Dystrophin isoform Dp71is present in lamellipodia and focal complexesin human astrocytoma cells U-373 MG

Carlos G. García-Tovar1,4, José Luna2, Raúl Mena2, Carlos I. Soto-Zárate1,4, Rafael Cortés1, Armando Pérez1,Gloria León-Avila3, Dominique Mornet5, Alvaro Rendón6, and José Manuel Hernández1*

Departments of 1 Cell Biology, 2 Physiology, Biophysics and Neurosciences, and 3 Genetics and Molecular Biology, CINVESTAV-IPN, Zacatenco,México,4 Morphology Unit, FES-Cuautitlán, Cuautitlán Izcalli, México,5INSERM U 128, IFR 24, Montpellier, France, and 6INSERM EMI 99-18, Strasbourg, France

Received 25 February 2002 and in revised form 5 June 2002 and 4 July 2002; accepted 4 July 2002

Summary

Dp71 is the most abundant product of the dmdgene in the brain. There are at least 2 isoforms derivedfrom alternative splicing of exon 78 (Dp71d, which contains exon 78 and Dp71f, the spliced isoform)but the precise localization and function of each isoform is still unknown. In the present study, wedemonstrate by RT-PCR that the Dp71f isoform is present in an astrocytoma cell line U-373 MG, and itssubcellular localization was determined in the cytoplasm, particularly in perinuclear areas, with loweramounts towards the periphery but increasing in the leader borders of lamellipodia and focal complexes.Double labeling indirect immunofluorescence showed that Dp71f colocalized with actin-like β−dystro-glycan and β-1 integrin. We also demonstrated by triple labeling that Dp71f was colocalized with actinand two members of integrin complexes, α-actinin and vinculin, in focal complexes. Ventral plasmamembranes were enriched and in those containing focal complex proteins, we found colocalization ofDp71f, actin and vinculin. It is concluded that U-373 MG cells express Dp71f as part of lamellipodia andfocal complex proteins, and possibly connected via distroglycan complexes to integrin complexes.

Key words: Dp71f – dystrophin – glycoprotein complex – integrin complex – cell adhesion

0065-1281/02/104/03-245 $ 15.00/0

Introduction

Dystrophin (Dp427) is the largest member of the spec-trin superfamily, one known function of which is to bindmembrane glycoproteins and cytoplasmic proteins,including their link with the actin network and sig-nalling molecules to regulate actin interactions(Hartwig, 1994). Dp is located on the subsarcolemmalsurface in skeletal muscle. Dp is an actin-binding pro-tein (Levine et al., 1992; Rybakova et al., 1996) associ-ated with 2 other proteins: dystroglycans and sarcogly-

cans-sarcospan, also called dystrophin-associated gly-coproteins (DAGs) to form the dystrophin-DAG com-plex (DGC; Crosbie et al., 1997; Yoshida et al., 2000),which also interacts with syntrophins-dystrobrevincomplexes. Dystrophin interacts directly with the dys-troglycan subcomplex which binds to laminin. Thus,dystrophin and dystroglycans form a link between theextracellular matrix and the actin network (Ervasti et al.,1990; Ervasti and Campbell, 1991, 1993; Susuki et al.,

*Correspondence to: Dr. José Manuel Hernández Hernández, Departamento de Biología Celular, Centro de Investigación y de Estudios Avan-zados del I.P.N., Av. I.P.N. 2508, Col. San Pedro Zacatenco, México, CP 07360; tel: *52 55 57473800; ext. 3989; fax: *52 55 57477081;e-mail: manolo@cell.cinvestav.mx

acta histochem. 104(3) 245–254 (2002)© Urban & Fischer Verlaghttp://www.urbanfischer.de/journals/actahist

1992; Susuki et al., 1994; Jung et al., 1995; Henry andCampbell, 1996, 1999; Rentschler et al., 1999). Dys-trophin defects result in 2 phenotypes: Duchenne mus-cular dystrophy (DMD) and Becker muscular dystrophy(BMD; Hoffman et al., 1987; Koenig et al., 1988; Ahnand Kunkel, 1993).

The dmd gene contains several promoters whichcode for different Dp products, full length and shorttranscripts, which are related with various non-muscu-lar pathological signs that are observed in DMD patients(Nudel et al., 1989; Bar et al., 1990; Boyce et al.,1991;Górecki et al., 1992; Huard and Tremblay, 1992; Hug-not et al., 1992; Lederfein et al., 1992; Ahn and Kunkel,1993; Byers et al., 1993; Tinsley et al., 1993; Nishio etal., 1994; D’Souza et al., 1995; Lidov et al., 1995). Eachtranscript encoding for Dp products can be spliced alter-natively to produce multiple isoforms (Feener et al.,1989). The most abundant non-muscular product(Dp71) is a short C-terminal 71-kDa protein (Bar et al.,1990; Hugnot et al., 1992; Lederfein et al., 1992). Dif-ferential splicing of exon 78 produces at least 2 Dp71isoforms. Dp71d is the isoform in which exon 78 is pre-sent and the dystrophin carboxyl terminus end is pre-served because the open reading frame is not affected.Another isoform, called the Dp71 founder sequence has31 amino acid residues (founder sequence) insertedinstead of the original last 13 amino acid residues. Thefounder sequence is exclusive for this isoform and ispresent when exon 78 is absent and the open readingframe is affected (Kramarcy et al., 1994; Austin et al.,1995). Recently, a subpopulation of truncated Dp71transcripts has been characterized; it was found to beaffected in exons 71 to 74 which were excised by splic-ing. These truncated Dp71 transcripts are also alterna-tively spliced in exon 78 (Austin et al., 2000).

