55Journal of Cell Science 112, 55-67 (1999)Printed in Great Britain © The Company of Biologists Limited 1998JCS7292

The M-cadherin catenin complex interacts with microtubules in skeletal

muscle cells: implications for the fusion of myoblasts

Ulrike Kaufmann 1, Joachim Kirsch 2, Andrej Irintchev 3, Anton Wernig 3 and Anna Starzinski-Powitz 1,*1Institut der Anthropologie und Humangenetik fuer Biologen, Johann-Wolfgang-Goethe-Universitaet Frankfurt, Siesmayerstrasse70, D-60054 Frankfurt/Main, Germany2Max Planck-Institut fuer Hirnforschung, Deutschordenstrasse 46, D-60528 Frankfurt/Main, Germany3Physiologisches Institut der Universitaet Bonn, Wilhelmstrasse 31, D-53111 Bonn, Germany*Author for correspondence (e-mail: starzinski-powitz@em.uni-frankfurt.d400.de)

Accepted 15 October; published on WWW 8 December 1998

M-cadherin, a calcium-dependent intercellular adhesionmolecule, is expressed in skeletal muscle cells. Its patternof expression, both in vivo and in cell culture as well asfunctional studies, have implied that M-cadherin isimportant for skeletal muscle development, in particularthe fusion of myoblasts into myotubes. M-cadherin formedcomplexes with the catenins in skeletal muscle cells similarto E-cadherin in epithelial cells. This suggested that themuscle-specific function of the M-cadherin catenin complexmight be mediated by additional interactions with yetunidentified cellular components, especially cytoskeletalelements. These include the microtubules which also havebeen implicated in the fusion process of myoblasts. Here wepresent evidence that the M-cadherin catenin complexinteracts with microtubules in myogenic cells by using threeindependent experimental approaches. (1) Analysis bylaser scan microscopy revealed that the destruction ofmicrotubules by nocodazole leads to an altered cell surfacedistribution of M-cadherin in differentiating myogeniccells. In contrast, disruption of actin filaments had littleeffect on the surface distribution of M-cadherin. (2) M-

cadherin antibodies coimmunoprecipitated tubulin fromextracts of nocodazole-treated myogenic cells but not ofnocodazole-treated epithelial cells ectopically expressingM-cadherin. Vice versa, tubulin antibodiescoimmunoprecipitated M-cadherin from extracts ofnocodazole-treated myogenic cells but not of nocodazole-treated M-cadherin-expressing epithelial cells. (3) M-cadherin and the catenins, but not a panel of controlproteins, were copolymerized with tubulin from myogeniccell extracts even after repeated cycles of assembly anddisassemly of tubulin. Moreover, neither M-cadherin norE-cadherin could be found in a complex withmicrotubules in epithelial cells ectopically expressing M-cadherin. Our data are consistent with the idea that theinteraction of M-cadherin with microtubules might beessential to keep the myoblasts aligned during fusion, aprocess in which both M-cadherin and microtubules havebeen implicated.

Key words: M-cadherin, Microtubule, Cytoskeleton

SUMMARY

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INTRODUCTION

Cadherins are members of a multigene family transmembrane, calcium-dependent intercellular adhesmolecules which influence morphological processes suchtissue development and maintenance (Takeichi, 1991; Geand Ayalon, 1992; Huber et al., 1996) and in particular testablishment of intercellular junctions (Boller et al., 198Geiger and Ginsberg, 1991). Mutational experiments as was NMR and X-ray analyses have revealed that the extraceldomains of the cadherins mediate homophilic intercelluadhesion (Ringwald et al., 1987; Shapiro et al., 1995; Nagaal., 1996). The cytoplasmic tails of the cadherins usuacomplex with the cytoplasmic proteins α-catenin, β-cateninand plakoglobin as has been shown e.g. for E-cadherin (Ozet al., 1989; Aberle et al., 1994; Butz and Kemler, 1994; Hinet al., 1994; Näthke et al., 1994), N-cadherin (Hirano et

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1992; Knudsen et al., 1995; Hertig et al., 1996) and M-cadher(Kuch et al., 1997).

A number of groups have demonstrated that most of thcadherins could only function if they were connected to thcatenins. Two distinct complexes have been identified, e.g. epithelial cells, cardiac myocytes and skeletal muscle cells: oconsisting of the respective cadherin, β-catenin and α-catenin,the second complex contained plakoglobin instead of β-catenin(Butz and Kemler, 1994; Aberle et al., 1994; Hinck et al., 1994Hertig et al., 1996; Kuch et al., 1997). In some cases, it coube shown that the cadherin-catenin complexes interact wicytoskeletal elements such as actin microfilaments anassociated proteins (Hirano et al., 1987). It has been shown tα-catenin is able to directly crosslink the actin filamentsthereby providing a molecular link from E-cadherin to the actincytoskeleton (Rimm et al., 1995). This is supported by analysshowing that α-catenin has significant amino acid sequenc

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homologies to the actin-binding domains of vinculi(Herrenknecht et al., 1991; Nagafuchi et al., 1991). Recenit was shown by in vivo and in vitro studies that vinculin caassociate with the cadherin-catenin complex by binding toα-catenin (Hazan et al., 1997; Weiss et al., 1998). The in vstudies have also indicated that vinculin competes with α-catenin for binding to β-catenin (Hazan et al., 1997)Furthermore, it was demonstrated that the α-catenin-vinculininteraction in the E-cadherin mediated assembly of the apjunctional complex in epithelial cells (Watabe-Uchida et a1998).

Alpha-actinin, an actin filament-crosslinking moleculmight provide an additional connection from the cadhecomplex to the cytoskeleton as has been indicated for cadherin-expressing embryonal lung fibroblasts (Knudsenal., 1995; Nieset et al., 1997). Thus far, no other element ofcytoskeletal filament network has been identified to physicainteract with the cadherins either directly or via the catenin

A particular member of the cadherin family, namely Mcadherin, has been identified in skeletal muscle cell lines in situ in prenatal and postnatal skeletal muscle of somorigin (Donalies et al., 1991; Rose et al., 1994; Irintchev et 1994; Bornemann and Schmalbruch, 1994). Functional assperformed in cell culture have implied a role for M-cadherduring fusion of mononucleated myoblasts into multinucleatmyotubes (Zeschnigk et al., 1995). In line with this have beobservations showing that M-cadherin expression wupregulated during myotube formation and declined afcompletion of this process (Pouliot et al., 1994; Kuch et a1997).

Most studies investigating the connection of the cadherwith the catenins and cytoskeletal proteins have beperformed with E-cadherin and N-cadherin. Very little known about the interaction of the M-cadherin catencomplex with the cytoskeletal network. The followinevidence, however, suggested that M-cadherin might intewith cytoskeletal elements which differ from those connectito the E-cadherin-catenin complex. Outside skeletal musM-cadherin has only been found in the synaptic glomerulithe cerebellum where it has been shown to constitute a spetype of adherens junction, the contactus adherens. Tjunction is morphologically similar to the zonula adherens epithelial cells, but in contrast to the latter, the contacadherens does not contain the proteins vinculin and α-actinin(Rose et al., 1995). In addition, actin microfilaments have been enriched in the contactus adherens junction, a feawhich would be typical for the zonula adherens (Gumbineral., 1988; Geiger and Ginsberg, 1991; Tsukita et al., 191993; Gumbiner, 1993). Since M-cadherin was able to focomplexes with the catenins in muscle cells (Kuch et al., 19and in the cerebellum (D. Winnekendonk et al., unpublishesimilar to E-cadherin, the specificity of the M-cadherinmediated cell-cell contact might include a connection of Mcadherin with cytoskeletal elements other than tmicrofilaments which interact with the cadherins via thcatenins.

