may anchor the SR to myofilaments or Z-bands during muscle contraction and relaxation.
- and
-sarcoglycan have critical and non-redundant roles for muscle function. Although the present findings appear to support this idea, further studies are needed to address these hypotheses. In conclusion, the present study demonstrates that
- and
-sarcoglycans are SR proteins, and that fact sheds light on the functions of sarcoglycans.
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Acknowledgments |
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Supported in part by grants from the Ichiro Kanehara Foundation and the TV YAMANASHI Science Development Fund.
Received for publication May 30, 2000; accepted December 1, 2000.
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