Dp71 is localized in the plasma membrane (Rapaportet al., 1993; Jung et al., 1993; Imamura and Ozawa,1998), synaptic vesicles and mitochondria (Jung et al.,1993), stress fiber-like structures (Howard et al., 1998,1999), and cytosol (Dechesne et al., 1995). However,only in a few studies specific markers for isoforms wereused. Blake et al. (1999) performed subcellular fraction-ation of the rat forebrain and found Dp71d in micro-somes, the soluble fraction, light membranes, synapticmembranes, postsynaptic densities and mitochondria.Claudepierre et al. (1999, 2000b) found Dp71f in thebody of cells of the ganglionic cell layer of retina and inMüller glial cells forming clusters in the perinuclearcytoplasm colocalized with actin filaments in areas withlarge amounts of Dp71f. Aleman et al. (2001) showed inhippocampal neurons and forebrain astrocytes Dp71fand Dp71d to be colocalized in the cytoplasm withGolgi complexes and neuronal nuclei, whereas Dp71dalso colocalized with neurofilaments. We found Dp71fto be present in the cytoplasm of primary brain culture

cells; this localization was more conspicuous near thenucleus, and diminished towards the periphery. Somecells showed this protein to be present in lamellipodia-like structures and focal complexes (Garcia-Tovar et al.,2001).

The presence of dystroglycan in combination withvarious short products of the dmdgene (such as Dp71,Dp116, Dp140, and Dp260) has been found in varioustissues including the central nervous system, peripheralnerves, brain, retina, epithelial cells and early embryon-ic tissues (Durbeej et al., 1998). Therefore, Dps and itsisoforms may form complexes that are similar to DGCfound in muscular tissues. As mentioned above, DGCparticipates in cell contacts with the extracellular matrixand therefore, DGC may interact with protein complex-es involved in adhesion processes.

In this respect, studies on the relationship betweensome of the Dp products have demonstrated and β1 andβ3 integrins, α-actinin, vinculin, talin, paxillin and focaladhesion kinase, which are associated with cell motilityand attachment (Vohra et al., 1998; Yoshida et al., 1996,1998; Hance et al., 1999). Actin also plays a key role ina number of these functions. Actin forms different struc-tures on the bases of polymerization and depolymeriza-tion dynamics: lamellipodia, filopodia, microspikes,stress fibers, arcs, focal adhesions and focal complexeswhich are specific types of focal adhesion present inborders of lamellipodia (Nobes and Hall, 1995; Small etal., 1998; Schoenwaelder and Burridge, 1999; Critch-ley, 2000). Actin binding proteins (ABP) play a pivotalrole in the generation, function and regulation of thesestructures and dystrophin products, such as ABPs, mayparticipate in events in which actin is involved.

In order to study the possible involvement of Dp71 incell motility and adhesion, we selected an original cellline that can adhere and in which Dp71f is expressed.On the basis of these characteristics, U-373 MG cellsare an appropriate cell model to study participation ofDp71f in adhesion in the brain. The U-373 MG cell line(ATCC HTB17) has been obtained from a human astro-cytoma, glioblastoma multiforme (GBM), the mostmalignant and common intracranial tumor (Burger etal., 1988). The cells are characterized by high motilityand they are able to produce extracellular matrix (ECM)proteins in vitro, by which the cells can modulate theirown microenvironment (Bouterfa et al., 1999).

In the present study using RT-PCR, we checkedwhether the Dp71f isoform is expressed in U-373 MGcells. Furthermore, we investigated with the use of dou-ble and triple immunofluorescence staining subcellulardistribution patterns of Dp71f, the actin network and themajor proteins involved in the integrin complex. Final-ly, ventral plasma membranes were prepared of thesecells after being fixed on coverslips, and proteins thatare colocalized in remaining adhesion zones were iden-

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ing to Austin et al. (1995), except for gel purification ofthe products). The products were analyzed in a 2%agarose gel. PCR was performed with total RNA extractas control product. DNA ladders were 0.5–10 kb in size(NE Biolabs, Beverly MA, USA) with the 3.0 kb beingthe most brilliant band according to the manufacturer.

cDNA screening using PCR

The 837 and 2296 primers were used to amplify DNAsegments flanking exon 78 of Dp71 cDNA. Amplifiedproducts were separated on 3% agarose-TBE gels con-taining ethidium bromide (Austin et al., 1995). DNAladders were 20–1000 bp in size (FMC Bioproducts,Vallensbaek Strand, Denmark), with 200 and 500 pbbeing the most brilliant bands according to the manu-facturer.

Double and triple immunofluorescence staining

Cultured cells grown on glass cover slips were fixed for20 min with 4% paraformaldehyde in PBS (unless indi-cated otherwise, all incubations were performed atroom temp and samples were washed twice with PBSafter each step). Afterwards, cells were permeabilizedwith 0.5% Triton X-100 in PBS for 5 min, then blockedwith 1% bovine serum albumin (BSA) for 20 min. Fordouble labeling, we used one of the following anti-bodies: 5F3, JAF or anti-β1-integrin, combined withTRITC-labeled phalloidin. In brief, cells were fixed,permeabilized, blocked as described above and incubat-ed overnight with primary undiluted Ab 5F3 or JAF ormouse anti-β-integrin mAb, diluted 1:100 in PBS at4(C, followed by incubation with FITC-labeled goatanti-mouse Abs for 5F3 or β1-integrin Abs or withFITC-labeled goat anti-rabbit antibody for JAF Ab,diluted 1:30 in PBS, for 60 min. Cells were then incu-bated with TRITC-labeled phalloidin (diluted 1:20 inPBS) for 20 min.

For triple labeling the cells were treated as above.The samples were then incubated overnight at 4 °C withthe primary Ab (anti-α-actinin or anti-vinculin, diluted1:20 in PBS); followed by incubation with CYTM5-labeled polyclonal goat anti-mouse IgG, diluted 1:30 inPBS, for 60 min. Cells were then incubated with undi-luted FITC-labeled 5F3 mAb for 2 h. Finally, cells wereincubated for 20 min with TRITC-labeled phalloidin,diluted 1:20 in PBS. All samples were mounted on glassslides using gelvatol (Monsanto, St. Louis MO, USA)containing 0.5% p-phenylenediamine.

