RESUMO
We previously showed that the cell-cell junction protein plakoglobin (PG) not only suppresses motility of keratinocytes in contact with each other, but also, unexpectedly, of single cells. Here we show that PG deficiency results in extracellular matrix (ECM)-dependent disruption of mature focal adhesions and cortical actin organization. Plating PGâ»/â» cells onto ECM deposited by PG+/â» cells partially restored normal cell morphology and inhibited PGâ»/â» cell motility. In over 70 adhesion molecules whose expression we previously showed to be altered in PGâ»/â» cells, a substantial decrease in fibronectin (FN) in PGâ»/â» cells stood out. Re-introduction of PG into PGâ»/â» cells restored FN expression, and keratinocyte motility was reversed by plating PGâ»/â» cells onto FN. Somewhat surprisingly, based on previously reported roles for PG in regulating gene transcription, PG-null cells exhibited an increase, not a decrease, in FN promoter activity. Instead, PG was required for maintenance of FN mRNA stability. PGâ»/â» cells exhibited an increase in activated Src, one of the kinases controlled by FN, a phenotype reversed by plating PGâ»/â» cells on ECM deposited by PG+/â» keratinocytes. PGâ»/â» cells also exhibited Src-independent activation of the small GTPases Rac1 and RhoA. Both Src and RhoA inhibition attenuated PGâ»/â» keratinocyte motility. We propose a novel role for PG in regulating cell motility through distinct ECM-Src and RhoGTPase-dependent pathways, influenced in part by PG-dependent regulation of FN mRNA stability.
Assuntos
Movimento Celular/fisiologia , Fibronectinas/metabolismo , Transdução de Sinais/fisiologia , gama Catenina/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Animais , Western Blotting , Movimento Celular/genética , Células Cultivadas , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Fibronectinas/genética , Técnica Indireta de Fluorescência para Anticorpo , Queratinócitos/citologia , Queratinócitos/metabolismo , Camundongos , Camundongos Knockout , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/genética , gama Catenina/genética , Proteínas rac1 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/metabolismo , Proteína rhoA de Ligação ao GTP/genéticaRESUMO
Darier's disease (DD) is an inherited autosomal-dominant skin disorder characterized histologically by loss of adhesion between keratinocytes. DD is typically caused by mutations in sarcoendoplasmic reticulum Ca(2+)-ATPase isoform 2 (SERCA2), a major regulator of intracellular Ca(2+) homeostasis in the skin. However, a defined role for SERCA2 in regulating intercellular adhesion remains poorly understood. We found that diminution of SERCA2 function by pharmacological inhibition or siRNA silencing in multiple human epidermal-derived cell lines was sufficient to disrupt desmosome assembly and weaken intercellular adhesive strength. Specifically, SERCA2-deficient cells exhibited up to a 60% reduction in border translocation of desmoplakin (DP), the desmosomal cytolinker protein necessary for intermediate filament (IF) anchorage to sites of robust cell-cell adhesion. In addition, loss of SERCA2 impaired the membrane translocation of protein kinase C α (PKCα), a known regulator of DP-IF association and desmosome assembly, to the plasma membrane by up to 70%. Exogenous activation of PKCα in SERCA2-deficient cells was sufficient to rescue the defective DP localization, desmosome assembly, and intercellular adhesive strength to levels comparable to controls. Our findings indicate that SERCA2-deficiency is sufficient to impede desmosome assembly and weaken intercellular adhesive strength via a PKCα-dependent mechanism, implicating SERCA2 as a novel regulator of PKCα signaling.