M-cadherin has been shown to be involved in the fusionmyoblasts into myotubes (Zeschnigk et al., 1995), a procwhich also requires extensive changes in components ofcytoskeletal network. Beside the observations that fusion winfluenced by the intermediate filaments desmin and vimen

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(Tao and Wallace, 1991; Li at al., 1994), other investigationhave implied that intact microtubules are important fomyogenic differentiation, in particular the elongation analignment of the pre-fusion myoblasts (Croop and Holtze1975; Antin et al., 1981; Toyama et al., 1982; Holtzer et a1985; Saitoh et al., 1988). In some of those experiments, thas been supported by the use of microtubule-destroyialkaloids (nocodazole, colcemid) or stabilizing agents (taxoBoth, microtubule-associated proteins such as MAP4 (Mangand Olmsted, 1996) and stabilizing modifications of thmicrotubular ends by an exchange of Tyr to Glu in the tubulmolecule, which increase during the myogenic differentiatioprocess, might also be central for the function of microtubulduring myotube formation (Bulinski et al., 1988; Gunderson al., 1989).

Taken together, it appears that M-cadherin on the one haand cytoskeletal components on the other are central elemein the myoblast fusion process. These observations suggeconnection of M-cadherin with the cytoskeletal network durinmyogenic differentiation.

In this paper we demonstrate the interaction of the Mcadherin catenin complex with microtubules in terminalldifferentiating (fusing) myogenic cells.

MATERIALS AND METHODS

Cell cultureMouse myoblasts, designated pmi28, were derived from a primaculture and used throughout all experiments (Irintchev et al., 1997aThese pmi28 myoblasts have been implanted in vivo and shownintegrate into and improve muscle contractile strength in syngenanimals (Irintchev et al., 1997a). Pmi28 cells have been propagain culture over 40 passages without loss of myogenic propertiesproliferative capacity (Irintchev et al., 1997a).

In the present experiments, pmi28 cells were grown in Hamnutrient mixture F10 (Gibco) supplemented with 20% fetal caserum (FCS; Costar) in 5% CO2 atmosphere at 37°C. To initiatemyogenic differentiation, growth medium was replaced with differentiation medium consisting of Dulbecco’s modified Eagle’(DMEM) medium (Gibco) with 10% horse serum (Costar). MDCKcells (ATCC CCL 34) were grown in DMEM-medium supplementewith 5% FCS.

AntibodiesPolyclonal antibodies against the extracellular domain of M-cadherwere affinity purified as described (Rose et al., 1994). Rabbit antiseagainst α-catenin and β-catenin were also raised against fusionproteins. The α-catenin expression vector contained α-catenin cDNAnucleotides 456 to 980 and the β-catenin peptide was encoded by β-catenin cDNA nucleotides 968 to 1,370. The rabbit antisera obtainwere affinity purified as described for M-cadherin. Plakoglobiantibodies were a gift from W. W. Franke, DKFZ Heidelberg (Roset al., 1995). DECMA-1 is a monoclonal antibody against E-cadher(Vestweber and Kemler 1985). DM 1A, the monoclonal antibodagainst α-tubulin, was purchased from Sigma. The monoclonaantibody MF 20 reacts with sarcomeric myosin (Bader et al., 1982The cytokeratin antibody was purchased from Progen and the desantibody from Boehringer Mannheim. DTAF-conjugated goat antrabbit/goat anti-rat/goat anti-mouse IgGs, Cy3-coupled goat anmouse/goat anti-rabbit IgG, and Texas Red-conjugated goat arabbit IgGs (Dianova) were employed as secondary antibodies. Acbundles were stained with FITC-labeled phalloidin (Sigma) at a finconcentration of 10 µg/ml.

57M-cadherin and microtubules

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Alkaloid treatment of pmi28 cells and MDCK-M-cadherintransfectantsPmi28 myoblasts and MDCK-M-cadherin transfectants were treafor 30 hours or less either with the actin filament-destroying alkalocytochalasin D (0.5 µg/ml) or with nocodazole (0.5 µg/ml)depolymerizing the microtubules (purchased from Sigma). Talkaloids were then removed from the cells and replaced by frmedium.

ImmunfluorescenceFor immunfluorescence staining, cells were plated onto collagen tS-coated glass coverslips (Boehringer Mannheim). For the majoof experiments, cells were rinsed in phosphate-buffered saline (Pand fixed in 4% paraformaldehyde at room temperature for minutes. After fixation, cells were permeabilized by incubation wi0.1% saponin or 0.1% NP 40 in PBS for 10 minutes. Fixation apermeabilization were done alternately in methanol at −20°C for 10minutes. For double-labelling experiments, cells were incubated wthe corresponding antibodies diluted in PBS/0.5% FCS at rotemperature for 30 minutes. After washing three times with PBbinding of the primary antibodies was detected by species-specfluorochrome-conjugated antibodies. (CY3 or DTAF diluted 1:250PBS/0.5% FCS.) Controls in the absence of primary antibodconfirmed the specifity of the immunolabelling. Fluorescence wdetected with a Zeiss Axiophot microscope (Zeiss). Pictures wtaken with a ×63 or ×100 objective. Photographs were taken usinKodak T-MAX film (400 ASA; Eastman Kodak, Rochester, NY).

Confocal microscopyOptical sections with a pixel size of 0.01 µm2 were recorded using aSarastro 2000 confocal microscope (IMAGESPACE softwaMolecular Dynamics, Sunnyvale, CA). A Gaussian filter with 3 × 3× 3 kernel size was applied to the primary data to eliminate statistnoise. In double-labelling experiments, both fluorescence channwere recorded simultaneously, and the signals were compared alevel of single optical sections.

Immunoprecipitation of the M-cadherin catenin complexand immunoblottingThe co-immunoprecipitations were performed as described for cadherin (Butz and Kemler, 1994) and M-cadherin (Kuch et al., 199Immunoprecipitated proteins were eluted from the Protein Sepharose with SDS-PAGE sample buffer at 100°C for 10 minute

For immunodetection, proteins were separated by SDS-PA(Laemmli et al., 1970) and transferred onto nitrocellulose membra(Biometra) using a fast blot apparatus (Biometra) and a transfer bucontaining: 150 mM glycine, 25 mM Tris-HCl, pH 8.8, and 10%methanol. Membranes were stained in 0.2% Ponceau S in trichloroacetic acid for 5 minutes followed by washing in distillewater to visually confirm protein transfer. After Ponceau S staininthe membranes were blocked with 4% (w/v) dried milk in TBSbuffer (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.2%Tween-20 fo1 hour. Nitrocellulose-membranes were incubated with a primaantibody for 1 hour followed by detection with alkaline phosphata(AP)-conjugated (1:1,000, Dinaova), or peroxidase-labeled secondantibodies (1:10,000, Sigma). Proteins of interest were detectedusing nitro blue tetrazolium and 5-bromo-4 chloro-3-indolyphosphate as substrates (NBT/BCIP) (Boehringer Mannheim) orenhanced chemiluminescence (ECL, Amersham).

Coimmunoprecipitation of M-cadherin and tubulin inpmi28 cells and MDCK-M-cadherin transfectantsDifferentiation induced pmi28 cells and MDCK-M-cadherintransfectants were treated for 30 hours with nocodazole (0.5 µg/ml).These immunprecipitations were carried out essentially as descrin the previous paragraph at 4°C (disassembly of microtubules) at 20°C (microtubules are partially assembled).

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Transfection of MDCK and pmi28 cellsThe cDNA of M-cadherin (Donalies et al., 1991; Kuch et al., 199was cloned into the eukaryotic expression vector pRc/CM(Invitrogen, San Diego, CA) Epithelial MDCK cells were thentransfected with the resultant vector by using the calcium phosphtechnique (Gorman, 1985). The M-cadherin cDNA sequence available in the EMBL data libary under accession no. M7454Transformed MDCK cells were selected by incubation in mediucontaining 800 µg/ml G418 (Gibco).