As controls, we used not related mAbs (having thecorresponding isotype of the specific mAbs used, nega-tive data not shown) and also we omitted the first anti-body in order to detect nonspecific staining of the sec-ondary antibodies.

tified. Our studies indicate participation of Dp71f in celladhesion in the brain as an intracellular molecule thatbinds integrin complexes to the actin network.

Material and methods

Reagents

Unless specified otherwise, all reagents were obtainedfrom Sigma (St. Louis MO, USA).

Antibodies

Monoclonal anti-Dp71f antibody (mAb) 5F3 and poly-clonal rabbit anti-β1-dystroglycan antibody (Ab) JAFhave been produced and characterized in detail by Fab-brizio et al. (1994) and Pons et al. (1994); mouse anti- β1-integrin mAb (Life Technologies, Rockville MD, USA),mouse anti-β-actinin mAb and TRITC-labeled phalloidinto detect F-actin were purchased from Sigma; mouse anti-vinculin mAb was obtained from Chemicon (TemeculaCA, USA). The secondary Abs, horseradish peroxidase-and FITC-labeled polyclonal goat anti-mouse IgG, FITC-and TRITC-labeled polyclonal goat anti-rabbit IgG andCYTM5-labeled polyclonal goat anti-mouse IgG wereobtained from Zymed (San Francisco CA, USA).

U-373 MG Cultures

U-373MG cells were cultured in DMEM containing10% fetal calf serum, 100 U/ml penicillin and strepto-mycin at 37 ºC 5% CO2 in a humid incubator.

RT-PCR

Total RNA was extracted from U-373MG cells using theguanidium-acid phenol method of Chomczynski andSacchi (1987). For RT-PCR experiments, we used pri-mers that were previously reported by Austin et al.(1995): 513 (5′-TGCATAGACGTGTAAAACCTGCC-3′; position 11541-11519 of human dystrophin/Dp71cDNA); 512 (5′-GAAGCTCACTCCTCCACTCG-TACC-3′; positioned 28 bp 5′ of initiator ATG); 741 (5′-TCTAGAATTCATGAGGGAACAGCTCAAAGG-3′;position at the initiator ATG of Dp71); 2296(5′-TCTAGAATTCTTA-TTCTGCTCCTTCTTC-3′ ;position 11352-11335 of dystrophin cDNA); 837(5′-CCTTCCCTAGTAGTTCAAGACG-3′; position11205–11223 of dystrophin cDNA). RT-PCR was per-formed using the reverse primer 513, and then 512 and513 primers were used to amplify Dp71 cDNA. Theamplified products were used as a template for addition-al PCR using the 741 and 2296 primers in order toobtain products containing the sequence encoding forDp71 isoforms (all experiments were performed accord-

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the oligonucleotide primers 837-2296 to amplify DNAsegments flanking exon 78 of Dp71. We obtained 2fragments of 160 and 128 bp, which is consistent withthe presence and absence of exon 78, respectively(Fig. 1B). As control, product amplification was notobserved when we employed total RNA as a templatefor PCR.

Localization of Dp71f, β-dystroglycan and β1-integrinin lamellipodia of U-373 MG cells

Dp71f was localized in the cytoplasm, but was moreabundant in perinuclear areas and diminished towardsthe periphery of the cells. However, labeling wasstronger in leader borders of lamellipodia and focalcomplexes (Fig. 2a). In order to detect any associationsof this isoform with the actin cytoskeleton, we per-formed double labeling. Cells labeled with 5F3 mAb(Fig. 2a) and TRITC-labeled phalloidin (Fig. 2b)showed Dp71f and actin in leader borders of lamellipo-dia and focal complexes. Colocalization was confirmedin merged confocal microscopical images (data notshown).

It has been reported that dystroglycan is a DGC com-ponent in nervous tissue that is able to bind to Dp71(Jung et al., 1995). To investigate whether dystroglycanis localized in U-373MG cells in a similar way as actinand Dp71f, we performed double labeling using JAF Abwith TRITC-labeled phalloidin. β-Dystroglycan ap-peared to be colocalized with actin in leader borders oflamellipodia and focal complexes (Fig. 2c, d).

Preparation of fluorescent 5F3 mAb

5F3 mAb was adjusted to a final concentration of10 mg/ml in 250 mM bicarbonate buffer, pH 9.5. FITCwas diluted in the same buffer, added to the mAb solu-tion (drop wise to a final concentration of 0.025–0.033mg/mg of protein) and incubated for 2 h at room tempwith constant agitation. In order to eliminate unboundlabeling products, the conjugate was filtered on a 5 cm× 1 cm Sephadex G-25 column (Amersham, Amersham,UK) previously equilibrated with PBS, pH 7.4.

Preparation of ventral plasma membranes

Ventral plasma membranes were prepared from U-373MG cells grown on glass coverslips using a modifi-cation of the lysis squirting technique (Nermut et al.,1991) as described by Cattelino et al. (1995). In brief,cells were grown on coverslips and then washed twicewith ice-cold water. After one min, cells were squirtedover by using a jet of ice-cold water from a water bottleand immediately fixed with 4% paraformaldehyde. Thenext steps were similar as those described above fortriple immunofluorescence labeling.

Confocal scanning laser microscopy

Confocal scanning laser microscopical analysis wasperformed using a MRC-600 (Bio-Rad, Watford, UK),mounted on a Nikon Diaphot (Nikon, Tokyo, Japan)epifluorescence microscope. Ten to 20 0.5–1-µm-thickconsecutive optical images in the Z-direction wereobtained. Images were collected with a ×60 immersionobjective. The 2 or 3 signals of double- or triple-labeledcells were obtained simultaneously. Images were pro-cessed using the Comos software program (Bio-Rad).Final images were obtained after processing them inConfocal Assistant (v. 4.02, copyright Todd Clark Brel-je, Minneapolis MN, USA) and PowerPoint software(v. 2000, Microsoft, Redmond WA, USA), and printedon an Epson Stylus 740i using 1440 dpi (Epson, MexicoS.A. de C.V., Mexico) and Epson photo paper(S041141; Epson, Long Beach CA, USA).