Assuntos
Doença de Darier/metabolismo , Desmoplaquinas/metabolismo , Proteína Quinase C-alfa/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Transdução de Sinais/fisiologia , Cálcio/metabolismo , Carcinoma de Células Escamosas , Adesão Celular/fisiologia , Comunicação Celular/fisiologia , Linhagem Celular Tumoral , Doença de Darier/patologia , Desmossomos/metabolismo , Desmossomos/patologia , Humanos , Filamentos Intermediários/metabolismo , Queratinas/metabolismo , Neoplasias Bucais , RNA Interferente Pequeno , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genéticaRESUMO
The ability of cells to modulate interactions with each other and the substrate is essential for epithelial tissue remodeling during processes such as wound healing and tumor progression. However, despite strides made in the field of proteomics, proteins involved in adhesion have been difficult to study. Here, we report a method for the enrichment and analysis of proteins associated with the basal surface of the cell and its underlying matrix. The enrichment involves deroofing the cells with 20 mM ammonium hydroxide and the removal of cytosolic and organellar proteins by stringent water wash. Proteomic profiling was achieved by LC-FTMS, which allowed comparison of differentially expressed or shared proteins under different cell states. First, we analyzed and compared the basal cell components of mouse keratinocytes lacking the cell-cell junction molecule plakoglobin with their control counterparts. Changes in the molecules involved in motility and invasion were detected in plakoglobin-deficient cells, including decreased detection of fibronectin, integrin beta(4), and FAT tumor suppressor. Second, we assessed the differences in basal cell components between two human oral squamous cell carcinoma lines originating from different sites in the oral cavity (CAL33 and UM-SCC-1). The data show differences between the two lines in the type and abundance of proteins specific to cell adhesion, migration, and angiogenesis. Therefore, the method described here has the potential to serve as a platform to assess proteomic changes in basal cell components including extracellular and adhesion-specific proteins involved in wound healing, cancer, and chronic and acquired adhesion-related disorders.
Assuntos
Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Queratinócitos/metabolismo , Espectrometria de Massas/métodos , Proteínas/genética , Proteínas/metabolismo , Hidróxido de Amônia , Animais , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Adesão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/metabolismo , Humanos , Hidróxidos/farmacologia , Queratinócitos/citologia , Queratinócitos/efeitos dos fármacos , Camundongos , Neoplasias Bucais/metabolismo , Neoplasias Bucais/patologia , Peptídeos/química , Peptídeos/metabolismo , gama Catenina/deficiência , gama Catenina/metabolismoRESUMO
The intermediate filament (IF)-binding protein desmoplakin (DP) is essential for desmosome function and tissue integrity, but its role in junction assembly is poorly understood. Using time-lapse imaging, we show that cell-cell contact triggers three temporally overlapping phases of DP-GFP dynamics: (1) the de novo appearance of punctate fluorescence at new contact zones after as little as 3 min; (2) the coalescence of DP and the armadillo protein plakophilin 2 into discrete cytoplasmic particles after as little as 15 min; and (3) the cytochalasin-sensitive translocation of cytoplasmic particles to maturing borders, with kinetics ranging from 0.002 to 0.04 microm/s. DP mutants that abrogate or enhance association with IFs exhibit delayed incorporation into junctions, altering particle trajectory or increasing particle pause times, respectively. Our data are consistent with the idea that DP assembles into nascent junctions from both diffusible and particulate pools in a temporally overlapping series of events triggered by cell-cell contact and regulated by actin and DP-IF interactions.
Assuntos
Actinas/metabolismo , Desmoplaquinas/metabolismo , Filamentos Intermediários/metabolismo , Citoesqueleto de Actina/metabolismo , Animais , Proteínas do Domínio Armadillo/metabolismo , Adesão Celular/fisiologia , Linhagem Celular , Citoplasma/enzimologia , Citoplasma/metabolismo , Desmossomos/metabolismo , Células Epiteliais/metabolismo , Regulação da Expressão Gênica , Humanos , Camundongos , Placofilinas/metabolismo , TransfecçãoRESUMO
By tethering intermediate filaments (IFs) to sites of intercellular adhesion, desmosomes facilitate formation of a supercellular scaffold that imparts mechanical strength to a tissue. However, the role IF-membrane attachments play in strengthening adhesion has not been directly examined. To address this question, we generated Tet-On A431 cells inducibly expressing a desmoplakin (DP) mutant lacking the rod and IF-binding domains (DPNTP). DPNTP localized to the plasma membrane and led to dissociation of IFs from the junctional plaque, without altering total or cell surface distribution of adherens junction or desmosomal proteins. However, a specific decrease in the detergent-insoluble pool of desmoglein suggested a reduced association with the IF cytoskeleton. DPNTP-expressing cell aggregates in suspension or substrate-released cell sheets readily dissociated when subjected to mechanical stress whereas controls remained largely intact. Dissociation occurred without lactate dehydrogenase release, suggesting that loss of tissue integrity was due to reduced adhesion rather than increased cytolysis. JD-1 cells from a patient with a DP COOH-terminal truncation were also more weakly adherent compared with normal keratinocytes. When used in combination with DPNTP, latrunculin A, which disassembles actin filaments and disrupts adherens junctions, led to dissociation up to an order of magnitude greater than either treatment alone. These data provide direct in vitro evidence that IF-membrane attachments regulate adhesive strength and suggest furthermore that actin- and IF-based junctions act synergistically to strengthen adhesion.
