American Journal ofPathology, Vol. 146, No. 1, January 1995Copyright © American Societyfor Investigative Pathology

Ultrastructural Localization of the C-Terminus ofthe 43-kd Dystrophin-Associated Glycoproteinand Its Relation to Dystrophin in Normal MurineSkeletal Myofiber

Yoshihiro Wakayama,* Seiji Shibuya,*Atsushi Takeda,t Takahiro Jimi,*Yoshiko Nakamura,t andHiroaki OnikitFrom the Division ofNeurology, Department ofMedicine, *Department of Clinical Pathology,t and Electron MicroscopeLaboratory,* Showa University Fujigaoka Hospital,Yokohama, Japan

We used single and double immunogold labelingelectron microscopy to investigate ultrastruc-tural localization of the C terminus of the 43-kddystropbin-associated glycoprotein (43-DAG)and its relationship to dystrophin in normal mu-rine skeletal myofibers. Single immunolabeling lo-calized the antibody against the C terminus of43-DAG to the inside surface of the muscle plasmamembrane and the sarcoplasmic side ofplasmamembrane invaginations. Double immunolabelingco-localized antibodies against dystrophin andthe Cterminus of43-DAG to the same site noted inthe single immunolabeling localization of43-DAG.In particular, dystrophin and the C-terminal 43-DAG antibody signals were often observed as dou-blets separated by less than 30 nm. We comparedthese results with those obtained from doubleimmunogold labeling with anti-dystrophin andanti-frspectrin, as weUl as anti-C-terminal 43-DAGand anti-frspectrin antibodies. The antibodiesagainst dystrophin and 13-spectrin, or 1-spectrinand 43-DAG, also co-localized to similar sites inskeletal muscle fibers. Signals of doubletforma-tions were noted but theirfrequency was signifi-cantly lower than the doubletfrequency of anti-dystropbin and anti-43-DAG antibodies. Theresults support the presence of dystrophin and43-DAG linkage at the inside surface ofthe murineskeletal muscle plasma membrane. (Am JPathol 1995, 146:189-196)

Dystrophin, the product of the Duchenne musculardystrophy gene,1-9 is a membrane cytoskeleton pro-tein anchored to the inner surface of the sarcolemmaof normal myofibers by dystrophin-associated glyco-proteins (DAGs) or dystrophin-associated proteins(DAPs).10 DAGs and DAPs have molecular masses of156, 59, 50, 43, 35, and 25 kd and are named 156-DAG, 59-DAP(A1), 50-DAG(A2), 43-DAG(A3), 35-DAG(A4), and 25-DAP(A5).1112 The 43-DAG as wellas the 50- and 35-DAGs and the 25-DAP are integralmembrane proteins, whereas the 59-DAP and 156-DAG are a peripheral membrane protein and an ex-tracellular peripheral membrane protein, respec-tively. The transmembrane 43-DAG and extracellular156-DAG, encoded by a single messenger RNA, aretranslated in-frame from a single 97-kd precursor pro-tein and are named dystroglycan.1314 The 156-DAGbinds to laminin of extracellular matrices in muscleand nonmuscle tissue,14 whereas 43-DAG was pre-viously thought to link DAGs indirectly with dystrophinby way of 59-DAP in normal skeletal muscle plasmamembrane. 12 However, recent biochemical data sug-gest that 43-DAG may interact directly with dystro-phin. 15We synthesized a peptide from the C terminus of

43-DAG and generated a sheep antibody againstthis region. This antibody was used to investigate thelocalization of 43-DAG and its relationship to dystro-phin at the ultrastructural level. The results were com-pared with those obtained from double immunolabel-ing experiments with anti-dystrophin and anti-,B-spectrin, and anti-43-DAG and anti-,3-spectrinantibodies.

Supported by Grant 5A-2-07 from the National Center for NervousMental and Muscular Disorders, Ministry of Health and Welfare,Japan.

Accepted for publication September 29, 1994.

Address reprint requests to Dr. Yoshihiro Wakayama, Division ofNeurology, Department of Medicine, Showa University FujigaokaHospital, 1-30 Fujigaoka, Aoba-ku, Yokohama 227, Japan.