Results

Expression of Dp71f in U-373 MG astrocytoma cells

The presence of Dp71f in U-373 MG cells was con-firmed by nested PCR. From a cDNA obtained with RT-PCR using the 513 primer, we amplified the ORFsequence of Dp71 by PCR in U-373 MG cells using theprimers 741-2296, and as expected, a 1.9 kb productwas obtained (Fig. 1A). In order to establish the pres-ence of the Dp71f isoform, we performed PCR using

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Fig. 1. Dp71f in U-373 MG cells. A. A 1.9 kb product (arrow) is pre-sent after PCR using the primers 741-2296 (lane 2); as a control,total RNA was used for PCR and no product amplification wasobtained (lane 3). B. mRNA of Dp71d with exon 78 (top arrow, 160bp product) and Dp71f without exon 78 (bottom arrow, 128 bp prod-uct) was detected by nested PCR using the 837 and 2296 primerswhich are flanking this exon of Dp71 cDNA (lane 2). A DNA ladder ispresent in lane 1 of A and B with the fragments indicated on the left.

Fig. 3. Triple stainingof Dp71f and actin withα-actinin or vinculin.U-373 MG cells weretriple labeled using theFITC-labelled antibodies5F3 MAb (a and e),TRITC-labelled phalloi-din (b and f) and anti-β-actinin (c) or anti-vinculin (g). Mergedimages are shown inpanels d and h. Arrowsindicate staining of theleader borders of lamel-lipodia and focal com-plexes. Bars, 10 µm.

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Fig. 2. Double staining of actin and Dp71f,β-dystroglycan or β1-integrin. U-373 MGcells were double labeled using 5F3 mAb (a),JAF Ab (c) or anti-β1-integrin Ab (e), in com-bination with TRITC-labeled phalloidin (b, dand f).Arrows indicate staining of leader bor-ders of lamellipodia and focal complexes. gand h, Controls in which cells were labeledonly with TRITC-labeled phalloidin (g in thegreen channel and h in the red channel). Bars,10 µm.

We performed double-labeling assays using anti-β1-integrin mAb and TRITC-labeled phalloidin to demon-strate possible involvement of Dp71f in adhesionevents. The localization pattern was similar to that ofDp71f andβ-dystroglycan in relation to focal complex-es (Fig. 2e, f).

Colocalization of Dp71f with α-actinin and vinculinin focal complexes of U-373 MG cells

To explore a possible interaction between DGC mem-bers with integrin complexes, we performed triplelabeling using specific markers for the focal complexproteins, α-actinin or vinculin, FITC-labeled 5F3 mAband TRITC-labeled phalloidin. Dp71f and actin colo-calized with α-actinin (Fig. 3a, b, c) and vinculin (Fig.3e, f, g). Merged images showed white areas corre-sponding to triple colocalization (Fig. 3d, h). To con-firm these results, we performed experiments in whichthe cells were eliminated leaving only the ventral plas-ma membranes with proteins associated to it, includingthose related with adhesion. When DGC members areinvolved in adhesion events, these proteins must be pre-sent in these ventral plasma membranes. Triple labelingwas performed with FITC-labeled 5F3 mAb, TRITC-labeled phalloidin and anti-vinculin antibody. Dp71f,actin and vinculin colocalized in leader borders oflamellipodia and focal complexes (Fig. 4).

Discussion

In the present report, we have shown that U-373 MG cellsexpress a Dp71f isoform and this protein is colocalizedwith actin, β-distroglycan, β1-integrin, α-actinin, andvinculin in lamellipodia and focal complexes.

Dp71 is the most abundant dmd gene product whichis also expressed in non-muscular cells and up date itsfunction has not been described. Several isoforms of Dpare produced by alternative splicing and each isoformmay perform a distinct function in different types ofcells or in one cell type. In a primary culture of rat braincells, we have found expression of 2 isoforms, Dp71dand Dp71f, that displayed different localization pat-

terns. We observed that the localization pattern in thecytoplasm was stronger near the nucleus, but Dp71fwas also present in borders of lamellipodia-like struc-tures of cells in motion. In addition, immunofluores-cence labeling was more intense in focal complexes ofthe same cells (Garcia-Tovar et al., 2001). The isoformDp71d was found to be related to stress fibers, as hasbeen previously reported (Howard et al., 1998, 1999).

In order to establish whether Dp71f is related withadhesion proteins, we selected as model a brain cell linethat shows high motility and a large number of adhesionevents. First, we confirmed the presence of Dp71f pro-tein and its mRNA by RT-PCR in U-373 MG cells,according to Austin et al. (1995, 2000). The distributionpattern of the protein (Garcia-Tovar et al., 2001) wassimilar to that in the primary brain cell culture but it wasalso found in leader borders of lamellipodia and focalcomplexes. This localization pattern coincides with thatreported by González et al. (2000), who described thatthe isoforms that do not contain exon 78 are exclusivelyfound in the cytoplasm, whereas the isoforms contain-ing this exon show a predominant nuclear localization.In addition, the isoform with exons 71 and 78 splicedshowed a punctuate labeling throughout the cytoplasmwhereas the cellular borders were labeled more intense-ly. On the other hand, Aleman et al. (2001) labeledastrocytes with the same antibody (5F3) and the cyto-plasmic localization is similar to that we have found,but these authors did not find labeling in focal complex-es. Differences with our results in primary brain cellcultures were only found in some cells in the primarycultures that were in motion.

There are reports indicating that the Dp71 isoformcolocalizes with dystroglycan, a member of DGC, andthis interaction may be similar to that found for Dp inmuscle. This assumption was made because reducedlevels of Dp71 directly affect dystroglycan levels (Junget al., 1995; Greenberg et al., 1996 and Claudepierre etal., 2000a), but distinct types of DGCs may be generat-ed in different tissues (Blake et al., 1999).