Assuntos
Actinas/metabolismo , Membrana Celular/metabolismo , Filamentos Intermediários/metabolismo , Citoesqueleto de Actina/metabolismo , Biotinilação , Caderinas/metabolismo , Adesão Celular , Linhagem Celular , Proteínas do Citoesqueleto/genética , Citoesqueleto/metabolismo , DNA Complementar/metabolismo , Desmogleínas , Desmoplaquinas , Desmossomos/metabolismo , Detergentes/farmacologia , Proteínas de Fluorescência Verde , Humanos , Queratinócitos/metabolismo , Ceratodermia Palmar e Plantar/metabolismo , L-Lactato Desidrogenase/metabolismo , Proteínas Luminescentes/metabolismo , Microscopia de Fluorescência , Ligação Proteica , Estrutura Terciária de Proteína , Fatores de Tempo , Transfecção , Células Tumorais CultivadasRESUMO
Plakophilin 2 (PKP2), a desmosome component, modulates the activity and localization of the small GTPase RhoA at sites of cell-cell contact. PKP2 regulates cortical actin rearrangement during junction formation, and its loss is accompanied by an increase in actin stress fibers. We hypothesized that PKP2 may regulate focal adhesion dynamics and cell migration. Here we show that PKP2-deficient cells bind efficiently to the extracellular matrix, but upon spreading display total cell areas ≈ 30% smaller than control cells. Focal adhesions in PKP2-deficient cells are ≈ 2 × larger and more stable than in control cells, and vinculin displays an increased time for fluorescence recovery after photobleaching. Furthermore, ß4 and ß1 integrin protein and mRNA expression is elevated in PKP2-silenced cells. Normal focal adhesion phenotypes can be restored in PKP2-null cells by dampening the RhoA pathway or silencing ß1 integrin. However, integrin expression levels are not restored by RhoA signaling inhibition. These data uncover a potential role for PKP2 upstream of ß1 integrin and RhoA in integrating cell-cell and cell-substrate contact signaling in basal keratinocytes necessary for the morphogenesis, homeostasis, and reepithelialization of the stratified epidermis.
Assuntos
Movimento Celular/fisiologia , Adesões Focais/fisiologia , Integrina beta1/genética , Integrina beta4/genética , Queratinócitos/fisiologia , Placofilinas/metabolismo , Linhagem Celular , Desmossomos/fisiologia , Células Epiteliais/citologia , Células Epiteliais/fisiologia , Humanos , Integrina beta1/metabolismo , Integrina beta4/metabolismo , Queratinócitos/citologia , Placofilinas/genética , Transdução de Sinais/fisiologia , Cicatrização/fisiologia , Proteína rhoA de Ligação ao GTP/genética , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
Although much is known about signaling factors downstream of Rho GTPases that contribute to epidermal differentiation, little is known about which upstream regulatory proteins (guanine nucleotide exchange factors [GEFs] or GTPase-activating proteins [GAPs]) are involved in coordinating Rho signaling in keratinocytes. Here we identify the GEF breakpoint cluster region (Bcr) as a major upstream regulator of RhoA activity, stress fibers, and focal adhesion formation in keratinocytes. Loss of Bcr reduced expression of multiple markers of differentiation (such as desmoglein-1 [Dsg1], keratin-1, and loricrin) and abrogated MAL/SRF signaling in differentiating keratinocytes. We further demonstrated that loss of Bcr or MAL reduced levels of Dsg1 mRNA in keratinocytes, and ectopic expression of Dsg1 rescued defects in differentiation seen upon loss of Bcr or MAL signaling. Taken together, these data identify the GEF Bcr as a regulator of RhoA/MAL signaling in keratinocytes, which in turn promotes differentiation through the desmosomal cadherin Dsg1.