To obtain E-cadherin-expressing pmi28 myoblasts, the cells wcotransfected with plasmid pBATEM2 expressing E-cadherin friendly gift from M. Takeichi, University of Kyoto, Nose et al., 1988Nagafuchi and Takeichi, 1988) and the plasmid pSV2neo (Southand Berg, 1982) containing the neomycin resistance gene. All pmE-cadherin transfectants were selected with (800 µg/ml) G418 (GibcoBRL).

Cyclic disassembly and assembly of tubulin in cellextracts by taxol and GTPMicrotubules obtained from pmi28 cells or MDCK-M-cadherintransfectants were enriched and purified by GTP/taxol precipitatias described by Vallee (1982, 1986). Pmi28 cells or MDCK-Mcadherin transfectants were washed twice with PBS. Cells wdislodged from the culture plates and sedimented at 2,000 rpm fominutes. The cell pellet was resuspended in 3 ml swelling solutionmM EGTA, 1 mM MgSO4, pH 6.6) to swell on ice for 15 minutes.The cells were then pelleted by centrifugation for 2 minutes at 2,0rpm and 2 ml of the supernatant was removed. At this stage, proteinhibitors (1 mM phenylmethylsulfonyl fluoride, 1 mM Na-vanadate1 mM Na-molybdate, 1 mM PMSF) were added to the pellet. Cewere lysed by homogenization in a Dounce homogenizer. Cell lywas monitored by phase-contrast-microscopy. 0.1 M PIPES-NaOpH 6.6, was added to the homogenate. Nuclei and debris wremoved by centrifugation (5 minutes at 2,000 rpm). The supernatwas recovered and centrifuged for 30 minutes by 15,000 rpm. In first round of tubulin polymerization, 1 mM GTP (Sigma) was addeto the supernatant. To receive the critical concentration of tubupolymerization, 100 µg of purified bovine brain tubulin (ICN) wereadded to the cytosolic extract. The mixture was incubated at 37°C15 minutes. Microtubules were pelleted by centrifugation in PEMbuffer (0.1 M piperazine-N,N′-bis(2-ethanesulfonicacid) (Pipes)-NaOH, 1 mM EGTA, 1 mM MgSO4, pH 6.6) containing a 10%sucrose cushion (30 minutes at 15,000 rpm). The assembmicrotubules were resuspended in 50 µl PEMF buffer. Afterpolymerization, microtubules were depolymerized with 2mM CaC2on ice for 15 minutes.

Second polymerization was induced by 4 mM EGTA and 1 mGTP at 37°C. Polymerization and depolymerization cycles werepeated three times to avoid cytoplasmic contaminations. For the polymerization step, microtubules were enriched by 4 mM EGTA,mM GTP. Stabilization of microtubules was induced by adding 20 µmtaxol (Sigma). The pellet contained the purified microtubules pmi28 cells or MDCK-M-cadherin transfectants. Pellet ansupernatant were resuspended in 5× SDS-buffer. Proteins of pellet andsupernatant were separated by SDS-PAGE and transferred onitrocellulose-membranes.

RESULTS

Determination of experimental paradigms for long-term alkaloid treatmentFor the experiments described in this paper, pmi28 myoblawere treated either with the actin filament-destroying alkalocytochalasin D or with nocodazole depolymerizing thmicrotubules. The alkaloid concentrations were chosen by

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Fig. 1.Distribution of M-cadherin in alkaloid-treated pmi28 myoblasts. Pmi28 myoblasts wereinduced to differentiate and either treated withcytochalasin D (cy: G,H) or nocodazole (no: C,D)or left untreated for the same time (co: A,B; E,F).Subsequently, pmi28 cells were fixed and co-immunostained with either M-cadherin (A,C) andtubulin (B,D) antibodies or with M-cadherinantibodies (E,G) and FITC-phalloidin (F,H).Depolymerization of microtubules by nocodazole(D) lead to a patchy staining pattern of M-cadherin (C). Cytochlasin D-treated (G) anduntreated (E) pmi28 cells showed a more uniformsurface staining of M-cadherin. Bars, 12 µm.

following procedure: cells were treated for 30 hours or lewith either cytochalasin D or nocodazole. After this, thalkaloids were removed from the cells and replaced by fremedium allowing differentiation for an additional 30 hoursThese tests revealed that a concentration of 0.5 µg/ml ofcytochalasin D and 0.5 µg/ml of nocodazole had destroyed thactin or microtubules system which was already visible afone hour of incubation (data not shown). The destruction of cytoskeleton was completely reversed within five hours afwithdrawal of the alkaloids from the culture medium, arevealed by immunolabeling. In agreement with published d(Saitoh et al., 1988), we also found that the fusion-inhibitineffect of the alkaloid treatment was reversible. Fusion occurwithin 30 hours after withdrawal of the alkaloids. Based on ttests described, 0.5 µg/ml of cytochalasin D and 0.5 µg/ml ofnocodazole were used in all subsequent experiments wpmi28 cells.

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Surface distribution of M-cadherin in alkaloid-treated pmi28 myoblastsPmi28 cells were induced to differentiate and kept in alkaloifor 30 hours. After fixation, the distribution of M-cadherin,tubulin and actin were analyzed by doubleimmunofluorescence-stainings using either M-cadherin (Fig1A,C) and tubulin (Fig. 1B,D) antibodies or, alternatively, M-cadherin antibodies (Fig. 1E,G) and fluorochrome-conjugatephalloidin to visualize actin (Fig. 1F,H). Treatment of the pmi28cells with nocodazole revealed a redistribution of M-cadheriafter depolymerization of tubulin from a rather uniform to amore patchy membrane- labelling (Fig. 1C). A uniformmembrane-labelling of M-cadherin was still observed after thdestruction of actin microfilaments although some patchstaining pattern was also visible (Fig. 1G). This suggested thin differentiating pmi28 cells, M-cadherin was more associatewith microtubules rather than with actin microfilaments.

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Fig. 2.M-cadherin redistribution in non-differentiating pmi28 myoblasts. Non-differentiating pmi28 myoblasts were treatedeither with cytochalasin D (cy: G,H) ornocodazole (no: C,D) for 2 hours or leftuntreated (co: A,B E,F). Cells were fixed andco-immunostained with M-cadherin (A,C) andtubulin (B,D) antibodies or, alternatively, withM-cadherin antibodies (E,G) and FITCphalloidin (F,H). The distribution of M-cadherin was altered in cytochalasin D-treatedcells (G) whereas nocodazole-treated cells (C)showed a rather uniform staining pattern similarto untreated cells (A). A similar effect was seenafter alkaloid treatment for 30 hours (notshown). Bars, 12 µm.

In contrast to these observations, M-cadherin redistributin non-differentiating pmi28 cells was more pronounced afdestruction of the microfilaments (Fig. 2G,H) than thmicrotubules (Fig. 2C,D).

M-cadherin immunolabelling of living alkaloid-treatedpmi28 cells reconfirmed that M-cadherin was indeed preson the cell surface (data not shown; Rose et al., 1994). Ththe effect of the alkaloids on the redistribution of M-cadherapplied to the membrane-bound M-cadherin and not to intracellular pool.

The treatment of pmi28 cells with nocodazole did not resin a redistribution of NCAM, another cell-cell adhesiomolecule (data not shown; Dickson et al., 1990) indicating thnot any protein on the cell surface will change location wnocodazole treatment.

