Assembly of Desmosomal Cadherins into Desmosomes is Isoform Dependent
2001; Elsevier BV; Volume: 117; Issue: 1 Linguagem: Inglês
10.1046/j.0022-202x.2001.01400.x
ISSN1523-1747
AutoresKen Ishii, Suzanne M. Norvell, Leslie J. Bannon, Evangeline V. Amargo, Lauren T. Pascoe, Kathleen J. Green,
Tópico(s)Skin and Cellular Biology Research
ResumoDesmosomes are intercellular adhesive junctions that exhibit cell- and differentiation-specific differences in their molecular composition. In complex epithelia, desmosomes contain multiple representatives of the desmosomal cadherin family, which includes three desmogleins and three desmocollins. Rules governing the assembly of desmosomal cadherin isoforms into desmosomes of different cell types are unknown. Here we compared the assembly properties of desmoglein 2 (Dsg2) and desmocollin 2 (Dsc2), which are widely expressed, with Dsg1 and Dsc1, which are expressed in the differentiated layers of complex epithelia, by introducing myc-tagged forms into simple and squamous epithelial cells that do not express Dsg1 or Dsc1. Dsc2.myc and Dsg2.myc assembled efficiently into desmosomes in every cell type in spite of significant shifts in the stoichiometric relationship between desmogleins and desmocollins. In contrast, Dsc1a.myc, Dsc1b.myc, and Dsg1.myc did not stably incorporate into desmosomes in any line. Coexpression of Dsc1a.myc or Dsc1b.myc and Dsg1.myc did not lead to their colocalization and failed to enhance incorporation of either cadherin into desmosomes. Dsg1.myc, but not Dsc1a, Dsc1b, disrupted desmosome assembly in a cell-type-specific manner, and disruption correlated with the recruitment of Dsg1.myc, but not Dsc1a or Dsc1b, into a Triton-insoluble pool. The plakoglobin:E-cadherin ratio decreased in Dsg1-expressing cells with disrupted desmosomes, but a decrease was also observed in a Dsc1a line. Thus, a modest reduction of plakoglobin associated with E-cadherin is apparently not sufficient to disrupt desmosome assembly. Our results demonstrate that desmosome assembly tolerates large shifts in cadherin stoichiometry, but is sensitive to isoform-specific differences exhibited by desmogleins and desmocollins. Desmosomes are intercellular adhesive junctions that exhibit cell- and differentiation-specific differences in their molecular composition. In complex epithelia, desmosomes contain multiple representatives of the desmosomal cadherin family, which includes three desmogleins and three desmocollins. Rules governing the assembly of desmosomal cadherin isoforms into desmosomes of different cell types are unknown. Here we compared the assembly properties of desmoglein 2 (Dsg2) and desmocollin 2 (Dsc2), which are widely expressed, with Dsg1 and Dsc1, which are expressed in the differentiated layers of complex epithelia, by introducing myc-tagged forms into simple and squamous epithelial cells that do not express Dsg1 or Dsc1. Dsc2.myc and Dsg2.myc assembled efficiently into desmosomes in every cell type in spite of significant shifts in the stoichiometric relationship between desmogleins and desmocollins. In contrast, Dsc1a.myc, Dsc1b.myc, and Dsg1.myc did not stably incorporate into desmosomes in any line. Coexpression of Dsc1a.myc or Dsc1b.myc and Dsg1.myc did not lead to their colocalization and failed to enhance incorporation of either cadherin into desmosomes. Dsg1.myc, but not Dsc1a, Dsc1b, disrupted desmosome assembly in a cell-type-specific manner, and disruption correlated with the recruitment of Dsg1.myc, but not Dsc1a or Dsc1b, into a Triton-insoluble pool. The plakoglobin:E-cadherin ratio decreased in Dsg1-expressing cells with disrupted desmosomes, but a decrease was also observed in a Dsc1a line. Thus, a modest reduction of plakoglobin associated with E-cadherin is apparently not sufficient to disrupt