Advertisement

Super-Resolution Microscopy Reveals Altered Desmosomal Protein Organization in Tissue from Patients with Pemphigus Vulgaris

      Pemphigus vulgaris (PV) is an autoimmune epidermal blistering disease in which autoantibodies (IgG) are directed against the desmosomal cadherin desmoglein 3. To better understand how PV IgG alters desmosome morphology and function in vivo, biopsies from patients with PV were analyzed by structured illumination microscopy, a form of superresolution fluorescence microscopy. In patient tissue, desmosomal proteins were aberrantly clustered and patient IgG colocalized with markers for lipid rafts and endosomes. Additionally, steady-state levels of desmoglein 3 were decreased and desmosomes were reduced in size in patient tissue. Desmosomes at blister sites were occasionally split, with PV IgG decorating the extracellular faces of split desmosomes. Desmosome splitting was recapitulated in vitro by exposing cultured keratinocytes both to PV IgG and to mechanical stress, demonstrating that splitting at the blister interface in patient tissue is due to compromised desmosomal adhesive function. These findings indicate that desmoglein 3 clustering and endocytosis are associated with reduced desmosome size and adhesion defects in tissue of patients with PV. Further, this study reveals that superresolution optical imaging is a powerful approach for studying epidermal adhesion structures in normal and diseased skin.

      Abbreviations:

      DP (desmoplakin), Dsg3 (desmoglein 3), IgG (antibodies), NH (normal human), PV (pemphigus vulgaris)