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190 Wakayama et alAJPJanuary 1995, Vol. 146, No. 1

Materials and Methods

Peptide Synthesis andAntibody Production

General procedures for peptide synthesis and anti-body generation were similar to those describedpreviously.16 Briefly, the synthetic peptide of theC-terminal end of the rabbit 43-DAG was predictedby amino acid residues 880-894.13 This peptide isidentical to the synthetic peptide used for rabbit anti-43-DAG antibody generated by Ibraghimov-Beskrovnaya et al,13 and thyroglobulin was conju-gated with this peptide. The antibody against thispeptide was generated in sheep. Solid phaseenzyme-linked immunosorbent assay was used todetermine the sheep polyclonal antibody titer, whichwas x72,000. In addition, the peptide of theC-terminal region of skeletal muscle-specificj3-spectrin was predicted by amino acid residues,17and the peptide (GKKDKEKRFSFFPKKK) identical tothat reported by Porter et al18 was synthesized. Theextra cysteine was added to the 3' terminus in thispeptide for conjugation with thyroglobulin. Antibodiesagainst this peptide were generated in both sheepand rabbit. An enzyme-linked immunosorbent assay

was used to determine the sheep and rabbit poly-clonal antibody titers, which were x 12,500 andx200,000, respectively.

Immunohistochemistry and Western BlotAnalysis of Antibodies

Western blot analysis of the quadriceps femorismuscles of normal mice (C57BL/1OScSn) was car-

ried out by a previously described method16 withminor modifications. Sodium dodecyl sulfate poly-acrylamide gel electrophoresis was performed with a5 to 20% gradient gel and a 5% homogeneous gel for43-DAG and f3-spectrin, respectively. The proteinwas transferred from the gel to a clear blot P mem-

brane (ATTO, Tokyo, Japan) sheet by horizontal elec-trophoresis at 108 mA for 90 minutes at room tem-perature.

For immunohistochemistry the quadriceps femorismuscles of normal mice were excised and immedi-ately frozen in isopentane cooled by liquid nitrogen.Frozen 6-p cross sections of the muscles were placedon coverslips and incubated with primary antipeptideantisera, diluted 1:200 for sheep anti-43-DAG, 1:200for rabbit anti-f3-spectrin, and 1:100 for sheep anti-3-spectrin antiserum. Indirect immunofluorescentstaining was performed according to methods pre-viously described.16

Immunoelectron Microscopy

Quadriceps femoris muscles from six mice were putin a U-shaped muscle clamp and immersed for fixa-tion in chilled 4% paraformaldehyde solution in 0.1mol/L phosphate buffer (pH 7.4) for 1 hour. The fixedmuscles were washed three times in phosphate-buffered saline and frozen in liquid nitrogen-cooledisopentane. The muscles were then cut into 1-3 pmsections in a cryostat and washed three times inphosphate-buffered saline. To eliminate nonspecificreactions, the slices were incubated for 30 minutes atroom temperature in phosphate-buffered saline con-

taining 5% normal donkey serum for single immuno-labeling with sheep anti-43-DAG antiserum and 5%normal donkey and goat sera for double immunola-beling. For immunolabeling experiments, we usedseveral polyclonal antibodies (Table 1) including rab-bit anti-6-10-dystrophin antibody (generous gifts ofDrs. Byers and Kunkel).7'19 For one series of doubleimmunolabeling experiments, we used two differentpolyclonal antibodies generated in different speciesof animals such as rabbit and sheep. The combina-tions of antibodies were 1), rabbit anti-6-10-dystrophin and sheep anti-43-DAG antibodies; 2),rabbit anti-6-10-dystrophin and sheep anti-,B-spectrin antibodies; and 3), sheep anti-43-DAG andrabbit anti-f-spectrin antibodies. For single anddouble immunolabeling experiments, dilution titers ofprimary antibodies are listed in Table 1. For singleimmunolabeling, diluted primary sheep anti-43-DAGantibody was applied to sections for 24 hours at 4 C.For double immunolabeling, the diluted primary

Table 1. Primary Antibodies

ConcentrationAntigen Antibody or dilution

Dystrophin cDNA residues 6,181-9,544 Fusion protein7 Rabbit polyclonal 1:100043-DAG amino acid residues 880-894 (C-terminus)13 Sheep polyclonal 1:200