desmosome assembly. Our results demonstrate that desmosome assembly tolerates large shifts in cadherin stoichiometry, but is sensitive to isoform-specific differences exhibited by desmogleins and desmocollins. desmocollin desmoglein intermediate filament Desmosomes are intercellular adhesive junctions that anchor the intermediate filament (IF) cytoskeleton to the plasma membrane, thus providing mechanical strength to a tissue (Green and Gaudry, 2000Green K.J. Gaudry C.G. Are desmosomes more than tethers for intermediate filaments?.Nature Rev Mol Cell Biol. 2000; 1: 208-216Crossref PubMed Scopus (309) Google Scholar). Although desmosomes are abundant in epithelia, particularly the epidermis, they can also be found in diverse cell types such as cardiac muscle, pia and arachnoid meninges, and follicular dendritic cells (Farquhar and Palade, 1963Farquhar M.G. Palade G.E. 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Identification of the ubiquitous human desmoglein, Dsg2, and the expression catalogue of the desmoglein subfamily of desmosomal cadherins.Exp Cell Res. 1994; 211: 391-399https://doi.org/10.1006/excr.1994.1103Crossref PubMed Scopus (196) Google Scholar;Nuber et al., 1995Nuber U.A. Schafer S. Schmidt A. Koch P.J. Franke W.W. The widespread human desmocollin Dsc2 and tissue-specific patterns of synthesis of various desmocollin subtypes.Eur J Cell Biol. 1995; 66: 69-74PubMed Google Scholar). Although considerable overlap is exhibited in the distribution of these isoforms in stratified tissues, their expression is clearly differentiation-dependent. For instance, in epidermis, Dsg3 and Dsc3 are found predominantly in the basal and suprabasal layers of stratified epithelia whereas Dsg1 and Dsc1 are found in the more differentiated layers (Nuber et al., 1995Nuber U.A. Schafer S. Schmidt A. Koch P.J. Franke W.W. The widespread human desmocollin Dsc2 and tissue-specific patterns of synthesis of various desmocollin subtypes.Eur J Cell Biol. 1995; 66: 69-74PubMed Google Scholar,Nuber et al., 1996Nuber U.A. Schafer S. Stehr S. Rackwitz H. Franke W.W. Patterns of desmocollin synthesis in human epithelia: immunolocalization of desmocollins 1 and 3 in special epithelial and in cultured cells.Eur J Cell Biol. 1996; 71: 1-13PubMed Google Scholar;North et al., 1996North A. Chidgey M.A.J. Clarke J.P. Bardsley W.G. Garrod D.R. Distinct desmocollin isoforms occur in the same desmosomes and show reciprocally graded distributions in bovine nasal epidermis.Proc Natl Acad Sci. 1996; 93: 7701-7705Crossref PubMed Scopus (86) Google Scholar;Chidgey et al., 1997Chidgey M.A.J. Yue K.K.M. Gould S. Byrne C. Garrod D.R. Changing pattern of desmocollin 3 expression accompanies epidermal organisation during skin development.Dev Dynamics. 1997; 210: 315-327Crossref PubMed Scopus (32) Google Scholar;King et al., 1997King I.A. Angst B.D. Hunt D.M. Kruger M. Arnemann J. Buxton R.A. Hierarchical expression of desmosomal cadherins during stratified epithelial morphogenesis in the mouse.Differentiation. 1997; 62: 83-96https://doi.org/10.1007/s002580050207Crossref PubMed Scopus (53) Google Scholar). A role for desmosomal cadherins in adhesion and tissue integrity is supported by the experimental ablation and mutation of Dsg, resulting in defects in the epidermis and its appendages in mice (Allen et al., 1996Allen E. Yu Q.