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nocodazole-treated C2C12 cells, another myogenic mouse cline. However, the effect in pmi28 cells, as compared to C2C1cells, was much more pronounced (data not shown). Molikely, this relates to the fact that pmi28 cells were establishein cell culture only two years ago and thus, are much closer the in vivo situation than C2C12 cells. These were establishin cell culture almost three decades ago (Yaffe and Saxl, 197

The M-cadherin catenin complexes in alkaloidtreated pmi28 cellsRecently, we have shown (Kuch et al., 1997) that M-cadherforms two types of complexes with the cytoplasmic catenins imyogenic cells: one consisting of both α-catenin and β-catenin; in the second complex β-catenin is replaced byplakoglobin. These data raised the question of whether thredistribution of M-cadherin in alkaloid-treated pmi28 cells

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Fig. 3.The M-cadherin catenin complexes in alkaloid treated cellsMyoblasts were induced to differentiation and treated withcytochalasin D (cy) or nocodazole (no) for 30 hours or were leftuntreated (co). Each of the cell lysates was immunoprecipitated wM-cadherin antibodies. The immunoprecipitates were collected wProtein A-coated Sepharose beads, subjected to SDS-PAGE andtransferred to nitrocellulose-membranes for immunoblotting with cadherin (A), α-catenin (B), β-catenin (C) and plakoglobin (D)antibodies. Untreated cells (co) nocodazole (no), cytochalasin D treated pmi28 cells. 1, 2, 3: controls without primary antibodies. 1untreated pmi28 cells; 2: nocodazole and 3: cytochalasin D treatepmi28 cells. The alkaloid treatment had no influence on thecomposition of the two different M-cadherin catenin complexes indifferentiating pmi28 cells.

could be caused by disruption of the M-cadherin catecomplexes. In order to test this, pmi28 cells were induceddifferentiate and treated with cytochalasin D or nocodazoleindicated. Immunoprecipitates were collected with M-cadheantibodies from the cell lysates, subjected to SDpolyacrylamide electrophoresis and analyzed immunoblotting. The immunoblots were probed witantibodies to either M-cadherin (Fig. 3A), α-catenin (Fig. 3B),β-catenin (Fig. 3C) or plakoglobin (Fig. 3D). In all samplethe respective proteins could be detected in the protein extraregardless of whether the M-cadherin immunoprecipitawere collected from control cells or alkaloid-treated celThus, the results of this experiment indicated that the cadherin catenin complexes were unaffected by alkaltreatment.

Coimmunoprecipitation of M-cadherin and tubulin inpmi28 cellsAnalysis of differentiating pmi28 cells by confocal laser scmicroscopy double-immunolabelled with antibodies to Mcadherin and tubulin indicated that both antigens colocalizin untreated (Fig. 4B) as well as in nocodazole-treated c(Fig. 4A). In both situations, colocalization was main

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observed along the cell membrane but not in the cytoplasmOne likely explanation of this observation was that short pieceof microtubules colocalizing with M-cadherin still existed afternocodazole treatment. Based on this and the observation tthe M-cadherin catenin complex was still intact in nocodazoletreated cells, we performed experiments in order to analyze tputative interaction of microtubules and the M-cadherincomplex by co-immunoprecipitation and subsequenimmunoblot analysis.

We found that it was impossible to get interpretable coimmunoprecipitation results from untreated pmi 28 cells withM-cadherin and tubulin antibodies because microtubules fora filamentous network. In addition, microtubules depolymerizat 4°C, a temperature at which immunoprecipitations arusually performed. Therefore, we prepared protein extracts differentiating, nocodazole-treated pmi28 cells at 20°C (Fig5A,B,E,F) and, for comparisons, at 4°C (Fig. 5C,D,G,H). Thelysates were incubated with antibodies against M-cadher(Fig. 5A,C,E,G) or tubulin (Fig. 5B,D,F,H) and the collectedimmunoprecipitates were analyzed. It should be noted that tconditions of the co-immunoprecipitation experiments werechosen according to those described for the characterizationthe E-cadherin catenin and M-cadherin catenin complexe(Butz and Kemler, 1994; Kuch et al., 1997).

The results of these experiments (Fig. 5) revealed that Mcadherin antibodies co-immunoprecipitated tubulin at 20°C bunot at 4°C. Consistent with this finding, tubulin antibodies coimmunoprecipitated M-cadherin at 20°C but not at 4°CControl immunoblots showed in each case that the respectiantigen, either M-cadherin or tubulin, was precipitated at 4°C(Fig. 5). Thus the co-immunoprecipitation data supported thidea that M-cadherin can indeed connect to the microtubulcytoskeleton in differentiating pmi28 cells.

Biochemical analysis of the association of the M-cadherin complex with the microtubulesTo further corroborate the findings of our co-immunoprecipitation experiments we analyzed the associatioof microtubules with the M-cadherin complex by biochemicatechniques. The approach chosen was the cyclpolymerization and depolymerization of microtubules in thepresence of GTP (two cycles) and GTP/taxol (third cyclethereby co-polymerizing microtubule-associated proteins oother proteins specifically binding to microtubules (Vallee1982; Manfredi et al., 1982; Vallee, 1986). After each cycle thsupernatant was discarded, the pellet resuspended adisassembly and assembly of microtubules repeated. After tlast cycle, pellets and supernatants were analysed immunoblots. For experimental details see Materials anMethods.

In order to test whether the association of the M-cadhericomplex with microtubules were cadherin and/or cell typespecific, the GTP/taxol microtubule polymerizations shown inFig. 6 were performed with myogenic pmi28 cells expressinthe non-muscle protein E-cadherin. For this, we established tcell line pmi28/Ecad which co-expressed E-cadherin with Mcadherin on the cell surface, upregulated myogenic marke(sarcomeric myosin, troponin T, myogenin) upon induction odifferentiation and was able to form myotubes (data noshown). In addition, it could be shown that E-cadherin formetwo complexes with the catenins in myogenic cells in the sam

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Fig. 4.Confocal laser scan microscope images of differentiatingpmi28 cells showing the distribution of M-cadherin and tubulin.Pmi28 cells were induced to differentiate for 30 hours and treatedthe same time with nocodazole. After fixation, the cells were co-immunostained with M-cadherin and tubulin antibodies which wervisualized with secondary antibodies labelled with Cy3 fluorochro(M-cadherin: shown in red) or DTAF fluorochrome (tubulin: shownin green). The yellow color indicates a colocalization of M-cadherand tubulin. The confocal images represent a single optical sectioIn untreated (B) and nocodazole-treated cells (A), M-cadherincolocalizes with tubulin monomers at the cell membrane. Bars, 2 µm.Note: the side length of one pixel corresponds to 0.1 µm which is thehighest resolution possible using our confocal microscope.

manner as in epithelial MDCK cells (not shown). Thus, for tparameters tested including those shown in the next paragrpmi28/Ecad cells were comparable to untransfected pmcells (data not shown).

Cell lysates were prepared from pmi28/Ecad cells at 30 a96 hours after induction of differentiation and frompmi28/Ecad myoblasts. The microtubular pellets fro

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differentiating pmi28/Ecad cells were analyzed as indicated Materials and Methods. Immunoblots of the pellets and thsupernatants of the GTP/taxol precipitates were probed wantibodies to M-cadherin (Fig. 6A), E-cadherin (Fig. 6B), α-catenin (Fig. 6C), β-catenin (Fig. 6D), plakoglobin (Fig. 6E),and tubulin (Fig. 6F). Antibodies to sarcomeric myosin (Fig6G) and desmin (Fig. 6H) were used as specificity controls the co-polymerization experiments. Lysates of untreated pmi2cells and BSA served as positive and negative controls for tantibody reactions, respectively. Both, M- and E-cadherin awell as the catenins and plakoglobin could be detected in tGTP/taxol-polymerized microtubule pellets and were absefrom the supernatants. However, sarcomeric myosin andesmin were undetectable in the microtubule pellet and in tsupernatant. These data are consistent with the assumption M-cadherin and its associated catenins are connected to microtubular network in differentiating skeletal muscle cellsThis interaction, however, was not specific for M-cadherisince E-cadherin could also be found in the GTP/taxoprecipitates.

Finally, it should be emphasized that the microtubulpolymerizations with lysates of untransfected pmi28 cells gavrise to comparable data (data not shown).