Synthetic peptidef-Spectrin (C-terminus of 270-kd muscle isoform)18 Rabbit polyclonal 1:200

Synthetic peptide Sheep polyclonal 1:100

Ultrastructural Localization of 43-DAG 191AJPJanuary 1995, Vol. 146, No. 1

sheep and rabbit antibodies were mixed and appliedtogether to the sections for 24 hours at 4 C. After thor-ough rinsing, the four secondary antibodies, 10-nmgold-labeled and 5-nm gold-labeled donkey anti-sheep antibodies (Bio Cell, Cardiff, UK) and 10-nmgold-labeled and 5-nm gold-labeled goat anti-rabbitantibodies (Amersham, Arlington Heights, IL) were di-luted 1:20 in phosphate-buffered saline. For singleimmunolabeling, 5-nm gold-labeled donkey anti-sheep secondary antibody was used. For double im-munolabeling, dystrophin was marked with 10-nmgold-labeled goat anti-rabbit secondary antibody.Also, 5-nm gold-labeled donkey anti-sheep second-ary antibody was used for labeling of 43-DAG and,3-spectrin. For the combined labeling of 43-DAG and,B-spectrin, 43-DAG was labeled with 10-nm gold-labeled donkey anti-sheep secondary antibody. Theappropriate combination of two diluted secondary an-

tibodies was prepared and applied together to thesections for 24 hours at 4 C and then washed fourtimes. Control sections were incubated with nonim-mune sheep serum or sheep and rabbit sera insteadof the respective primary antibodies. The antibody-labeled and control muscle samples were addition-ally fixed in chilled 2.5% glutaraldehyde solution in 0.1mol/L phosphate buffer (pH 7.4) for 30 minutes. Thesesamples were post-fixed in chilled 2% OS04 solutionfor 1 hour, dehydrated in an ascending series of etha-nol and propylene oxide, and embedded in Epon. Theunstained ultrathin sections were observed under theelectron microscope.The percentage of doublet formation of 5-nm gold

particles with 10-nm gold particles within 30 nm was

calculated as follows. Electron micrographs were

taken at random of plasma membrane areas (at least3 p of plasma membrane length per myofiber) of 10muscle fibers from each of 6 mice in individual groupsof three different double immunolabeled combina-tions. Printing was done at a final magnification ofx 160,000. For statistical analysis, the prints from 180muscle fiber plasma membranes of 18 mice in threecombinations of double immunolabeling experimentswere coded and mixed. Then the percentage of 5-nmgold particles associated with 10-nm gold particleswithin 30 nm versus all 5-nm gold particles with andwithout 10-nm gold particles within 30 nm was cal-culated in prints from each fiber. After finishing all cal-culations, the prints of 180 muscle fiber plasma mem-branes were decoded, and the mean percentage ofeach mouse and the group mean percentage of 6mice were calculated for each group. These pro-

cesses were performed in muscle samples stainedby three different sets of the two different antibodieslisted in Table 2. The group means of percentages of

Table 2. Doublet Formation

Antibody combination (group mean + SE)*

Dystrophin and 43-DAG 27.2 ± 4.1Dystrophin and 1-spectrin 14.2 ± 1.843-DAG and 3-spectrin 9.6 ± 1.2

*Percentage of doublets of 5-nm particles with 10-nm particleswithin 30 nm versus 5-nm particles at the inside surface of muscleplasmalemma in the three antibody combinations shown. Statisti-cal analysis by two-tailed t-test: dystrophin and 43-DAG comparedwith dystrophin and f3-spectrin, P < 0.05; and compared with 43-DAG and ,B-spectrin, P < 0.01; dystrophin and ,B-spectrin com-pared with 43-DAG and f3-spectrin, P > 0.1.

5-nm and 10-nm gold particle doublets were statis-tically compared by the two-tailed t-test (Table 2). Inaddition, we performed reciprocal immunolabelingexperiments in which dystrophin was labeled with5-nm gold-labeled goat anti-rabbit secondary anti-body, and 10-nm gold-labeled donkey anti-sheepsecondary antibody was used for labeling 43-DAGand f-spectrin in the quadriceps femoris muscles of6 mice in a dystrophin and 43-DAG group and a dys-trophin and 1-spectrin group, respectively. Prints from120 muscle fiber plasma membranes of 12 mice werecoded and mixed in two groups. Other processes in-cluding statistical analysis were similar to those de-scribed above.