-C. Fuchs E. Mice expressing a mutant desmosomal cadherin exhibit abnormalities in desmosomes, proliferation, and epidermal differentiation.J Cell Biol. 1996; 133: 1367-1382Crossref PubMed Scopus (133) Google Scholar;Koch et al., 1997Koch P.J. Mahoney M.G. Ishikawa H. et al.Targeted disruption of the pemphigus vulgaris antigen (desmoglein 3) gene in mice causes loss of keratinocyte cell adhesion with a phenotype similar to pemphigus vulgaris.J Cell Biol. 1997; 137: 1091-1102Crossref PubMed Scopus (363) Google Scholar), and by the existence of human autoimmune and inherited blistering diseases that target the desmosomal cadherins (Amagai and Citi, 1994Amagai M. Molecular mechanisms of pemphigus diseases.in: Citi S. Molecular Mechanisms of Epithelial Cell Junctions: from Development to Disease. R.G. Landes Co, Austin1994: 245-257Google Scholar;Amagai et al., 1994Amagai M. Hashimoto T. Shimizu N. Nishikawa T. Absorption of pathogenic autoantibodies by the extracellular domain of pemphigus vulgaris antigen (Dsg3) produced by baculovirus.J Clin Invest. 1994; 94: 59-67Crossref PubMed Scopus (288) Google Scholar;Stanley, 1995Stanley J.R. Autoantibodies against adhesion molecules and structures in blistering skin diseases.J Exp Med. 1995; 181: 1-4Crossref PubMed Scopus (68) Google Scholar;Rickman et al., 1999Rickman L. Simrak D. Stevens H.P. et al.N-terminal deletion in a desmosomal cadherin causes the autosomal dominant skin disease striate palmoplantar keratoderma.Hum Mol Genet. 1999; 8: 971-976https://doi.org/10.1093/hmg/8.6.971Crossref PubMed Scopus (172) Google Scholar). Both Dsg and Dsc are required for conferring adhesive properties on normally nonadherent L929 cell fibroblasts, and it has been suggested that these cadherins can interact in a heterophilic manner (Chitaev and Troyanovsky, 1997Chitaev N.A. Troyanovsky S.M. Direct Ca2+-dependent heterophilic interaction between desmosomal cadherins, desmoglein and desmocollin, contributes to cell-cell adhesion.J Cell Biol. 1997; 138: 193-201Crossref PubMed Scopus (188) Google Scholar;Marcozzi et al., 1998Marcozzi C. Burdett I.D.J. Buxton R.S. Magee A.I. Coexpression of both types of desmosomal cadherin and plakoglobin confers strong intercellular adhesion.J Cell Sci. 1998; 111: 495-509PubMed Google Scholar;Tselepis et al., 1998Tselepis C. Chidgey M. North A. Garrod D. Desmosomal adhesion inhibits invasive behavior.Proc Natl Acad Sci. 1998; 95: 8064-8069Crossref PubMed Scopus (152) Google Scholar). The precise requirements necessary for adhesion are not fully understood, however (Kowalczyk et al., 1996Kowalczyk A.P. Borgwardt J.E. Green K.J. Analysis of desmosomal cadherin-adhesive function and stoichiometry of desmosomal cadherin-plakoglobin complexes.J Invest Dermatol. 1996; 107: 293-300Crossref PubMed Scopus (77) Google Scholar), and it seems likely that adhesion depends on parameters such as the stoichiometric relationship between Dsc, Dsg, and plakoglobin. In addition, the preferred binding partners in vivo are totally unknown. The cytoplasmic tails of the desmosomal cadherins share certain similarities, but also exhibit important differences. These domains link the plasma membrane to the IF cytoskeleton through a complex of proteins. This complex includes plakoglobin, which binds directly to the ICS domain found in all Dsg and the a form of Dsc, and desmoplakin, which binds to plakoglobin and anchors IF to the desmosomal plaque (Kouklis et al., 1994Kouklis P.D. Hutton E. Fuchs E. Making a connection: direct binding between keratin intermediate filaments and desmosomal proteins.J Cell Biol. 1994; 127: 1049-1060Crossref PubMed Scopus (240) Google Scholar;Bornslaeger et al., 1996Bornslaeger E.B. Corcoran C.M. Stappenbeck T.S. Green K.J. Breaking the connection: displacement of the desmosomal plaque protein desmoplakin from cell–cell interfaces disrupts anchorage of intermediate filament bundles and alters intercellular junction assembly.J Cell Biol. 1996; 134: 985-1001Crossref PubMed Scopus (175) Google Scholar;Kowalczyk et al., 1997Kowalczyk A.P. Bornslaeger E.A. Borgwardt J.E. et al.The amino-terminal domain of desmoplakin binds to plakoglobin and clusters desmosomal cadherin-plakoglobin complexes.J Cell Biol. 1997; 139: 773-784Crossref PubMed Scopus (189) Google Scholar;Smith