Analysis of the cell type specificity of theassociation of M-cadherin with microtubulesIn order to test how M-cadherin would associate withcytoskeletal components in a non-muscle environmenMDCK cells, a canine kidney epithelial cell lineendogenously expressing E-cadherin, was transformed wan M-cadherin expression plasmid (Kuch et al., 1997). ThMDCK/Mcad transformants were either treated withcytochalasin D or nocodazole for 30 hours, respectively, oleft untreated, and analyzed by double immunofluorescenusing antibodies to M-cadherin and E-cadherin (Fig. 7). Iaddition, cell lysates were prepared from MDCK/Mcadtransformants and used for GTP/taxol precipitations (Fig. 8as well as coimmunoprecipitation experiments (Fig. 9).

The double-immunofluorescence of MDCK/Mcad cells withrabbit antibodies to M-cadherin and mouse monoclonantibody DECMA-1 to E-cadherin (Vestweber and Kemler1985) revealed that treatment of MDCK/Mcad cells withcytochalasin D abolished the uniform membrane-labelling oboth E-cadherin and M-cadherin and gave rise to a more patcstaining pattern (Fig. 7E and F). In contrast, depolymerizatioof microtubules had almost no influence on the staining patteof M-cadherin and E-cadherin in MDCK/Mcad transformant(Fig. 7C and D) when compared to the untreated controls (F7A and B). These experiments suggested that E-cadherin aM-cadherin were rather associated with the actimicrofilaments than with the microtubules in M-cadherintransfected MDCK cells.

Biochemical analysis of the association of E-cadherin anM-cadherin in MDCK/Mcad cells with cytoskeletalcomponents showed that neither M-cadherin nor E-cadheco-polymerized with microtubules in the GTP/taxolpolymerization assay (Fig. 8A and B). However, the proteinusually forming complexes with E- and M-cadherin inMDCK/Mcad cells, namely α-catenin, β-catenin andplakoglobin, sedimented with the microtubules (Fig8C,D,E,F). The intermediate filament protein cytokeratin wa

at

eme

inn.

62 U. Kaufmann and others

Fig. 5. Coimmunoprecipitation of M-cadherin and tubulin from pmi28 cells. Cells were induced to differentiation and treated with nocodazolefor 30 hours. Immunoprecipitation (IP): cell lysates were incubated with M-cadherin (A,C; E,G) or tubulin (B,D; F,H) antibodies either at 20°C(A,B,E,F) or at 4°C (C,D,G,H). CO: negative control in which M-cadherin or tubulin antibodies were replaced by PBS. CE: total cell extract;served as positive controls for M-cadherin and tubulin antibodies. The immunoprecipitates were separated by SDS polyacrylamide gelelectrophoresis and immunoblots were prepared. These were probed either with M-cadherin (A,B,C,D) or tubulin antibodies (E,F,G,H). M-cadherin antibodies coimmunoprecipitated tubulin at 20°C (E) but not at 4°C (G). Vice versa, tubulin antibodies coimmunoprecipitated M-cadherin only at 20°C. (B).

Fig. 6.Enrichment ofmicrotubules and microtubule-associated proteins inpmi28/Ecad cells by GTP-induced polymerization andtaxol stabilization. Fromextracts of pmi28/Ecad cells,ectopically expressing E-cadherin, microtubules werepolymerized and enriched byGTP and taxol according to theexperimental design describedin Materials and Methods.Supernatant (S) and pellets (P)from each preparation wereresolved by SDS-PAGE andelectroblotted ontonitrocellulose membranes. Theblots were probed with M-cadherin (A), E-cadherin (B),α-catenin (C), β-catenin (D)plakoglobin (E), tubulin (F),MF20 (G), and desmin (H)antibodies. Pellet (P) andsupernatants (S) were preparedfrom untreated myoblasts (P0,S0) and from pmi28 cells after30 hours (P30, S30) and 96hours (P96, S96) ofdifferentiation. Controls: totallysates of MDCK (M), pmi28cells (pm), pmi28/E-cadherintransfectants (K8) and bovine-serum-albumin (BSA). M-cadherin, E-cadherin, α-catenin, β-catenin plakoglobin and tubulin were found in thepellet of each preparation but not in the supernatant. Sarcomeric myosin (G) and desmin (H) were undetectable in the microtubule pellet and inthe supernatant.

63M-cadherin and microtubules

w

-

inheer

inlly

ntncn

Fig. 7. Distribution of E-cadherin and M-cadherin inMDCK/M-cadherintransfectants. MDCK/M-cadherin transfectantsectopically expressing M-cadherin, were treated withcytochalasin D (cy: E,F) ornocodazole for 30 hours(no: C,D) or left untreated(co: A,B). Cells were fixedand stained with either E-cadherin (A,C,E) or M-cadherin antibodies (B,D,F).Destruction of microtubulesand microfilaments wasconfirmed by jointlylabelling the cells withtubulin antibodies or FITC-phalloidin (not shown).Cytochalasin D abolishedthe membrane-labelling ofE-cadherin and M-cadherinleading to a more patchystaining pattern (cy: E,F). Incontrast, depolymerizationof microtubules had noinfluence on the stainingpattern of E-cadherin andM-cadherin in MDCK/M-cad cells (no: C,D). Bars: 20µm (D); 12 µm (F).

not found in the pellets (Fig. 8G; negative control). Theresults are in agreement with the observation thdepolymerization of microtubules had no effect on the surfadistribution of cadherins in MDCK/Mcad cells (Fig. 7)Furthermore, our results are also in line with published dimplying that in epithelial cells, an β-catenin pool outside theadhesion complex could be able to interact indirectly wmicrotubules via the tumor suppressor molecule APC whhas been shown to interact both, with β-catenin andmicrotubules (Munemitsu et al., 1994; Hülsken et al., 1994

Cell lysates of MDCK/Mcad cells were incubated witeither E-cadherin (Fig. 9E,G,I,M), M-cadherin (Fig9A,C,L,O) or tubulin (Fig. 9B,D,F,H,K,N) antibodies at 20°CImmunoprecipitates were collected and immunoblots preparThese immunoblots were probed with antibodies to Mcadherin (Fig. 9A-D), E-cadherin (Fig. 9E-H) and tubulin (Fi9I-O). The data of this experiment confirmed that there wasassociation of M-cadherin nor E-cadherin with thmicrotubular network in MDCK/Mcad cells. Theimmunofluorescence data (Fig. 7) and the immunoprecipitatdata (Fig. 9) suggested that E-cadherin and M-cadherin

seatce.ata

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ionhad

similar levels of expression in the MDCK/M-cadherin-transfected cells implying that our results were not due to lolevel expression of M-cadherin.

DISCUSSION

In this paper we present evidence for the interaction of Mcadherin with microtubules in myogenic pmi28 cells. Inaddition, we found that in a myogenic environment, E-cadhercould also associate with microtubules suggesting that tassociation of microtubules is mainly determined by thcellular environment and less by the cadherin itself. Foexample, epithelial MDCK cells lack the appropriateenvironment for the attachment of microtubules to a cadhercatenin complex and thus, neither E-cadherin nor ectopicaexpressed M-cadherin could interact with microtubules.