Results

Western Blot Analysis andImmunofluorescence

Western blot analysis showed that sheep antiserumagainst 43-DAG reacted with a protein of molecularmass of 43 kd in muscle extracts of mice, whereasrabbit and sheep antisera against skeletal muscle-specific f3-spectrin reacted with a 270-kd protein(Figure 1).

By immunohistochemistry, these three antiseraagainst 43-DAG and skeletal muscle-specificf-spectrin strongly and clearly stained the cross-sectioned myofiber surface membranes in normalmice (Figure 2).

Immunoelectron MicroscopicObservations

Single immunolabeling electron microscopy of Epon-embedded muscle samples of normal mice stainedby the sheep anti-C-terminal 43-DAG antiserumshowed the presence of 5-nm gold particles just in-side the plasma membranes (Figure 3), and gold par-ticles were also observed inside the myofiber a short

192 Wakayama et alA,/Pjanuar 1995, Vol. 146, No. I

Figure 1. Western blots o/' 4.3-DAG (A) anid-s3spectrin (B ) in tmuiisclc' tissue frotml norn1ialamuse. Ilectrophoresis and blottinig were perJbrmned as described in Mltaterials and Methods.Immunostaining was nith sheep anti-43-DAGantisernm (11:1000) anid with rabbit (1:1000)anid sheep ( 1:600) aniti- 3-spectrin antisera. The43-DAG banid is observed at 43-kd mnolectularwveight (A), anid the -3 spectrin banid is noted at270-kd molecular weight in both sheep (1B lane1) anid rabbit (B. lane 2) antisera. A'umbers tothe lefi inidicate mnolciliar- miasses n/standards(Bio-Rcd Laboratorics, Richmnondcl, CA).

Figure 2. Imniunohistochemical staining withsheep anti-43-DAG (A), r-abbit anti- -.spectrin(B). andcl sheep canti- (-spectrin (C) anitisera inniuiiscle tissue fromt normial mouse. immunlore-

actioni o?f all anitiserai is observed at the surfticemiiemnbr-anies of mn-y(ofibens in normal notouse.

Mfagnification, X 70.

Figure 3. Electron micrograph of sheep anti-

43-DAG antiseruni binding in normal mlrinsteniYofiber. The 5- nnii gold imnmnlitioreactioni par-ticles are seeni at the iniside slutface of' theiuiscle plasna membrane. Magnification,x 104,000.

distance from the plasma membranes. Plasma mem-brane invagination and vesicular structures were fre-quently present in subsarcolemmal areas. In thesestructures, the 5-nm gold particles were present on

the sarcoplasmic side. Double immunolabeling elec-tron microscopy disclosed that signals against rabbitanti-6-10-dystrophin and sheep anti-C-terminal 43-DAG antibodies co-localized at the inside surface of

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Ultrastructural Localization of 43-DAG 193AJPJanuary 1995, Vol. 146, No. 1

muscle plasma membranes. These signals were alsopresent on the cytoplasmic side of plasmalemmal in-vaginations and vesicular structures associated withmuscle plasma membranes (Figure 4). In some in-stances, the signals for dystrophin, 43-DAG, or3-spectrin were present in a costameric structure, but

doublet formations were often observed. In particular,the signals of rabbit anti-6-10-dystrophin and sheep

Figure 4. Electron micrographs of double immunostaining uithsheep anti-43-DAG and rabbit anti-6-10-dystrophin antibodies innormal murine myofibers. Both 5-nm gold particles (43-DAG) and10-nm gold particles (dystrophin) are present at the inside surface ofnormal myofibers. Both sized particles arefrequently associated witheach other within 30 nm distance. Arrow in A shows the close asso-

ciation of the two different sized particles. MT, nmitochondria. Magni-fication, x 104,000 (A and B) and x 130,000 (C).