and Fuchs, 1998Smith E.A. Fuchs E. Defining the Interactions between intermediate filaments and desmosomes.J Cell Biol. 1998; 141: 1229-1241Crossref PubMed Scopus (198) Google Scholar). The sequence differences exhibited by the desmosomal cadherin cytoplasmic domains and possible distinct preferences for other potential binding partners, such as the plakophilins (Kowalczyk et al., 1999Kowalczyk A.P. Hatzfeld M. Bornslaeger E.A. et al.The head domain of plakophilin-1 binds to and enhances its recruitment to desmosomes: implications for cutaneous disease.J Biol Chem. 1999; 274: 18145-18148https://doi.org/10.1074/jbc.274.26.18145Crossref PubMed Scopus (118) Google Scholar;Hatzfeld et al., 2000Hatzfeld M. Haffner C. Schulze K. Vinzens U. The function of plakophilin 1 in desmosome assembly and actin filament organization.J Cell Biol. 2000; 149: 209-222Crossref PubMed Scopus (131) Google Scholar), suggest a complexity of protein–protein interactions that would further increase the diversity of desmosome structure and function. It has been demonstrated that multiple Dsc and Dsg isoforms can coexist in a single junction (North et al., 1996North A. Chidgey M.A.J. Clarke J.P. Bardsley W.G. Garrod D.R. Distinct desmocollin isoforms occur in the same desmosomes and show reciprocally graded distributions in bovine nasal epidermis.Proc Natl Acad Sci. 1996; 93: 7701-7705Crossref PubMed Scopus (86) Google Scholar). It remains to be determined, however, whether one desmosomal cadherin can substitute for another in desmosome assembly. In this study, we directly compare the assembly behaviors of full-length tagged forms of the ubiquitous desmosomal cadherins, Dsg2 and Dsc2, to Dsg1, Dsc1a, and Dsc1b by introducing them into A431 cells, MDCK cells, and SCC9 cells, which all normally express Dsg2 and Dsc2 but not Dsg1 or Dsc1. Our data demonstrate that ectopic Dsg2 and Dsc2 efficiently assemble into desmosomes in cells that endogenously express these isoforms even though their expression leads to a significant shift in the ratio of total Dsg to Dsc. In contrast, Dsc1a, Dsc1b, and Dsg1 did not incorporate efficiently into desmosomes in these same cell types even when expressed at levels comparable to or lower than ectopic Dsg2 and Dsc2. Furthermore, even modest levels of Dsg1.myc, but not Dsc1a or Dsc1b, result in desmosome disruption and IF detachment in A431 cells. Together these data demonstrate that epithelial cells can tolerate wide ranges in levels of endogenous desmosomal cadherins, whereas low levels of heterotypic cadherins not only do not incorporate into desmosomes, but also can, in certain cells, disrupt them. For full-length c-myc epitope tagged human Dsc1a and Dsc1b, cDNA clones were isolated from lambda gt11 and lambda ZAPII libraries generated from normal human keratinocytes (a gift from Dr. John Stanley) and clones were constructed using an overlap extension strategy. Nucleotide numbers correspond to those previously reported (Accession z34522) (Theis et al., 1993Theis D.G. Koch P.J. Franke W.W. Differential synthesis of type 1 and type 2 desmocollin mRNAs in human stratified epithelia.Int J Dev Biol. 1993; 37: 101-110PubMed Google Scholar); nucleotides 191–2676/2723–2921 are assigned to the full-length Dsc1a and nucleotides 191–2713 to the Dsc1b coding sequence, respectively. p489 (1–1206) was partially digested with EcoRV, digested to completion with SmaI, and ligated to an internal EcoRV site and an EcoRV site in the polylinker of p483 (669–2530) to generate p512 (1–2530) comprising the 5′ part of Dsc1. For c-myc tagged Dsc1b, a c-myc tag was added to the 3′ end of the cDNA by polymerase chain reaction (PCR) using primers T7 and KI4, consisting of BamHI site, a stop codon, c-myc epitope, and the C-terminal portion of Dsc1b (5′-CGG GAT CCT ACA AGT AAT ATT