Assuntos
Diferenciação Celular , Desmogleína 1/metabolismo , Queratinócitos/metabolismo , Proteínas Proteolipídicas Associadas a Linfócitos e Mielina/metabolismo , Proteínas Proto-Oncogênicas c-bcr/metabolismo , Transdução de Sinais , Proteína rhoA de Ligação ao GTP/metabolismo , Linhagem Celular Tumoral , Humanos , Queratinócitos/citologia , RNA Mensageiro/metabolismoRESUMO
Desmosomal cadherins, desmogleins (Dsgs) and desmocollins, make up the adhesive core of intercellular junctions called desmosomes. A critical determinant of epithelial adhesive strength is the level and organization of desmosomal cadherins on the cell surface. The Dsg subclass of desmosomal cadherins contains a C-terminal unique region (Dsg unique region [DUR]) with unknown function. In this paper, we show that the DUR of Dsg2 stabilized Dsg2 at the cell surface by inhibiting its internalization and promoted strong intercellular adhesion. DUR also facilitated Dsg tail-tail interactions. Forced dimerization of a Dsg2 tail lacking the DUR led to decreased internalization, supporting the conclusion that these two functions of the DUR are mechanistically linked. We also show that a Dsg2 mutant, V977fsX1006, identified in arrhythmogenic right ventricular cardiomyopathy patients, led to a loss of Dsg2 tail self-association and underwent rapid endocytosis in cardiac muscle cells. Our observations illustrate a new mechanism desmosomal cadherins use to control their surface levels, a key factor in determining their adhesion and signaling roles.
Assuntos
Desmogleína 2/química , Desmogleína 2/metabolismo , Adesão Celular , Desmogleína 2/genética , Humanos , Mutação , Propriedades de Superfície , Células Tumorais CultivadasRESUMO
The desmosomal cadherins, desmogleins (Dsgs) and desmocollins (Dscs), comprise the adhesive core of intercellular junctions known as desmosomes. Although these adhesion molecules are known to be critical for tissue integrity, mechanisms that coordinate their trafficking into intercellular junctions to regulate their proper ratio and distribution are unknown. We demonstrate that Dsg2 and Dsc2 both exhibit microtubule-dependent transport in epithelial cells but use distinct motors to traffic to the plasma membrane. Functional interference with kinesin-1 blocked Dsg2 transport, resulting in the assembly of Dsg2-deficient junctions with minimal impact on distribution of Dsc2 or desmosomal plaque components. In contrast, inhibiting kinesin-2 prevented Dsc2 movement and decreased its plasma membrane accumulation without affecting Dsg2 trafficking. Either kinesin-1 or -2 deficiency weakened intercellular adhesion, despite the maintenance of adherens junctions and other desmosome components at the plasma membrane. Differential regulation of desmosomal cadherin transport could provide a mechanism to tailor adhesion strength during tissue morphogenesis and remodeling.
Assuntos
Desmocolinas/metabolismo , Desmogleína 2/metabolismo , Desmossomos/metabolismo , Cinesinas/metabolismo , Membrana Celular/metabolismo , Humanos , Junções Intercelulares/metabolismo , Cinesinas/deficiência , Microtúbulos/metabolismo , Ligação Proteica , Transporte Proteico , Células Tumorais Cultivadas , Gravação em VídeoRESUMO
Plakophilin 2 (PKP2), an armadillo family member closely related to p120 catenin (p120ctn), is a constituent of the intercellular adhesive junction, the desmosome. We previously showed that PKP2 loss prevents the incorporation of desmosome precursors enriched in the plaque protein desmoplakin (DP) into newly forming desmosomes, in part by disrupting PKC-dependent regulation of DP assembly competence. On the basis of the observation that DP incorporation into junctions is cytochalasin D-sensitive, here we ask whether PKP2 may also contribute to actin-dependent regulation of desmosome assembly. We demonstrate that PKP2 knockdown impairs cortical actin remodeling after cadherin ligation, without affecting p120ctn expression or localization. Our data suggest that these defects result from the failure of activated RhoA to localize at intercellular interfaces after cell-cell contact and an elevation of cellular RhoA, stress fibers, and other indicators of contractile signaling in squamous cell lines and atrial cardiomyocytes. Consistent with these observations, RhoA activation accelerated DP redistribution to desmosomes during the first hour of junction assembly, whereas sustained RhoA activity compromised desmosome plaque maturation. Together with our previous findings, these data suggest that PKP2 may functionally link RhoA- and PKC-dependent pathways to drive actin reorganization and regulate DP-IF interactions required for normal desmosome assembly.