anti-C-terminal 43-DAG antibodies were often ob-served in a form of doublet with intervals less than 30nm (Figure 4A-C). Muscle samples stained with rab-bit anti-6-10-dystrophin and sheep anti-f3-spectrin orrabbit anti-f3-spectrin and sheep anti-43-DAG anti-bodies also disclosed similar ultrastructural localiza-tion (Figures 5 and 6); doublet formation was ob-served in both cases. All of the immunocontrol musclespecimens were devoid of 5-nm and/or 10-nm goldparticles (Figure 7). Statistiqal analysis of doublet for-mation demonstrated a significantly higher percent-age of doublets with the combination of anti-dystrophin and anti-43-DAG antibodies than in otherantibody combinations (Table 2). Moreover, in the re-ciprocal labeling experiments, in which dystrophinwas labeled with 5-nm gold particle antibody and 43-DAG and ,B-spectrin were labeled with 10-nm goldparticle antibody, doublet formation was also signifi-cantly higher with the combination of anti-dystrophinand anti-43-DAG antibodies (25.1 ± 4.1% (groupmean ± SE) doublet formation of 5-nm particles with10-nm particles within 30 nm versus all 5-nm particlesat the inside surface of muscle plasmalemma) com-pared with the combination of anti-dystrophin andanti-3-spectrin antibodies (7.3 + 1.7%; P < 0.01).

DiscussionBiochemical studies have demonstrated a large oli-gomeric complex of sarcolemma glycoprotein, tightlylinked to dystrophin, which includes cytoskeletal(dystrophin and 59 kd), transmembrane (50, 43, 35,and 25 kd), and extracellular (156 kd) components.12Among these, the complete amino acid sequenceof 156-DAG and 43-DAG in rabbit13 and human14skeletal muscles have been deduced from the cDNAsequence. 156-DAG and 43-DAG of the dystrophin-glycoprotein complex are called a- and 3-dystro-glycan, respectively, although both are encoded bythe dystroglycan gene and translated from a singlemRNA.14 Post-translational modifications of a 97-kdprecursor protein produces two mature 156-kd and43-kd proteins.

The human and rabbit dystroglycans contain 895amino acids with an overall sequence identity of93%. 14 The 43-kd components of rabbit and humandystroglycans are highly conserved with identicalamino acid sequences in the transmembrane domainand the intracellular C-terminal region.14 Thereforethe amino acid sequence in the transmembrane do-main and the intracellular C-terminal region of mousedystroglycan may be identical to that of the rabbit andhuman. This speculation is supported by the fact that

194 Wakayama et alAJP january 1995, Vol. 146, No. 1

Figure 5. Electron oilicrograph of douible im-munostaining uwith sheep anti-43-DAG andrabbit anti413spectrin antisera in normal mu-rine mnofiber. Both 5-nm gold particles (13-spec-tinl) and 10-nm gold particles (43-DAG) arepresent at the inside surface of normal mvofi-ber. Magniflicationi, X 104, 000.

Figure 6. Electron miicrograph qf douible imW-munostaining with sheep anti-,1-spectrin andrabbit anti-610-distrophin antibodies in nor-mnal murine myofiber. Both 5-nm gold particles(1-3spectrin) and 10-nm gold particles (dystro-phini) are present at the inside slurface of nor-mnal mvofiber Both sized particles are occasioni-allv associatced u'ith each other within 30 nmdistance buit the association is less frequentthan that in sheep atnti-4.3-DAG and rabbitaniti-6-10-distrophini antibodies. Magnifica-tion, X104,000.

Figure 7. Electron micrograph of'imnniunocon-trol staining uith normal sheep serum in nor-mal mvofiber in u'hich signals of 43-DAG areabsent. Magnification, X 104, 000.

our sheep antibody against the C-terminal region ofrabbit 43-DAG readily recognized the 43-kd compo-nent of normal mouse skeletal muscle plasma mem-brane in light microscopic studies and stained theinside surface of normal mouse skeletal myofibers atthe ultrastructural level.

During review of this manuscript, we noted an elec-tron microscopic study by Cullen et al20 in which sig-nals of a monoclonal antibody against a syntheticpeptide of the cytoplasmic domain of 43-DAG local-ized to the muscle plasma membrane. To our knowl-edge, the present study is the first report of ultrastruc-tural co-localization of antibodies against theC-terminal region of 43-DAG and dystrophin in skel-etal myofibers.