CAG AAA TGA GCT TTT GCT CCA CAT TTT TAA TCA GAG TG-3′) on p426 (2380–2893). The resulting PCR product was subcloned into pBluescript-SK (Stratagene, La Jolla, CA) at BamHI site to generate p846. To extend a fragment to overlap with p512, PCR was carried out on a mixture of the MscI fragment of p483 (1650–2502) and NspI fragment of p846 (containing the downstream portion of Dsc1b after 2482) using primers KI9, consisting of the internal Dsc1 sequence (5′-GGA ACA TAG AAG AAA AGG-3′), and KI4. The resulting PCR product was digested with NdeI and BamHI and ligated to p512 to obtain the full length of Dsc1b with a myc tag (p853). p853 was digested with SalI and BamHI and subcloned into the SalI/BamHI site of the β-actin expression vector (p104). The resulting plasmid was named p855. To generate a c-myc tagged Dsc1a, sequential PCR was used to excise 2677–2722 from Dsc1 sequences, followed by extension and addition of a c-myc tag at the 3′ end of Dsc1a. First, PCR was carried out on p426 with primers T7 and KI1, complement of 2724–2740 joined to 2663–2677 (5′-CCA CAC AAA TAC ACC TTT TCG CCA AGC CGA GG-3′), and primers KI2, nucleotides 2663–2677 joined to 2724–2740 (5′-CCT CGG CTT GGC GAA AAG GTG TAT TTG TGT GG-3′), and KI5, containing a BamHI site, a stop codon, c-myc epitope, and the C-terminal portion of Dsc1a (5′-CGG GAT CCT ATT TCT TGA TGC ATG TCT TTG CTA ATG TCC TAA ATT TGG GTT C-3′). The subsequent PCR on a mixture of these two products with primers T7 and KI3 (5′-CGG GAT CCT ACA AGT CCT CTT CAG AAA TGA GCT TTT GCT CCA CTT TCT TGA TGC ATG TC-3′) was used to add the myc tag on the 3′ end. The resulting PCR product was subcloned into pBluescript-SK at BamHI to generate p861, which encodes the 3′ region of Dsc1a with myc tag. To extend the overlap region with p512, PCR was used on a mixture of the MscI fragment of p483 (1650–2502) and the NspI fragment of p861 (2482–2908) with primers KI9 and KI5. PCR was performed on this PCR product with primers KI9 and KI3. The resulting PCR product was digested with NdeI and BamHI and ligated to p512 to generate the full length of Dsc1a with a myc tag (p853). The c-myc tagged Dsc1a was ligated into the SalI/BamHI site of the β-actin expression vector (p104), and termed p854. The Dsc1a.myc and Dsc1b.myc clones were sequenced and compared with the published sequence, a clone obtained from Dr. Takashi Hashimoto (Hashimoto et al., 1997Hashimoto T. Kiyokawa C. Mori O. et al.Human desmocollin 1 (Dsc1) is an autoantigen for the subcorneal pustular dermatosis type of IgA pemphigus.J Invest Dermatol. 1997; 109: 127-131Crossref PubMed Scopus (181) Google Scholar), and other available databases. The sequence of the Dsc1a and Dsc1b clones used in this work differed by two and one different amino acids, respectively, from Accession z34522, but these were both present in sequences in another available clone (Accession X72925) (King et al., 1993King I.A. Arnemann J. Spurr N.K. Buxton R.S. Cloning of the cDNA (DSC1) coding for human type 1 desmocollin and its assignment to chromosome 18.Genomics. 1993; 18: 185-194https://doi.org/10.1006/geno.1993.1453Crossref PubMed Scopus (45) Google Scholar) and are thus likely to represent polymorphisms. Full-length c-myc epitope tagged human Dsc2a was removed from p390 (Kowalczyk et al., 1994Kowalczyk A.P. Palka H.L. Luu H.H. Nilles L.A. Anderson J.E. Wheelock M.J. Green K.J. Posttranslational regulation of plakoglobin expression: influence of the desmosomal cadherins on plakoglobin metabolic stability.J Biol Chem. 1994; 269: 31214-31223Abstract Full Text PDF PubMed Google Scholar) using SalI and HindIII sites and then ligated to a purified, SalI/HindIII digested β-actin expression vector; the resulting plasmid used for these studies was named p476. A cDNA construct encoding human Dsg2 in pBluescript