Dystroglycan is expressed in the brain (Górecki et al.,1994) and has adhesive properties (Matsumura et al.,1997). These findings suggest that dystroglycan partici-pates in adhesive functions. We have performed double

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Fig. 4. Triple staining of Dp71f, actin and vinculinin ventral plasma membranes. U-373 MG cellswere triple labeled using the FITC-labeled antibod-ies 5F3 Mab (a), TRITC-labeled phalloidin (b) andanti-vinculin (c). Arrows indicate staining on themembranes that remained after treatment andcorrespond to lamellipodia and focal complexes.Bar, 10 µm.

exon 78 and Dp71f without exon 78) and the type ofcell. In neuronal cells, differences in the C-terminalcause different localization patterns of the isoforms andthus may induce formation of other complexes withspecific functions for each one, according to the iso-form and its location. This assumption is in agreementwith the findings of Crawford et al. (2000) who showedthat alternative splicing in the C-terminus of Dps regu-lates the composition of DGC and that proteins relatedto each product or isoform may be different.

Experiments have to be performed to determine thepresence and localization of other members of DGC,since the sarcoglycan subcomplex, the other member ofDGC, is required to stabilize dystroglycan in the sar-colemma (Roberds et al., 1993). In contrast with dystro-glycan, some members of the sarcoglycan subcomplex(α- and γ-sarcoglycans) are expressed only in striatedmuscle but others (β- and δ-sarcoglycan) are expressedin smooth and cardiac muscle as well, and in non-mus-cular tissues. Recently, a new sarcoglycan protein, ε-sarcoglycan, homologous to α-sarcoglycan, which formpart of the sarcoglycan subcomplex has been describedwhich is expressed in smooth and cardiac muscle and innon-muscular tissues, including brain (Ettinger et al.,1997; McNally et al., 1998; Straub et al., 1999, Claude-pierre et al., 2000a). DGC and integrin complexes arelinked in adhesion areas and the sarcoglycan subcom-plex in brain may function as a stabilizator of the dys-troglycan subcomplex (like in muscle). Sarcospan, theother DGC component, in turn, can form a complexwith sarcoglycan and dystrobrevin (Crosbie et al., 1997,1999; Chan et al., 1998; Araishi et al., 1999; Yoshida etal., 2000). It is a member of the tetraspanning superfam-ily, that facilitates interaction between DGC and inte-grin complexes (Maecker et al., 1997).

In conclusion, Dp71f shows a cytoplasmic localiza-tion in U-373 MG cells, it is more abundant in perinu-clear areas and is present in lower amounts towards theperiphery. Levels of Dp71f are elevated in the leaderborders of lamellipodia and focal complexes. In theseareas, the protein colocalizes with β-dystroglycan (amember of the DGC complex), β1-integrin, α-actininand vinculin (members of focal adhesion complexes).We propose that Dp71f forms part of a DGC complex asin muscle cells and is involved in adhesion functions.

Acknowledgments

This study was supported in part by The European EconomicCommunity (EI-CT93-0098) and by a CONACYT scholar-ship (85542) granted to the first author. We are grateful toFortunato Mote, David Pérez, Armando Sánchez and IvanVargas for their valuable technical support, and Esther Cid forher secretarial support. We also thank Imelda Saldaña, Alber-to Martínez, Arnulfo Apango, Pedro Hernández and José LuisZarco for assistance in bibliographic services.

labeling to investigate whether Dp71f can form DGC byestablishing colocalization of dystroglycan, actin andDp71f. We found dystroglycan to be colocalized withactin in lamellipodia and focal complexes. This findingsuggests that actin in U-373 MG cells forms a complexwith DGC members (dystroglycan and Dp). In thisrespect, Howard et al. (1998) described an actin bindingsite for Dp71 that is similar as that for Dp in muscle;therefore, Dp71 may form complexes with dystroglycanthus establishing a bridge between the extracellularmatrix and actin. This hypothesis is in agreement withthe data of Claudepierre et al. (2000a).

Focal complexes have been described to be linkedwith integrin complexes and the presence of both DGCand members of the integrin family in these areas hasbeen reported (Vohra et al., 1998; Yoshida et al., 1996and 1998; Hance et al., 1999). Moreover, Cavaldesi etal. (1999) reported that dystroglycan forms a complexwith Grb2 and focal adhesion kinase in bovine brain;these proteins are related to signal transduction andassembling of focal adhesion. Russo et al. (2000) con-firmed these findings and reported interactions betweenGrb2 and β-dystroglycan via the SH3 domain.

Integrins are not known to interact directly with dys-troglycan, but they do bind to members of the lamininfamily and are colocalized with dystroglycan in certaincell types (Henry and Campbell, 1999). It has also beenreported that integrins are linked with the formation ofthe laminin-based matrix in basement membranes(Sasaki et al., 1998). We confirmed the presence of β1-integrin in focal complexes in U-373 MG cells thatcolocalize with actin. Colocalization of Dp71f and actinwith α-actinin or vinculin (2 members of focal adhe-sions complexes) was observed. This result is in agree-ment with those of Yoshida et al. (1996 and 1998),Vohra et al. (1998), Hance et al. (1999), Cavaldesi et al.(1999) and Russo et al. (2000), who found similar asso-ciations between DGC and integrin proteins.

To confirm the relationship between Dp71f and focaladhesion, experiments were performed with ventralplasma membranes. We observed Dp71f in the adhesionzones to be colocalized with actin and vinculin. Littledifference was found in the labeling patterns of Dp71fand actin (linear in the border of lamellipodia andincreased staining in focal complex areas) in compari-son with that obtained for vinculin (punctuate, princi-pally in focal complex areas).