From our findings, which were based on three independetechniques (co-immunolocalizations of M-cadherin and tubuliin alkaloid-treated cells, coimmunoprecipitations and cycliGTP/taxol co-polymerizations), we derived the model show

64

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l.,yf

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ns

U. Kaufmann and others

Fig. 8.Enrichment of microtubules and microtubule-associated proteins in MDCK/M-cadherin-transfectants(2/II) by GTP-induced polymerization and taxolstabilization. Microtubules from MDCK-cell extractswere polymerized and enriched by GTP and taxol.Supernatant (S) and pellets (P) were resolved by SDS-PAGE and electroblotted onto nitrocellulose membranes.The blots were probed with M-cadherin (A), E-cadherin(B), α-catenin (C), β-catenin (D), plakoglobin (E),tubulin (F), and cytokeratin (G) antibodies. Controls weretotal lysates of MDCK-cells (M), pmi28 myoblasts (MB),MDCK-M-cad cells (2/II) and bovine serum albumin(BSA). Neither E-cadherin nor M-cadherin co-polymerized with microtubules. Cytokeratin wasundetectable (G) in the microtubule pellets (P) and in thesupernatants (S).

in Fig. 10. In this model, M-cadherin is complexed with thcatenins and plakoglobin as demonstrated (Kuch et al., 19Fig. 3). Furthermore, cutting of the microtubules (e.g. nocodazole) would not destroy the M-cadherin catencomplex but would allow its movement in the membrane agreement with Fig. 1C and Fig. 3). In contrast, destructionactin filaments by cytochalasin D would not result in suchredistribution (movement) of the M-cadherin catenin compl(in agreement with Fig. 1G and Fig. 3) which would be kein place by its connection to the microtubular network.

In agreement with our data, the myogenic but not tepithelial environment provides a cadherin-dependemicrotubule-associated factor(s) (cdMAF) connecting the Mcadherin catenin complex to the microtubular system. indicated in the model (Fig. 10), the mechanism by whicdMAF mediates this interaction is entirely open. Onpossibility would be that cdMAF binds directly to thcytoplasmic domain of M-cadherin. Alternatively, one of thcatenins might interact with cdMAF thereby providing thmolecular link between the M-cadherin catenin complex athe microtubules. Also, it is unclear whether or not cdMAinteracts directly with the microtubules or is bridged by anothmolecule.

In contrast to other cell types such as epithelial cells (Rimet al., 1995) or fibroblasts (Knudsen et al., 1995), the posslinkage of the microfilaments with the M-cadherin catencomplex in myogenic cells has not been studied in detailindicated by a questionmark in the diagram (Fig. 10Therefore, it is not known whether α-actinin or vinculin are

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involved in connecting these two systems or whether α-cateninmight bind directly to the microfilaments.

In conclusion, our model is in line with previous reportswhich have shown that the microtubules aligned with thelongated membrane of the differentiating muscle cel(Warren, 1974; Tassin et al., 1985; Holtzer et al., 1985; Kanet al., 1991).

One important question concerns the function of the Mcadherin catenin complex-microtubule interaction duringdifferentiation of skeletal muscle cells. As already mentionein the introduction, the microtubules seem to establish and/to keep the polarized, elongated shape of the differentiatioinduced myogenic cells. Obviously, this is a critical step fothe alignment of pre-fusion myoblasts. Another important stefor alignment is of course intercellular recognition ofmyoblasts. Previous data from our group (Zeschnigk et a1995) supported the notion that M-cadherin is most likelinvolved in the recognition step since functional pertubation oM-cadherin prevented myotube formation. By putting theresults of this paper in the context of these previous data, omight conclude that M-cadherin placed the microtubules otwo adjacent myoblasts in parallel with each other.

What mechanisms might be involved in such a process alignment? As has been shown by several groups, myotuformation required profound changes of any of the cytoskeletfilament systems (Toyama et al., 1982; Holtzer et al., 198Tassin et al., 1985; Lin et al., 1994). In addition to thetopological reorganization of microtubules, it has beeproposed that the transition of the tyrosinylated tubulin toward

65M-cadherin and microtubules

Fig. 9. Immunoprecipitation of M-cadherin, E-cadherin and tubulin in MDCK/M-cadherin-transfectants. MDCK/M-cadherin transfectants weretreated with nocodazole for 30 hours. Cell lysates were incubated with M-cadherin (A,C; L,O), E-cadherin (E,G; I,M) or tubulin antibodies(B,D; F,H; K,N) at 20°C (A,B,E,F; I,K,L) or at 4°C (C D,G,H; M,N,O) and analyzed on immunoblots. These were probed either with M-cadherin (A,B,C,D), E-cadherin (E,F,G,H) or tubulin antibodies (I,K,L,M,N,O). Neither M-cadherin nor E-cadherin could becoimmunoprecipitated with tubulin antibodies, and vice versa.

Fig. 10.Model of a possible interaction of M-cadherin with the cytoskeleton during muscle celldifferentiation. (A) In myoblasts, microtubulesradiate from the centrosomal region to the cellmembrane. (B) After induction of differentiation,myoblasts align and their microtubules orientateparallel to the longitudinal axis. This is mediatedby M-cadherin catenin complexes which areconnected to the microtubules via a cadherin-dependent microtubule-associated factor(s)(cdMAF) provided by the differentiation-induced, myogenic environment. As indicated bythe questionmarks, the mechanism by whichcdMAF mediates this interaction is entirely open.One possibility would be that cdMAF bindsdirectly to the cytoplasmic domain of M-cadherin. Alternatively, one of the catenins mightinteract with cdMAF thereby establishing themolecular link between the M-cadherin catenincomplex and the microtubules. Also, it is unclearwhether or not cdMAF interacts directly with themicrotubules or is bridged by another molecule.In contrast to other cell types such as epithelialcells or fibroblasts, the possible linkage of themicrofilaments with the M-cadherin catenin complex in myogenic cells has not been studied in detail as indicated by the questionmark in thediagram. Therefore, it is not known whether α-actinin or vinculin are involved in connecting these two systems or whether α-catenin mightbind directly to the microfilaments. Based on this model, cutting of the microtubules (e.g. by nocodazole) would not destroy the M-cadherincatenin complex but would allow diffusion in the plane-membrane of the plasma. In contrast, destruction of actin filaments by cytochalasin Dwould not result in such a redistribution (movement) of the M-cadherin catenin complex which would be kept in place by its connection to themicrotubular network. (C) After cell fusion: in polynucleated myotubes, microtubules are still in parallel with the cell membrane.

A B C

M-cadherin

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?

cdMAF

?

?

cdMAF

?

?

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microtubules

α-cateninβ-catenin

/plakoglobin

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66

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U. Kaufmann and others

the glutaminated tubulin might be an important event in tgeneration of stabilized microtubules in differentiatinmyogenic cells (Gundersen et al., 1989). It remains to studied, however, whether such post-translationmodifications of tubulin are relevant for the connection of tM-cadherin catenin complex to microtubules.

The microtubule-associated protein 4 (MAP4) has also besuggested to play a role in the generation of a microtubuarray giving myogenic cells a polarized shape (Mangan aOlmsted, 1996). There is, however, no experimental evidethat MAP4 was involved in the establishment of the Mcadherin-catenin-microtubule array.

Finally, it should be tested whether the function of the Mcadherin catenin complex might be regulated by its interactwith the microtubules. This would be in analogy to recent da(Bershadsky et al., 1996; Enomoto et al., 1996) showing tmicrotubules are involved in regulating the integrin-dependesignalling cascade in fibroblasts.

Related to the latter complex, it might be interesting to nothat the transmembrane protein meltrin α has also beenimplicated in the regulation of myoblast fusion, possibly the fusion process itself (Yagami-Hiromasa et al., 1995). far, there is no evidence for a cross-talk between tmechanisms postulated above and the action of meltrin α. Itwould be reasonable to speculate, however, that the fusprocess itself requires a cross-talk between an adhescytoskeletal network system and other fusion-regulatimolecules such as meltrin.

We thank A. Krapohl for preparing the first MDCK/Mcadtransformants, K. Weber for his critical reading of the manuscript,Becker for technical help and E. Kiessling and H. Ziegler fphotographs. This work was supported through grants of the DeutsForschungsgemeinschaft to A.S.P. (SFB 169/C8 and SFB 474/B2A.K. (Ki 339/4-2 and SFB 474/B7) and to A.W. (We 859/7), the Fonder Chemischen Industrie to A.S.P. and the Deutsche Stifterverbthrough a Schilling professorship to A.K.

REFERENCES

Aberle, H., Butz, S., Stappert, J., Weissig, H., Kemler, R. and Hoschuetzky,H. (1994). Assembly of the cadherin-catenin complex in vitro witrecombinant proteins. J. Cell Sci. 107, 3655-3663.