R3 BptII SR- was a gift from Drs W. Franke and S. Schafer. We termed this plasmid p594. To generate a C-terminal myc epitope tag on Dsg2, a PCR using p594 as template was performed. A primer consisting of the internal Dsg2 sequence termed SN8-(5′ GCC AAT GCA GAG AAA GTA AC 3′) and a primer consisting of three restriction sites, AatII, BamHI, XhoI, a stop codon, c-myc epitope, and the C-terminal portion of Dsg2 termed SN9-(5′ GTG ACG TCT GAT CCC TCG AGC TAC AAG TCC TCT TCA GAA ATG AGC TTT TGC TCC ACG GAG TAA GAA TGC TG 3′) were used to generate a C-terminally c-myc epitope tagged Dsg2 cDNA fragment. This fragment was purified and digested with AatII and BglII and then ligated back into AatII/BglII digested and purified p594 containing the remaining Dsg2 N-terminal region. The resulting full-length C-terminally c-myc epitope tagged human Dsg2 in pBluescript R3 BptII SR- was termed p685. Myc-tagged Dsg2 was removed from p685 using BamHI and was ligated to BamHI digested β-actin (p104) to generate the final expression vector, which was termed p726. c-myc epitope tagged Dsg1 cDNA and c-myc epitope tagged plakoglobin cDNA were described previously (Kowalczyk et al., 1994Kowalczyk A.P. Palka H.L. Luu H.H. Nilles L.A. Anderson J.E. Wheelock M.J. Green K.J. Posttranslational regulation of plakoglobin expression: influence of the desmosomal cadherins on plakoglobin metabolic stability.J Biol Chem. 1994; 269: 31214-31223Abstract Full Text PDF PubMed Google Scholar;Norvell and Green, 1998Norvell S.M. Green K.J. Contributions of extracellular and intracellulaar domains of full length and chimeric cadherin molecules to junction assembly in epithelial cells.J Cell Sci. 1998; 111: 1305-1318Crossref PubMed Google Scholar). Parental MDCK cells (a gift from Dr. Peter Kopp) were cultured in Dulbecco's modified Eagle's medium (DMEM) (Mediatech, Hernden, VA) containing 10% fetal bovine serum (FBS), 100 units penicillin per ml, and 100 µg per ml streptomycin. The oral squamous cell carcinoma line SCC9 was obtained from Dr. Jim Rheinwald. Parental SCC9 cells were cultured in DMEM/F-12 (Life Technologies, Rockville, MD) containing 10% FBS, 100 units penicillin per ml, and 100 µg per ml streptomycin. To obtain mass-selected cells, cells were cotransfected with calcium phosphate precipitates containing each myc-tagged desmosomal cadherin vector and selected under 400 µg per ml of G418 sulfate (Mediatech) for SCC9 cells and 500 µg per ml of G418 for MDCK cells. For Dsg1.myc, the plasmid was cotransfected with neomycin-resistant expression vector. A431 epithelial cells were a gift from Dr. M. Wheelock, University of Toledo. Parental A431 cells were cultured in DMEM containing 10% FBS, 100 units per ml penicillin, and 100 µg per ml streptomycin. Stable cell lines expressing myc-tagged desmosomal cadherin or control cell lines expressing neomycin resistance (Neo) were generated as described previously (Norvell and Green, 1998Norvell S.M. Green K.J. Contributions of extracellular and intracellulaar domains of full length and chimeric cadherin molecules to junction assembly in epithelial cells.J Cell Sci. 1998; 111: 1305-1318Crossref PubMed Google Scholar). Mass-selected cells expressing both Dsg1.myc and Dsc1-myc were generated by re-transfection of line 18 expressing Dsg1.myc with Dsc1a-myc (p854) or Dsc1b-myc (p855) plasmid, and they were selected using 500 µg per ml G418 sulfate with 1 µg per ml puromycin (Sigma Chemical, St. Louis, MO) to maintain Dsg1.myc expression. Stable cell lines expressing full-length Dsg1.myc and plakoglobin (Dsg1.myc + Pg) were generated by re-transfection of line 18 expressing Dsg1.myc with Pg.myc (p330), and were selected using 1 µg per ml puromycin (Sigma Chemical), to maintain Dsg1.myc expression, and 700 µg per ml G418 sulfate (Mediatech), to select for plakoglobin expression. Control Dsg1 + Neo lines were generated by re-transfection of Dsg1.myc (18) with the β-actin promoter driving the neomycin resistance marker. The following mouse monoclonal antibodies were used in this study: DP2.15, against the desmoplakin I/II rod domains (Cowin et al., 1985Cowin P. Kapprell H.