We propose that Dp71f and dystroglycan, as part ofDGC, are more diffusely distributed in the leader borderof lamellipodia, because they are involved in initialadhesion and may form the adhesion focus in parallelwith integrin complexes. Localization of Dp productsand isoforms can be different; this can be due to thecontrol of the expression of isoforms that is dependenton the alternative splicing (for example, Dp71d with

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References

Ahn AH, and Kunkel LM (1993) The structural and function-al diversity of dystrophin. Nat Genet 3: 283–291

Aleman V, Osorio B, Chavez O, Rendon A, Mornet D, andMartinez D (2001) Subcellular localization of Dp71 dys-trophin isoforms in cultured hippocampal neurons andforebrain astrocytes. Histochem Cell Biol 115: 243–254

Araishi K, Sasaoka T, Imamura M, Noguchi S, Hama H, Wak-abayashi E, Yoshida M, Hori T, and Ozawa E (1999) Lossof the sarcoglycan complex and sarcospan leads to muscu-lar dystrophy in β-sarcoglycan-deficient mice. Hum MolGenet 8: 1589–1598

Austin RC, Howard PL, D’Souza VN, Klamut HJ, and RayPN (1995) Cloning and characterization of alternativelyspliced isoforms of Dp71. Hum Mol Genet 4: 1475–1483

Austin RC, Morris GE, Howard PL, Klamut HJ, and Ray PN(2000) Expression and synthesis of alternatively splicedvariants of Dp71 in adult human brain. Neuromusc Disord10: 187–193

Bar S, Barnea E, Levy Z, Neuman S, Yaffe D, and Nudel U(1990) A novel product of the Duchenne muscular dystrophygene which greatly differs from the known isoforms in itsstructure and tissue distribution. Biochem J 272: 557–560

Blake DJ, Hawkes R, Benson MA, and Beesley PW (1999)Different dystrophin-like complexes are expressed in neu-rons and glia. J Cell Biol 147: 645–657

Bouterfa H, Darlapp AR, Klein E, Pietsch T, Roosen K, andTonn JC (1999) Expression of different extracellular matrixcomponents in human brain tumor and melanoma cells inrespect to variant culture conditions. J Neuro-oncol 44:23–33

Boyce FM, Beggs AH, Feener C, and Kunkel LM (1991)Dystrophin is transcribed in brain from a distant upstreampromoter. Proc Natl Acad Sci USA88: 1276–1280

Burger PC, Heinz ER, Shibata T, and Kleihues P (1988) Topo-graphic anatomy and CT correlations in the untreatedglioblastoma multiforme. J Neurosurg 68: 698–704

Byers TJ, Lidov HGW, and Kunkel LM (1993) An alternativedystrophin transcript specific to peripheral nerve. NatGenet 4: 77–81

Cattelino A, Longhi R, and de Curtis I (1995) Differential dis-tribution of two cytoplasmic variants of α6β1 integrinlaminin receptor in the ventral plasma membrane ofembryonic fibroblasts. J Cell Sci 108: 3067–3078

Cavaldesi M, Macchia G, Barca S, Defilippi P, Tarone G, andPetrucci TC (1999) Association of the dystroglycan com-plex isolated from bovine brain synaptosome with proteinsinvolved in signal transduction. J Neurochem 72:1648–1655

Chan Y, Bonnemann CG, Lidov HGW, and Kunkel LM(1998) Molecular organization of sarcoglycan complex inmouse myotubes in culture. J Cell Biol 143: 2033–2044

Chomczynski P, and Sacchi N (1987) Single-step method ofRNA isolation by acid guanidinium thiocyanate-phenol-cloroform extraction. Anal Biochem 162: 156–159

Claudepierre T, Rodius F, Frasson M, Fontaine V, Picaud S,Dreyfus H, Mornet D, and Rendon A (1999) Differentialdistribution of dystrophins in rat retina. Invest OphtalmolVis Sci 40: 1520–1529

Claudepierre T, Dalloz C, Mornet D, Matsumura K, Sahel J,and Rendon A (2000a) Characterization of the intermolecu-lar associations of the dystrophin-associated glycoproteincomplex in retinal Müller glial cells. J Cell Sci 113:3409–3417

Claudepierre T, Mornet D, Pannicke T, Forster V, Dalloz C,Bolaños F, Sabel J, Reichenbach A, and Rendon A (2000b)Expression of Dp71 in Müller glial cells: a comparisonwith utrophin- and dystrophin-associated-proteins. InvestOphtalmol Vis Sci 41: 294–304

Crawford GE, Faulkner JA, Crosbie RH, Campbell KP,Froehner SC, and Chamberlain JS (2000) Assembly of thedystrophin-associated protein complex does not require thedystrophin COOH-terminal domain. J Cell Biol 150:1399–1409

Critchley DR (2000) Focal adhesions–the cytoskeletal con-nection. Curr Opin Cell Biol 12: 133–139

Crosbie RH, Heighway J, Venzke DP, Lee JC, and CampbellKP (1997) Sarcospan, the 25 kDa transmembrane compo-nent of the dystrophin-glycoprotein complex. J Biol Chem272: 31221–31224

Crosbie RH, Lebakken CS, Holt KH, Venzke DP, Straub V,Lee JC, Grady RM, Chamberlain JS, Sanes JR, and Camp-bell KP (1999) Membrane targeting and stabilization ofsarcospan is mediated by the sarcoglycan subcomplex. JCell Biol 145: 153–165

D’Souza VN, Nguyen TM, Morris GE, Karges W, PillersDAM, and Ray PN (1995) A novel dystrophin isoform isrequired for normal retinal electrophysiology. Hum MolGenet 4: 837–842

Dechesne CJ, Griffoin JM, Mayeux V, Fabbrizio E, and PonsF (1995) Identification and immunocytochemical localiza-tion of utrophin and dystrophin products in the rat periph-eral vestibular system. Auditory Neurosci 1: 341–349

Durbeej M, Henry MD, and Campbell KP (1998) Dystrogly-can in development and disease. Curr Opin Cell Biol 10:594–601

Ervasti JM, Ohlendieck K, Kahl SD, Gaver MG, and Camp-bell KP (1990) Deficiency of a glycoprotein component ofthe dystrophin complex in dystrophic muscle. Nature 345:315–319