Assuntos
Actomiosina/metabolismo , Desmossomos/metabolismo , Placofilinas/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Actinas/metabolismo , Animais , Caderinas/metabolismo , Cateninas/metabolismo , Comunicação Celular , Linhagem Celular , Linhagem Celular Tumoral , Citoesqueleto/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Junções Intercelulares/metabolismo , Microscopia de Fluorescência , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Cadeias Leves de Miosina/metabolismo , Placofilinas/genética , Ligação Proteica , Proteína Quinase C/metabolismo , Interferência de RNA , Transdução de Sinais , delta CateninaRESUMO
Regulation of classic cadherins plays a critical role in tissue remodeling during development and cancer; however, less attention has been paid to the importance of desmosomal cadherins. We previously showed that EGFR inhibition results in accumulation of the desmosomal cadherin, desmoglein 2 (Dsg2), at cell-cell interfaces accompanied by inhibition of matrix metalloprotease (MMP)-dependent shedding of the Dsg2 ectodomain and tyrosine phosphorylation of its cytoplasmic domain. Here, we show that EGFR inhibition stabilizes Dsg2 at intercellular junctions by interfering with its accumulation in an internalized cytoplasmic pool. Furthermore, MMP inhibition and ADAM17 RNAi, blocked shedding and depleted internalized Dsg2, but less so E-cadherin, in highly invasive SCC68 cells. ADAM9 and 15 silencing also impaired Dsg2 processing, supporting the idea that this desmosomal cadherin can be regulated by multiple ADAM family members. In contrast, ADAM10 siRNA enhanced accumulation of a 100-kDa Dsg2 cleavage product and internalized pool of Dsg2. Although both MMP and EGFR inhibition increased intercellular adhesive strength in control cells, the response to MMP-inhibition was Dsg2-dependent. These data support a role for endocytic trafficking in regulating desmosomal cadherin turnover and function and raise the possibility that internalization and regulation of desmosomal and classic cadherin function can be uncoupled mechanistically.
Assuntos
Proteínas ADAM/metabolismo , Desmogleína 2/metabolismo , Desmossomos/metabolismo , Endocitose/fisiologia , Receptores ErbB/metabolismo , Isoenzimas/metabolismo , Proteínas ADAM/genética , Animais , Adesão Celular/fisiologia , Linhagem Celular Tumoral , Desmogleína 2/genética , Receptores ErbB/genética , Humanos , Junções Intercelulares/metabolismo , Isoenzimas/genética , Metaloproteinases da Matriz/genética , Metaloproteinases da Matriz/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Plakophilins (PKPs) are armadillo family members related to the classical cadherin-associated protein p120(ctn). PKPs localize to the cytoplasmic plaque of intercellular junctions and participate in linking the intermediate filament (IF)-binding protein desmoplakin (DP) to desmosomal cadherins. In response to cell-cell contact, PKP2 associates with DP in plaque precursors that form in the cytoplasm and translocate to nascent desmosomes. Here, we provide evidence that PKP2 governs DP assembly dynamics by scaffolding a DP-PKP2-protein kinase C alpha (PKC alpha) complex, which is disrupted by PKP2 knockdown. The behavior of a phosphorylation-deficient DP mutant that associates more tightly with IF is mimicked by PKP2 and PKC alpha knockdown and PKC pharmacological inhibition, all of which impair junction assembly. PKP2 knockdown is accompanied by increased phosphorylation of PKC substrates, raising the possibility that global alterations in PKC signaling may contribute to pathogenesis of congenital defects caused by PKP2 deficiency.