-P. Franke W.W. The complement of desmosomal plaque proteins in different cell types.J Cell Biol. 1985; 101: 1442-1454Crossref PubMed Scopus (132) Google Scholar); KSB17.2, against keratin 18 (Sigma Chemical); anticytokeratin peptide 8 (Sigma Chemical); 6D8, against Dsg2 (Wahl et al., 1996Wahl J.K. Sacco P.A. McGranahan-Sadler T.M. Sauppe L.M. Wheelock M.J. Johnson K.R. Plakoglobin domains that define its association with the desmosomal cadherins and the classical cadherins: identification of unique and shared domains.J Cell Sci. 1996; 109: 1143-1154Crossref PubMed Google Scholar); 7G6, against Dsc2 (Wahl et al., 1996Wahl J.K. Sacco P.A. McGranahan-Sadler T.M. Sauppe L.M. Wheelock M.J. Johnson K.R. Plakoglobin domains that define its association with the desmosomal cadherins and the classical cadherins: identification of unique and shared domains.J Cell Sci. 1996; 109: 1143-1154Crossref PubMed Google Scholar); HECD-1, against E-cadherin; U100, against Dsc1 (Research Diagnostics, Flanders, NJ). The following rabbit polyclonal antibodies were used: 795, against E-cadherin (a gift from Drs R. Marsh and R. Brackenbury); 2026, against the c-myc epitope tag, referred to here as poly myc (a gift from Dr. J. Stanley); NW161, against desmoplakin (Bornslaeger et al., 1996Bornslaeger E.B. Corcoran C.M. Stappenbeck T.S. Green K.J. Breaking the connection: displacement of the desmosomal plaque protein desmoplakin from cell–cell interfaces disrupts anchorage of intermediate filament bundles and alters intercellular junction assembly.J Cell Biol. 1996; 134: 985-1001Crossref PubMed Scopus (175) Google Scholar). Also, 982, a human pemphigus autoantibody against Dsg1 (Kowalczyk et al., 1995Kowalczyk A.P. Anderson J.E. Borgwardt J.E. Hashimoto T. Stanley J.R. Green K.J. Pemphigus sera recognize conformationally sensitive epitopes in the amino-terminal region of desmoglein-1.J Invest Dermatol. 1995; 105: 147-152Crossref PubMed Scopus (75) Google Scholar), and 1407, a chicken antiplakoglobin antibody, were used (Gaudry et al.C.A. Gaudry, H.L. Palka, R.L. Dusek, A.C. Huen, M.J. Khandekar, L.G. Hudson, K.J. Green: Tyrosine phosphorylated plakoglobin is associated with desmogleins but not desmoplakin after EGFR activation, J Biol Chem, in press (published online 5/3/01)Google Scholar). Cells were plated on glass coverslips for 2 d and were then rinsed in complete phosphate-buffered saline (PBS) and fixed in methanol for 2 min at -20°C. The following antibodies were used: 2026 poly myc (1:700), 1407 (1:100), KSB17.2 (1:200), 982 (1:100), NW161 (1:100), DP2.15 (1:200). Alexa Fluor conjugated secondary antibodies (Molecular Probes, Eugene, OR) or the fluorescein or rhodamine conjugated secondary antibodies (Kirkegaard and Perry Laboratories, Gaithersburg, MD) were used. Marina Blue antirabbit IgG conjugate (Molecular Probes) was used for triple labeling. Processed coverslips were mounted in Vinol (Air Products and Chemicals, Allentown, PA) and examined with a Leitz DMR microscope. Photographs were taken on a Leitz orthomat E camera using T-MAX 100 or Ektachrome film (Kodak, Rochester, NY), or images were captured using a Hamamatsu Orca digital camera and Improvision Openlab software. Cells were fractionated into Triton-soluble and Triton-insoluble pools using coimmunoprecipitation buffer [1% Triton X-100, 145 mM NaCl, 10 mM Tris-HCl, pH 7.4, 5 mM ethylenediamine tetraacetic acid, 2 mM ethyleneglycol-bis(β-aminoethyl
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