      Introduction

      The desmosome is an intercellular junction that mediates robust adhesion between neighboring cells (
      • Berika M.
      • Garrod D.
      Desmosomal adhesion in vivo.
      ,
      • Kowalczyk A.P.
      • Green K.J.
      Structure, function, and regulation of desmosomes.
      ). Desmosomes are present in all epithelia, but are especially prominent in the skin and the heart (
      • Berika M.
      • Garrod D.
      Desmosomal adhesion in vivo.
      ,
      • Desai B.V.
      • Harmon R.M.
      • Green K.J.
      Desmosomes at a glance.
      ). These tissues experience a high degree of mechanical stress, and for this reason, desmosome dysfunction leads to skin and heart defects that are often characterized by tissue fragility (
      • Al-Jassar C.
      • Bikker H.
      • Overduin M.
      • et al.
      Mechanistic basis of desmosome-targeted diseases.
      ,
      • Cirillo N.
      150th anniversary series: desmosomes in physiology and disease.
      ,
      • Kottke M.D.
      • Delva E.
      • Kowalczyk A.P.
      The desmosome: cell science lessons from human diseases.
      ,
      • Thomason H.A.
      • Scothern A.
      • McHarg S.
      • Garrod D.R.
      Desmosomes: adhesive strength and signalling in health and disease.
      ). Desmosomes comprise desmosomal cadherins, which engage in extracellular adhesive interactions, and cytoplasmic plaque proteins, including plakoglobin, plakophilin, and desmoplakin (DP), which mediate linkages to the intermediate filament cytoskeleton. Desmogleins (Dsg) and desmocollins are desmosomal transmembrane proteins and members of the cadherin superfamily of adhesion molecules. The armadillo family protein plakoglobin links the cytoplasmic tail of the cadherins to DP, an intermediate filament binding protein and member of the plakin family of cytolinkers. Plakophilin, also an armadillo protein, facilitates lateral interactions between desmosomal cadherin complexes and further strengthens the plaque (
      • Desai B.V.
      • Harmon R.M.
      • Green K.J.
      Desmosomes at a glance.
      ,
      • Kowalczyk A.P.
      • Green K.J.
      Structure, function, and regulation of desmosomes.
      ). This architectural arrangement couples desmosomal cadherin adhesive interactions to the intermediate filament cytoskeleton, thereby establishing an integrated adhesive and cytoskeletal network that extends throughout a tissue (
      • Kowalczyk A.P.
      • Green K.J.
      Structure, function, and regulation of desmosomes.
      ).
      Desmosome assembly and disassembly dynamics must be precisely controlled to allow not only for strong adhesion and tissue integrity, but also for plasticity during processes such as wound healing and development (
      • Nekrasova O.
      • Green K.J.
      Desmosome assembly and dynamics.
      ). Alterations in desmosome dynamics are thought to contribute to desmosome disruption and loss of adhesion in disease states, including the autoimmune blistering disease pemphigus vulgaris (PV) (
      • Kitajima Y.
      New insights into desmosome regulation and pemphigus blistering as a desmosome-remodeling disease.
      ,
      • Kitajima Y.
      150(th) anniversary series: desmosomes and autoimmune disease, perspective of dynamic desmosome remodeling and its impairments in pemphigus.
      ). Pemphigus is a family of potentially fatal bullous diseases caused by autoantibodies directed against the extracellular domain of desmosomal cadherins (
      • Amagai M.
      The molecular logic of pemphigus and impetigo: the desmoglein story.
      ,
      • Amagai M.
      Autoimmune and infectious skin diseases that target desmogleins.
      ). Patients with PV produce IgG autoantibodies that target Dsg3, or both Dsg3 and Dsg1 (
      • Amagai M.
      The molecular logic of pemphigus and impetigo: the desmoglein story.
      ). PV IgG binding to the desmosomal cadherins results in the loss of keratinocyte adhesion, termed acantholysis, between the basal and spinous layers of the epidermis (
      • Amagai M.
      Autoimmune and infectious skin diseases that target desmogleins.
      ). Clinically, the disease presents as painful mucosal erosions and epidermal blisters (
      • Kneisel A.
      • Hertl M.
      Autoimmune bullous skin diseases. Part 1: Clinical manifestations.
      ). Although it has been established that anti-Dsg3 antibodies are sufficient to cause disease, the precise pathomechanisms by which PV IgG cause loss of adhesion and blister formation are not fully understood.
      Much of what we know about PV pathomechanisms comes from in vitro work in which cultured keratinocytes are exposed to PV IgG. Previous experiments by our group and others using cell culture models have revealed that PV IgG cause aberrant clustering of cell surface Dsg3, leading to increased endocytosis and decreased steady-state levels of Dsg3 (
      • Calkins C.C.
      • Setzer S.V.
      • Jennings J.M.
      • et al.
      Desmoglein endocytosis and desmosome disassembly are coordinated responses to pemphigus autoantibodies.
      ,
      • Iwatsuki K.
      • Takigawa M.