Recently, several investigators, principally usingconfocal microscopy18'21-23 have shown a dystro-phin microperiodicity loosely associated with cos-tameres.24 However, we were unable to consistentlyidentify costameres in muscle tissues labeled for dys-trophin, 43-DAG, or ,-spectrin. Observations ofthicker sections with a high voltage (accelerating volt-age 200 kV) electron microscope may clarify thispoint.The most interesting finding of this study was the

frequent association of the signal of the C-terminal43-DAG with the rod and cysteine-rich domains ofdystrophin. The finding was noted as doublets of dif-ferent sized gold particles at distances within 30 nm.This association also occured at lower frequency in

Ultrastructural Localization of 43-DAG 195AJPJanuary 1995, Vol. 146, No. 1

the same subcellular domain with other membranecytoskeleton combinations such as dystrophin and3-spectrin. This latter finding implies that dystrophinand j3-spectrin are mingled with each other in the sub-plasmalemmal cytoskeletal meshwork.

With regard to the linkage between dystrophin andDAGs, Ervasti and Campbell presented a molecularmodel12 in which the extreme C-terminal region ofdystrophin binds to 59-DAP and to the glycoproteincomplex composed of the integral membrane pro-teins 50-DAG, 43-DAG, and 35-DAG at the cytoplas-mic surface of the sarcolemma. Subsequently, Suzukiet al25 showed clearly that DAG bound to a locuswithin the cysteine-rich domain and the first half of theC-terminal domain of dystrophin. The results of thepresent study are consistent with recent biochemicalevidence implying that the C-terminal domain of 43-DAG links directly with the cysteine-rich and the firsthalf of the C-terminal domain of dystrophin.15 Addi-tional studies such as freeze etching electron micros-copy of the double immunogold-labeled musclesamples with anti-43-DAG and anti-dystrophin anti-bodies may throw further light on the complex mo-lecular interactions of dystrophin with DAGs.

AcknowledgmentsThe authors thank Drs. T. J. Byers and L. M. Kunkelfor their generous gift of anti dystrophin 6-10 anti-body. We also thank Dr. D. L. Schotland (Departmentof Neurology, University of Pennsylvania) andDr. A. Simpson (Showa Medical Association) for helpwith the manuscript, Mrs. U. Suzuki and Mrs. E.Horikawa for their skillful technical assistance, andMrs. T. Mitani for typing the manuscript.

References1. Hoffman EP, Brown Jr RH, Kunkel LM: Dystrophin: the

protein product of the Duchenne muscular dystrophylocus. Cell 1987, 51:919-928

2. Zubrzycka-Gaarn EE, Bulman DE, Karpati G, BurghesAHM, Belfall B, Klamut HJ, Talbot J, Hodges RS, RayPN, Worton RG: The Duchenne muscular dystrophygene product is localized in sarcolemma of humanskeletal muscle. Nature 1988, 333:466-469

3. Arahata K, Ishiura S, Ishiguro T, Tsukahara T, SuharaY, Eguchi C, Ishihara T, Nonaka I, Ozawa E, Sugita H:Immunostaining of skeletal and cardiac muscle sur-face membrane with antibody against Duchenne mus-cular dystrophy peptide. Nature 1988, 333:861-863

4. Bonilla E, Samitt CE, Miranda AF, Hays AP, SalviatiG, DiMauro S, Kunkel LM, Hoffman EP, RowlandLP: Duchenne muscular dystrophy: deficiency of dys-

trophin at the muscle cell surface. Cell 1988, 54:447-452

5. Watkins SC, Hoffman EP, Slayter HS, Kunkel LM: Im-munoelectron microscopic localization of dystrophin inmyofibers. Nature 1988, 333:863-866

6. Cullen MJ, Walsh J, Nicholson LVB, Harris JB: Ultra-structural localization of dystrophin in human muscleby using gold immunolabelling. Proc R Soc Lond Biol1990, 240:197-210

7. Byers TJ, Kunkel LM, Watkins SC: The subcellular dis-tribution of dystrophin in mouse skeletal, cardiac, andsmooth muscle. J Cell Biol 1991, 115:411-421

8. De Bleecker JL, Engel AG, Winkelmann JC: Localiza-tion of dystrophin and /-spectrin in vacuolar myopa-thies. Am J Pathol 1993, 143:1200-1208