Ervasti JM, and Campbell KP (1991) Membrane organizationof the dystrophin-glycoprotein complex. Cell 66: 1121–1131

Ervasti JM, and Campbell KP (1993) A role for the dys-trophin-glycoprotein complex as a transmembrane linkerbetween laminin and actin. J Cell Biol 122: 809–823

Ettinger AJ, Feng G, and Sanes JR (1997) Epsilon–sarcogly-can, a broadly expressed homologue of the gene mutated inlimb-girdle muscular dystrophy 2D. J Biol Chem 272:32534–32538

Fabbrizio E, Nudel U, Hugon G, Robert A, Pons F, and Mor-net D (1994) Characterization and localization of a 77 kDaprotein related to the dystrophin gene family. Biochem J299: 359–365

Feener CA, Koenig M, and Kunkel LM (1989) Alternativesplicing of human dystrophin mRNA generates isoforms atthe carboxy terminus. Nature 338: 509–511

Garcia-Tovar CG, Perez A, Luna J, Mena R, Osorio B, Ale-man V, Mondragon R, Mornet D, Rendon A, and Hernan-dez JM (2001) Biochemical and histochemical analysis of

252 Garcia-Tovar et al.

acta histochemica 104, 3 (2002)

71 kDa dystrophin isoform (Dp71f) in rat brain. Acta His-tochem 103: 209–224

González E, Montañez C, Ray PN, Howard PL, García-SierraF, Mornet D, and Cisneros B (2000) Alternative splicingregulates the nuclear or cytoplasmic localization of dys-trophin Dp71. FEBS Lett 482: 209–214

Górecki DC, Monaco AP, Derry JMJ, Walker AP, BarnardEA, and Barnard PJ (1992) Expression of four alternativedystrophin transcripts in brain regions regulated by differ-ent promoters. Hum Mol Genet 1: 505–510

Górecki DC, Derry JMJ, and Barnard EA (1994) Dystro-glycan: brain localization and chromosome mapping in themouse. Hum Mol Genet 3: 1589–1597

Greenberg DS, Schatz Y, Levy Z, Pizzo P, Yaffe D, and NudelU (1996) Reduced levels of dystrophin associated proteinsin the brains of mice deficient for Dp71. Hum Mol Gen 5:1299–1303

Hance JE, Fu SY, Watkins SC, Beggs AH, and Michalak M(1999) α-actinin-2 is a new component of the dystrophin-glycoprotein complex. Arch Biochem Biophys 365:216–222

Hartwig JH (1994) Introduction and subfamily 1: the spectrinfamily. In: Sheterline P (Ed) Protein profile, actin-bindingproteins 1: spectrin superfamily. Vol 1, issue 7. AcademicPress, London, UK, pp 711–749

Henry MD, and Campbell KP (1996) Dystroglycan: an extra-cellular matrix receptor linked to the cytoskeleton. CurrOpin Cell Biol 8: 625–631

Henry MD, and Campbell KP (1999) Dystroglycan inside andout. Curr Opin Cell Biol 11: 602–607

Hoffman EP, Brown RH, and Kunkel LM (1987) Dystrophin:the protein product of the Duchenne muscular dystrophylocus. Cell 51: 919–928

Howard PL, Klamut HJ, and Ray PN (1998) Identification ofa novel actin binding site within the Dp71 dystrophin iso-form. FEBS Lett 441: 337–341

Howard PL, Dally GY, Ditta SD, Austin RC, Worton RG,Klamut HJ, and Ray PN (1999) Dystrophin isoforms Dp71and Dp427 have distinct roles in myogenic cells. MuscleNerve 22: 16–27

Huard J, and Tremblay JP (1992) Localization of dystrophinin the Purkinje cells of normal mice. Neurosci Lett 137:105–108

Hugnot JP, Gilgenkrantz H, Vincent N, Chafey P, Morris GE,Monaco AP, Berwald-Netter Y, Koulakoff A, Kaplan JC,Kahn A, and Chelly J (1992) Distal transcript of the dys-trophin gene initiated from an alternative first exon andencoding a 75-kDa protein widely distributed in nonmuscletissues. Proc Natl Acad Sci USA89: 7506–7510

Imamura M, and Ozawa E (1998) Differential expression ofdystrophin isoforms and utrophin during dibutyryl-cAMP-induced morphological differentiation of rat brain astro-cytes. Proc Natl Acad Sci USA95: 6139–6144

Jung D, Filliol D, Metz-Boutigue MH, and Rendon A (1993)Characterization and subcellular localization of the dys-trophin-protein 71 (Dp71) from brain. Neuromusc Disord3: 515–518

Jung D, Yang B, Meyer J, Chamberlain JS, and Campbell KP(1995) Identification and characterization of the dystrophinanchoring site on β-dystroglycan. J Biol Chem 270:27305–27310

Koenig M., Monaco AP, and Kunkel LM (1988) The com-plete sequence of dystrophin predicts a rod-shapedcytoskeletal protein. Cell 53: 219–228

Kramarcy NR, Vidal A, Froehner SC, and Sealock R (1994)Association of utrophin and multiple dystrophin shortforms with the mammalian Mr 58,000 dystrophin-associat-ed protein (Syntrophin). J Biol Chem 269: 2870–2876

Lederfein D, Levy Z, Augier N, Mornet D, Morris G, Fuchs 0,Yaffe D, and Nudel U (1992) A 71-kilodalton protein is amajor product of the Duchenne muscular dystrophy gene inbrain and other nonmuscle tissues. Proc Natl Acad SciUSA89: 5346–5350

Levine BA, Moir AJG, Patchell VB, and Perry SV (1992)Binding sites involved in the interaction of actin with N-terminal region of dystrophin. FEBS Lett 298: 44–48

Lidov HGW, Selig S, and Kunkel LM (1995) Dp140: a novel140 kDa CNS transcript from the dystrophin locus. HumMol Genet 4: 329–335