Assuntos
Desmossomos/enzimologia , Placofilinas/metabolismo , Proteína Quinase C-alfa/metabolismo , Linhagem Celular , Desmoplaquinas/metabolismo , Desmossomos/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Humanos , Modelos Biológicos , Transporte Proteico/efeitos dos fármacos , Serina/metabolismo , Transdução de Sinais/efeitos dos fármacos , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
Although a number of cell adhesion proteins have been identified as caspase substrates, the potential role of differentiation-specific desmosomal cadherins during apoptosis has not been examined. Here, we demonstrate that UV-induced caspase cleavage of the human desmoglein 1 cytoplasmic tail results in distinct 17- and 140- kDa products, whereas metalloproteinase-dependent shedding of the extracellular adhesion domain generates a 75-kDa product. In vitro studies identify caspase-3 as the preferred enzyme that cleaves desmoglein 1 within its unique repeating unit domain at aspartic acid 888, part of a consensus sequence not conserved among the other desmosomal cadherins. Apoptotic processing leads to decreased cell surface expression of desmoglein 1 and re-localization of its C terminus diffusely throughout the cytoplasm over a time course comparable with the processing of other desmosomal proteins and cytoplasmic keratins. Importantly, whereas classic cadherins have been reported to promote cell survival, short hairpin RNA-mediated suppression of desmoglein 1 in differentiated keratinocytes protected cells from UV-induced apoptosis. Collectively, our results identify desmoglein 1 as a novel caspase and metalloproteinase substrate whose cleavage likely contributes to the dismantling of desmosomes during keratinocyte apoptosis and also reveal desmoglein 1 as a previously unrecognized regulator of apoptosis in keratinocytes.
Assuntos
Apoptose , Caspases/metabolismo , Desmogleína 1/fisiologia , Regulação Enzimológica da Expressão Gênica , Queratinócitos/enzimologia , Sítios de Ligação , Western Blotting , Caspase 3 , Diferenciação Celular , Linhagem Celular Tumoral , Citoplasma/metabolismo , DNA Complementar/metabolismo , Desmogleína 1/metabolismo , Desmossomos/metabolismo , Doxiciclina/farmacologia , Humanos , Indóis/farmacologia , Queratinócitos/metabolismo , Microscopia de Fluorescência , Mutação , Regiões Promotoras Genéticas , Ligação Proteica , Estrutura Terciária de Proteína , RNA/química , Retroviridae/genética , Fatores de Tempo , Transfecção , Raios UltravioletaRESUMO
Desmoglein 1 (Dsg1) is a component of desmosomes present in the upper epidermis and can be targeted by autoimmune antibodies or bacterial toxins, resulting in skin blistering diseases. These defects in tissue integrity are believed to result from compromised desmosomal adhesion; yet, previous attempts to directly test the adhesive roles of desmosomal cadherins using normally non-adherent L cells have yielded mixed results. Here, two complementary approaches were used to better resolve the molecular determinants for Dsg1-mediated adhesion: (1) a tetracycline-inducible system was used to modulate the levels of Dsg1 expressed in L cell lines containing desmocollin 1 (Dsc1) and plakoglobin (PG) and (2) a retroviral gene delivery system was used to introduce Dsg1 into normal human epidermal keratinocytes (NHEK). By increasing Dsg1 expression relative to Dsc1 and PG, we were able to demonstrate that the ratio of Dsg1:Dsc1 is a critical determinant of desmosomal adhesion in fibroblasts. The distribution of Dsg1 was organized at areas of cell-cell contact in the multicellular aggregates that formed in these suspension cultures. Similarly, the introduction of Dsg1 into NHEKs was capable of increasing the aggregation of single cell suspensions and further enhanced the adhesive strength of intact epithelial sheets. Endogenous Dsc1 levels were also increased in NHEKs containing Dsg1, providing further support for the coordination of these two desmosomal cadherins in regulating adhesive structures. These Dsg1-mediated effects on intercellular adhesion were directly related to the presence of an intact extracellular domain as ETA, a toxin that specifically cleaves this desmosomal cadherin, inhibited adhesion in both fibroblasts and keratinocytes. Collectively, these observations demonstrate that Dsg1 promotes the formation of intercellular adhesion complexes and suggest that the relative level of Dsg and Dsc expressed at the cell surface regulates this adhesive process.