9. Wakayama Y, Shibuya S, Jimi T, Takeda A, Oniki H:Size and localization of dystrophin molecule: immuno-electron microscopic and freeze etching studies ofmuscle plasma membranes of murine skeletal myofi-bers. Acta Neuropathol 1993, 86:567-577

10. Ervasti JM, Ohlendieck K, Kahl SD, Gaver MG, Camp-bell KP: Deficiency of a glycoprotein component of thedystrophin complex in dystrophic muscle. Nature1990, 345:315-319

11. Yoshida M, Ozawa E: Glycoprotein complex anchor-ing dystrophin to sarcolemma. J Biochem 1990, 108:748-752

12. Ervasti JM, Campbell KP: Membrane organization ofthe dystrophin-glycoprotein complex. Cell 1991, 66:1121-1131

13. lbraghimov-Beskrovnaya 0, Ervasti JM, Leveille CJ,Slaughter CA, Sernett SW, Campbell KP: Primarystructure of dystrophin-associated glycoproteins link-ing dystrophin to the extracellular matrix. Nature 1992,355:696-702

14. lbraghimov-Beskrovnaya 0, Milatovich A, Ozcelik T,Yang B, Koepnick K, Francke U, Campbell KP: Humandystroglycan: skeletal muscle cDNA, genomic struc-ture, origin of tissue specific isoforms and chromo-somal localization. Hum Mol Genet 1993, 2:1651-1657

15. Suzuki A, Yoshida M, Hayashi K, Mizuno Y, HagiwaraY, Ozawa E: Molecular organization at the glyco-protein-complex-binding site of dystrophin: threedystrophin-associated proteins bind directly to thecarboxy-terminal portion of dystrophin. Eur J Biochem1994, 220:283-292

16. Wakayama Y, Jimi T, Takeda A, Misugi N, Kumagai T,Miyake S, Shibuya S: Immunoreactivity of antibodiesraised against synthetic peptide fragments predictedfrom mid-portions of dystrophin cDNA. J Neurol Sci1990, 97:241-250

17. Winkelmann JC, Costa FF, Linzie BL, Forget BG: f3spectrin in human skeletal muscle: tissue-specific dif-ferential processing of 3' f3 spectrin pre-mRNA gener-ates a ,1 spectrin isoform with a unique carboxyl termi-nus. J Biol Chem 1990, 265:20449-20454

18. Porter GA, Dmytrenko GM, Winkelmann JC, Bloch RJ:

196 Wakayama et alAJPJanuary 1995, Vol. 146, No. 1

Dystrophin colocalizes with ,B-spectrin in distinct sub-sarcolemmal domains in mammalian skeletal muscle.J Cell Biol 1992, 117:997-1005

19. Lidov HGW, Byers TJ, Watkins SC, Kunkel LM: Local-ization of dystrophin to postsynaptic regions of centralnervous system cortical neurons. Nature 1990, 348:725-728

20. Cullen MJ, Walsh J, Nicholson LVB: Immunogold lo-calization of the 43-kDa dystroglycan at the plasmamembrane in control and dystrophic human muscle.Acta Neuropathol 1994, 87:349-354

21. Masuda T, Fujimaki N, Ozawa E, Ishikawa H: Confocallaser microscopy of dystrophin localization in guineapig skeletal muscle fibers. J Cell Biol 1992, 119:543-548

22. Minetti C, Beltrame F, Marcenaro G, Bonilla E: Dystro-

phin at the plasma membrane of human muscle fibersshows a costameric localization. Neuromusc Disord1992, 2:99-109

23. Straub V, Bittner RE, L6ger JJ, Voit T: Direct visualiza-tion of the dystrophin network on skeletal muscle fibermembrane. J Cell Biol 1992, 119:1183-1191

24. Pardo JV, Siliciano JD, Craig SW: A vinculin-containing cortical lattice in skeletal muscle: trans-verse lattice elements ('costameres') mark sites of at-tachment between myofibrils and sarcolemma. ProcNatl Acad Sci USA 1983, 80:1008-1012

25. Suzuki A, Yoshida M, Yamamoto H, Ozawa E:Glycoprotein-binding site of dystrophin is confined tothe cysteine-rich domain and the first half of thecarboxy-terminal domain. FEBS Lett 1992, 308:154-160