Maecker HT, Todd SC, and Levy S (1997) The tetraspaninsuperfamily: molecular facilitators. FASEB J 11: 428–442

Matsumura K, Chiba A, Yamada H, Fukuta-Ohi H, Fujita S,Endo T, Kobata A, Anderson LVB, Kanasawa I, CampbellKP, and Shimizu T (1997) A role of dystroglycan inSchwannoma cell adhesion to laminin. J Biol Chem 272:13904–13910

McNally EM, Ly CT, and Kunkel LM (1998) Human ε-sarco-glycan is highly related to α-sarcoglycan (adhalin), thelimb girdle muscular dystrophy 2D gene. FEBS Lett 422:27–32

Nermut MV, Eason P, Hirst EMA, and Kellie S (1991)Cell/substratum adhesions in RSV-transformed rat fibro-blasts. Exp Cell Res 193: 382–397

Nishio H, Takeshima Y, Narita N, Yanagawa H, Susuki Y,Ishikawa Y, Ishikawa Y, Minami R, Nakamura H, and Mat-suo M (1994) Identification of a novel first exon in thehuman dystrophin gene and of a new promoter locatedmore than 500 kb upstream of the nearest known promoter.J Clin Invest 94: 1037–1042

Nobes CD, and Hall A (1995) Rho, rac, and cdc42 GTPasesregulate the assembly of multimolecular focal complexesassociated with actin stress fibers, lamellipodia, and filopo-dia. Cell 81: 53–62

Nudel U, Zuk D, Einat P, Zeelon E, Levy Z, Neuman S, andYaffe D (1989) Duchenne muscular dystrophy gene prod-uct is not identical in muscle and brain. Nature 337: 76–78.

Pons F, Robert A, Fabbrizio E, Hugon G, Califano JPC,Feherentz JA, Martinez J, and Mornet D (1994) Utrophinlocalization in normal and dystrophin-deficient heart. Cir-culation 990: 369–374

Rapaport D, Greenberg DS, Tal M, Yaffe D, and Nudel U(1993) Dp71, the non-muscle product of the Duchennemuscular dystrophy gene is associated with the cell mem-brane. FEBS Lett 328: 197–202

Rentschler S, Linn H, Deinenger K, Bedford MT, Espanel X,and Sudol M (1999) The WW domain of dystrophinrequires EF-hands region to interact with beta-dystrogly-can. Biol Chem 380: 431–442

Roberds SL, Ervasti JM, Anderson RD, Ohlendieck K, KahlSD, Zoloto D, and Campbell KP (1993) Disruption of thedystrophin-glycoprotein complex in the cardiomyopathichamster. J Biol Chem 268: 11496–11499

Dp71f in focal complexes of U373 MG cells 253

acta histochemica 104, 3 (2002)

Russo K, Stasio E, Macchia G, Rosa G, Brancaccio A, andPetrucci TC (2000) Characterization of the β-dystroglycan-growth factor receptor 2 (Grb2) interaction. BiochemBipophys Res Commun 274: 93–98

Rybakova IN, Amann KJ, and Ervasti JM (1996) A newmodel for the interaction of dystrophin with F-actin. J CellBiol 135: 661–672

Sasaki T, Forsberg E, Bloch W, Addicks K, Fässler R, andTimpl R (1998) Deficiency of β1 integrins in teratomainterferes with basement membrane assembly and laminin-1 expression. Exp Cell Res 238: 70–81

Schoenwaelder SM, and Burridge K (1999) Bidirectional sig-naling between the cytoskeleton and integrins. Curr OpinCell Biol 11: 274–286

Small JV, Rottner K, Kaverina I, and Anderson KI (1998)Assembling an actin cytoskeleton for cell attachment andmovement. Biochim Biophys Acta 1404: 271–281

Straub V, Ettinger AJ, Durbeej M, Venzke DP, Cutshall S, SanesJR, and Campbell KP (1999) α-Sarcoglycan replaces α-sarcoglycan in smooth muscle to form a unique dystrophin-glycoprotein complex. J Biol Chem 274: 27989–27996

Suzuki A, Yoshida M, Yamamoto H, and Ozawa E (1992)Glycoprotein-binding site of dystrophin is confined to thecystein-rich domain and the first half of the carboxy-termi-nal domain. FEBS Lett 308: 154–160

Suzuki A, Yoshida M, Hayashi K, Mizuno Y, Hagiwara Y, andOzawa E (1994) Molecular organization at the glycopro-

tein-complex-binding site of dystrophin. Eur J Biochem220: 283–292

Tinsley JM, Blake DJ, and Davies KE (1993) Apo-dys-trophin-3: a 2.2 kb transcript from the DMD locus encod-ing the dystrophin glycoprotein binding site. Hum MolGenet 2: 521–524

Vohra MS, Komiyama M, Hayakawa K, Obinata T, and Shi-mada Y (1998) Subcellular localization of dystrophin andvinculin in cardiac muscle fibers and fibers of the conduc-tion system of the chicken ventricle. Cell Tissue Res 294:137–143

Yoshida T, Hanada H, Iwata Y, Pan Y, and Shigekawa M(1996) Expression of a dystrophin-sarcoglycan complex inserum-deprived BC3H1 cells and involvement of α-sarco-glycan in substrate attachment. Biochem Biophys ResCommun 225: 11–15

Yoshida T, Pan Y, Hanada H, Iwata Y, and Shigekawa M(1998) Bidirectional signaling between sarcoglycans andthe integrin adhesion system in cultured L6 myocytes. JBiol Chem 273: 1583–1590

Yoshida M, Hama H, Ishikawa-Sakurai M, Imamura M,Mizuno Y, Araishi K, Wakabayashi-Takai E, Noguchi S,Sasaoka T, and Ozawa E (2000) Biochemical evidence forassociation of dystrobrevin with the sarcoglycan-sarcospancomplex as a basis for understanding sarcoglycanopathy.Hum Mol Genet 9: 1033–1040

254 Garcia-Tovar et al.

acta histochemica 104, 3 (2002)