Antin, P., Forry-Schaudies, S., Friedman, T. M., Tapscott, S. J. andHoltzer, H. (1981). Taxol induces postmitotic myoblasts to assembinterdigitating microtubule-myosin arrays that exclude actin-filaments.J.Cell Biol. 90, 300-308.

Bader, D., Masaki, T. and Fishman, D. (1982). Immunochemical analysis ofmyosin heavy chain during avian myogenesis in vivo and in vitro. J. CellBiol. 95, 763-770.

Bershadsky, A., Chausovsky, A., Becker, E., Lyubimova, A. and Geiger, B.(1996). Involvement of microtubules in the control of adhesion-dependsignal transduction. Curr. Biol. 6, 1279-1289.

Boller, K., Vestweber, D. and Kemler, R. (1985). Cell-adhesion-moleculeuvomorulin is localized in the intermediate junctions of adult intestinepithelial cells. J. Cell Biol. 100, 327-332.

Bornemann, A. and Schmalbruch, H. (1994). Immunocytochemistry of M-cadherin in mature and regenerating rat muscle. Anat. Rec. 239, 119-125.

Bulinski, J. C., Richards, J. E. and Piperno, G. (1988). Posttranslationalmodifications of alpha tubulin: detyrosination and acetylation differentiapopulations of interphase microtubules in cultured cells. J. Cell Biol. 106,1213-1220.

Butz, S. and Kemler, R. (1994). Distinct cadherin-catenin complexes in C(2+)-dependent cell-cell adhesion. FEBS Lett. 355, 195-200.

hegbeal

he

enlarnd

nce-

-ionta

hatnt

te

inSohe

ionion-ng

S.orche

), todsand

h

le

ent

al

te

a

Croop, J. and Holtzer, H. (1975). Response of myogenic and fibrogenic cellsto cytochalasin B and to colcemid. J. Cell Biol. 65, 271-285.

Dickson, G., Peck, D., Moore, S. E., Barton, C. H. and Walsh, F. S. (1990).Enhanced myogenesis in NCAM-transfected mouse myoblasts. Nature 344,348-351.

Donalies, M., Cramer, M., Ringwald, M. and Starzinski-Powitz, A. (1991).Expression of M-cadherin, a member of the cadherin multigene familcorrelates with differentiation of skeletal muscle cells. Proc. Nat. Acad. Sci.USA 88, 8024-8028.

Enomoto, T. (1996). Microtubule disruption induces the formation of actinstress fibers and focal adhesions in cultured cells: possible involvementrho signal cascade. Cell Struct. Funct. 21, 317-326.

Geiger, B. and Ginsberg, D. (1991). The cytoplasmatic domain of adherens-type junctions. Cell Motil. Cytoskel. 20, 1-6.

Geiger, B. and Ayalon, O. (1992). Cadherins. Annu. Rev. Cell Biol. 8, 307-332. Gorman, C. (1985). In DNA Cloning: A Practical Approach. (ed. D. M.

Glover), pp. 143-190. IRL Press, Oxford.Gumbiner, B., Stevenson, B. and Grimaldi, A. (1988). The role of the cell

adhesion molecule uvomorulin in the formation and maintenance of thepithelial junctional complex. J. Cell Biol. 107, 1575-1587.

Gumbiner, B. M. (1993). Proteins associated with the cytoplasmatic surfacof adhesion molecules. Neuron11, 551-564.

Gundersen, G. G., Khawaja, S. and Bulinski, J. C. (1989). Generation of astable, posttranslationally modified microtubule array is an early event myogenic differentiation. J. Cell Biol. 109, 2275-2288.

Harlow, E. and Lane, D. (1988). Antibodies: A Laboratory Manual. ColdSpring Harbor Laboratory Press, New York.

Hazan, R. B., Kang, L., Rose, S., Borgen, P. I., Rimm, D. L. (1997). Vinculinis associated with the E-cadherin adhesion complex. J. Cell Biol. 272,32448-32453.

Herrenknecht, K., Ozawa, M., Eckerskorn, C., Lottspeich, F. M., Lenterand Kemler, R. (1991). The uvomorulin-anchorage protein α-catenin is avinculin homologue. Proc. Nat. Acad. Sci. USA 88, 9156-9160.

Hertig, C. M., Butz, S., Koch, S., Eppenberger-Ebehardt, M., Kemler, R.and Eppenberger, H. M. (1996). N-cadherin in adult rat cardiomyocytesin culture. J. Cell Sci. 109, 11-20.

Hinck, L., Näthke, I. S., Papkoff, J. and Nelson, W. J. (1994). Dynamics ofcadherin-catenin complex formation: novel protein interactions anpathways of complex assembly. J. Cell Biol. 125, 1327-1340.

Hirano, S., Nose, A., Hatta, K., Kawakami, A. and Takeichi, M. (1987).Calcium-dependent cell-cell adhesion molecules (cadherins): subclaspecificities and possible involvement of actin bundles. J. Cell Biol. 105,2501-2510.

Hirano, S., Kimoto, N., Shimoyama, Y., Hirohashi S. and Takeichi, M.(1992). Identification of a neural α-catenin as a key regulator of cadherinfunction and multicellular organization. Cell 70, 293-301.

Holtzer, H., Forry-Schaudies, S., Dlugosz, A., Antin, P. and Dubyak, G.(1985). Interactions between intermediate filaments, microtubules, amyobibrils in fibrogenic and myogenic cells. Ann. NY Acad. Sci. 455, 106-125.

Huber, O., Bierkamp, C. and Kemler, R. (1996). Cadherins and catenins indevelopment. Curr. Opin. Cell Biol. 8, 685-691.

Hülsken, J., Birchmeier, W. and Behrens, J. (1994). E-cadherin and APCcompete for the interaction with β-catenin and the cytoskeleton J. Cell Biol.126, 2061-2069.

Irintchev, A., Zeschnigk, M., Starzinski-Powitz, A. and Wernig, A. (1994).Expression pattern of M-cadherin in normal, denervated and regeneratmouse muscle. Dev. Dynam. 199, 326-337.

Irintchev, A., Langer, M. Zweyer, M., Theisen, R. and Wernig, A. (1997a).Functional improvement of damaged adult mouse muscle by implantatiof primary myoblasts. J. Physiol. 500, 775-785.

Irintchev, A., Langer, M., Zweyer, M. and Wernig, A. (1997b). Myoblasttransplantation in the mouse: what cells do we use. Basic Appl. Myologie 7,161-166.

Kano, Y., Fujimaki, N. and Ishikawa, H. (1991). The distribution andarrangement of microtubules in mammalian skeletal muscle fibers. CellStruct. Funct. 16, 251-261.

Knudsen, K. A., Soler, A. P., Johnson, K. R. and Wheelock, M. J. (1995).Interaction of α-actinin with the cadherin/catenin cell-cell adhesion complexvia α-catenin. J. Cell Biol. 130, 67-77.

Kuch, C., Winnekendonk, D., Butz, S., Unvericht, U., Kemler, R. andStarzinski-Powitz, A. (1997). M-cadherin-mediated cell adhesion andcomplex formation with the catenins in myogenic mouse cells. Exp. CellRes. 232. 331-338.

67M-cadherin and microtubules

ringr

by

rly

ic

s

on

or

f

.

:

Laemmli, W. (1970). Cleavage of structural proteins during assembly of thead of bacteriophage T 4. Nature 227, 680.

Li, H., Choudhary S. K., Milner, D. J., Munir, M. I., Kuisk, I. R. andCapetanaki, Y. (1994). Inhibition of desmin expression blocks myoblasfusion and interferes with the myogenic regulators MyoD and myogeninJ.Cell Biol. 124, 827-841.

Lin, Z., Lu, M. H., Schultheiss, T., Choi, J., Holtzer, S., DiLullo, C.,Fishman, D. and Holtzer, H. (1994). Sequential appearence of musclespecific proteins in myoblasts as a function of time after cell divisioevidence for a conserved myoblast differentiation program in skelemuscle. Cell Motil. Cytoskel. 29, 1-19.

Manfredi, J. J., Parness, J. and Horwitz, S. B. (1982). Taxol binds to cellularmicrotubules. J. Cell Biol. 94, 688-696.

Mangan, M. E. and Olmsted, J. B. (1996). A muscle-specific variant ofmicrotubule-associated protein 4 (MAP4) is required in myogenesDevelopment122, 771-781.

Munemitsu, S., Souza, B., Müller, O. Albert, I., Rubinfeld, B. and Polakis,P. (1994). The APC gene product associates with microtubules in vivo apromotes their assembly in vitro. Cancer Res. 54, 3676-3681.

Näthke, I. S., Hinck., Swedlow, J. R., Papkoff J. and Nelson, J. W. (1994).Defining interactions and distribuitions of cadherin and catenin complein polarized epithelial cells. J. Cell Biol. 125, 1341-1352.

Nagafuchi, A. and Takeichi, M. (1988). Cell binding function of E-cadherinis regulated by the cytoplasmatic domain. EMBO J. 7, 3679-3684.

Nagafuchi, A., Takeichi, M. and Tsukita, S. (1991). The 102 kd cadherin-associated protein: similarity to vinculin and posttranscriptional regulatiof expression. Cell 65, 849-857.

Nagar, B., Overduin, M., Ikura, M. and Rini, J. M. (1996). Structural basisof calcium-induced E-cadherin rigidification and dimerization. Nature 380,360-364.

Nieset, J. E., Redfield, A. R., Jin, F., Knudsen, K., Johnson, K. R. andWheelock, M. J. (1997). Characterization of the interactions of α-cateninwith α-actinin and β-catenin/plakoglobin. J. Cell Sci. 110, 1013-1022.

Nose, A., Nagafuchi, A. and Takeichi, M. (1988). Expressed recombinantcadherins mediate cell sorting in model systems. Cell 54, 993-1001.

Ozawa, M., Baribault, H. and Kemler, R. (1989). The cytoplasmaticdomain of the cell adhesion molecule uvomorulin associates with thindependent proteins structurally related in different species. EMBO J. 8,1711-1717.

Pouliot, Y., Gravel, M. and Holland, P. C. (1994). Developmental regulationof M-cadherin in the terminal differentiation of skeletal myoblasts. Dev.Dynam. 200, 305-312.

Rimm, D. l., Koslov, E. R., Kebriaei, P., Cianci, C. D. and Morrow, J. S.(1995). Alpha 1(E)-catenin is an actin-binding and-bundling protemediating the attachment of F-actin to the membrane adhesion compProc. Nat. Acad. Sci. USA 92, 8813-8817.

Ringwald, M., Schuh, R., Vestweber, D., Eistetter, H., Lottspeich, F., Engel,J., Dölz, R., Jähnig, F., Epplen, J., Mayer, S., Müller, C. and Kemler, R.(1987). The structure of the cell adhesion molecule uvomorulin. Insiginto the molecular mechanism of Ca2+ dependent cell adhesion. EMBO J.6, 3647-3653.

Rose, O., Rohwedel, J., Reinhardt, S., Bachmann, M., Cramer, M., Rotter,M., Wobus, A. and Starzinski-Powitz, A. (1994). Expression of M-cadherin protein in myogenic cells during prenatal mouse development differentiation of embryonic stemm cells in culture. Dev. Dynam. 201, 245-259.

Rose, O., Grund, C., Reinhardt, S., Starzinski-Powitz, A. and Franke, W.

he

t.

-n:tal

is.

nd

xes

on

ree

inlex.

hts

and

W. (1995). Contactus adhaerens, a special type of plaque-bearing adhejunction containing M-cadherin, in the granule layer of the cerebellaglomerulus. Proc. Nat. Acad. Sci. USA92, 6022-6026.

Saitoh, O., Arai, T. and Obinata, T. (1988). Distribution of microtubules andother cytoskeletal filaments during myotube elongation as revealed fluorescence microscopy. Cell Tissue Res. 252, 263-273.

Shapiro, L., Fannon, A. M., Kwong, P. D., Thompson, A., Lehmann, M.S., Grübel, G., Legrand, I. P. Als-Nielsen, J., Colman D. R. andHendrickson, W. A. (1995). Structural basis of cell-cell adhesion bycadherins. Nature 374, 327-336.

Southern, P. J. and Berg, P. (1982). Transformation of mammalian cells toantibiotic resistance with a bacterial gene under control of the SV40 earegion promotor. J. Mol. Appl. Genet. 1, 327-341.

Takeichi, M. (1991). Cadherin cell adhesion receptors as a morphogenregulator. Science 251, 1451-1455.

Tao, J. and Wallace, I. (1991). Site-specific antibodies block kinase Aphosphorylation of desmin in vitro and inhibit incorporation of myoblastinto myotubes. Cell Motil. Cytoskel. 19, 109-120.

Tassin, A. M., Maro, B. and Bornens, M. (1985). Fate of microtubule-organizing centers during myogenesis in vitro. J. Cell Biol. 100, 35-46.

Toyama, Y., Forry-Schaudies, S., Hoffmann, B. and Holtzer, H. (1982).Effects of taxol and colcemid on myofibrillogenesis. Proc. Nat. Acad. Sci.USA 79, 6556-6560.

Tsukita, S., Tsukita, S., Nagafuchi, A. and Yonemura, S. (1992). Molecularlinkage between cadherins and actin filaments in cell-cell adhesijunctions. Curr. Opin. Cell Biol.4, 834-839.

Tsukita, S., Itoh, M., Nagafuchi, A., Yonemura, S. and Tsukita, S. (1993).Submembranous junctional plaque proteins include potential tumsuppressor molecules. J. Cell Biol. 123, 1049-1053.

Vallee, R. B. (1982). A taxol-dependent procedure of the isolation omicrotubules and microtubule-associated proteins (MAPs). J. Cell Biol. 92,435-442.

Vallee, R. B. (1986). Purification of brain micotubules and micotubuleassociated protein 1 using taxol. Meth. Enzymol.134, 104-115.

Vestweber, D. and Kemler, R. (1985). Identification of a putative celladhesion domain of uvomorulin. EMBO J. 4, 3393-3398.

Warren, R. H. (1974). Microtubule organization in elongating muscle cellsJ. Cell Biol. 63, 550-566.

Weiss, E. E., Kroemker, M., Rüdiger, A. H., Jockusch, B. M. and Rüdiger,M. (1998). Vinculin is part of the cadherin-catenin junctional complexcomplex formation between α-catenin and vinculin. J. Cell Biol. 141, 755-764.

Watabe-Uchida, M., Uchida, N., Imamura, Y., Nagafuchi, A., Fujimoto,K., Uemura, T., Vermeulen, S., van Roy, F., Adamson, E. D. andTakeichi, M. (1998). Alpha-catenin-vinculin interaction functions toorganize the apical junctional complex in epithelial cells. J. Cell Biol. 143,847-857.

Yaffe, D. and Saxl, O. (1977). Serial passaging and differentiation ofmyogenic cells isolated from dystrophic mouse muscle. Nature 270, 725-727.

Yagami-Hiromasa, T., Sato, T., Kurisaki, T., Kamijo, K., Nabeshima, Y.and Fujisawa-Sehara, A. (1995). A metalloprotease-disintegrinparticipating in myoblast fusion. Nature 377, 652-656.

Zeschnigk, M., Kozian, D., Kuch, C., Schmoll, M. and Starzinski-Powitz,A. (1995). Involvement of M-cadherin in fusion and terminal differentiationof myogenic cells. J. Cell Sci. 108, 2973-2981.