      • Imaizumi S.
      • Yamada M.
      In vivo binding site of pemphigus vulgaris antibodies and their fate during acantholysis.
      ,
      • Iwatsuki K.
      • Han G.W.
      • Fukuti R.
      • et al.
      Internalization of constitutive desmogleins with the subsequent induction of desmoglein 2 in pemphigus lesions.
      ,
      • Jolly P.S.
      • Berkowitz P.
      • Bektas M.
      • et al.
      p38MAPK signaling and desmoglein-3 internalization are linked events in pemphigus acantholysis.
      ,
      • Mao X.
      • Sano Y.
      • Park J.M.
      • Payne A.S.
      p38 MAPK activation is downstream of the loss of intercellular adhesion in pemphigus vulgaris.
      ,
      • Mao X.
      • Li H.
      • Sano Y.
      • Gaestel M.
      • Mo Park J.
      • Payne A.S.
      MAPKAP kinase 2 (MK2)-dependent and -independent models of blister formation in pemphigus vulgaris.
      ,
      • Patel H.P.
      • Diaz L.A.
      • Anhalt G.J.
      • Labib R.S.
      • Takahashi Y.
      Demonstration of pemphigus antibodies on the cell surface of murine epidermal cell monolayers and their internalization.
      ,
      • Saito M.
      • Stahley S.N.
      • Caughman C.Y.
      • et al.
      Signaling dependent and independent mechanisms in pemphigus vulgaris blister formation.
      ,
      • Sato M.
      • Aoyama Y.
      • Kitajima Y.
      Assembly pathway of desmoglein 3 to desmosomes and its perturbation by pemphigus vulgaris-IgG in cultured keratinocytes, as revealed by time-lapsed labeling immunoelectron microscopy.
      ). In response to PV IgG in vitro, Dsg3 and other desmosomal components rearrange into streaks, or linear arrays, which extend perpendicularly from cell-cell borders (
      • Jennings J.M.
      • Tucker D.K.
      • Kottke M.D.
      • et al.
      Desmosome disassembly in response to pemphigus vulgaris IgG occurs in distinct phases and can be reversed by expression of exogenous Dsg3.
      ). These structures appear to be associated with lipid raft-mediated endocytosis of the Dsg3-PV IgG complex (
      • Delva E.
      • Jennings J.M.
      • Calkins C.C.
      • Kottke M.D.
      • Faundez V.
      • Kowalczyk A.P.
      Pemphigus vulgaris IgG-induced desmoglein-3 endocytosis and desmosomal disassembly are mediated by a clathrin- and dynamin-independent mechanism.
      ,
      • Stahley S.N.
      • Saito M.
      • Faundez V.
      • Koval M.
      • Mattheyses A.L.
      • Kowalczyk A.P.
      Desmosome assembly and disassembly are membrane raft-dependent.
      ). Furthermore, multiple signaling pathways have been implicated in PV pathogenesis, including tyrosine kinases and p38MAPK-dependent pathways (
      • Getsios S.
      • Huen A.C.
      • Green K.J.
      Working out the strength and flexibility of desmosomes.
      ,
      • Kitajima Y.
      150(th) anniversary series: desmosomes and autoimmune disease, perspective of dynamic desmosome remodeling and its impairments in pemphigus.
      ,
      • Koga H.
      • Tsuruta D.
      • Ohyama B.
      • et al.
      Desmoglein 3, its pathogenecity and a possibility for therapeutic target in pemphigus vulgaris.
      ,
      • Sharma P.
      • Mao X.
      • Payne A.S.
      Beyond steric hindrance: the role of adhesion signaling pathways in the pathogenesis of pemphigus.
      ,
      • Waschke J.
      The desmosome and pemphigus.
      ). There is also substantial evidence suggesting that steric hindrance of Dsg3-mediated adhesion causes keratinocyte acantholysis. For example, the majority of IgG isolated from patients with PV are directed against the amino terminal domain of Dsg3, a region of cadherins known to mediate adhesion (
      • Amagai M.
      • Karpati S.
      • Prussick R.
      • Klaus-Kovtun V.
      • Stanley J.R.
      Autoantibodies against the amino-terminal cadherin-like binding domain of pemphigus vulgaris antigen are pathogenic.
      ,
      • Di Zenzo G.
      • Di Lullo G.
      • Corti D.
      • et al.
      Pemphigus autoantibodies generated through somatic mutations target the desmoglein-3 cis-interface.
      ,
      • Patel S.D.
      • Chen C.P.
      • Bahna F.
      • Honig B.
      • Shapiro L.
      Cadherin-mediated cell-cell adhesion: sticking together as a family.
      ,
      • Sekiguchi M.
      • Futei Y.
      • Fujii Y.
      • Iwasaki T.
      • Nishikawa T.
      • Amagai M.
      Dominant autoimmune epitopes recognized by pemphigus antibodies map to the N-terminal adhesive region of desmogleins.
      ,
      • Shapiro L.
      • Weis W.I.
      Structure and biochemistry of cadherins and catenins.
      ). Further, monoclonal Dsg3 antibodies that target the adhesive interface cause loss of adhesion in cultured keratinocytes and blistering in mouse models of disease (
      • Payne A.S.
      • Ishii K.
      • Kacir S.
      • et al.
      Genetic and functional characterization of human pemphigus vulgaris monoclonal autoantibodies isolated by phage display.
      ,
      • Tsunoda K.
      • Ota T.
      • Aoki M.
      • et al.
      Induction of pemphigus phenotype by a mouse monoclonal antibody against the amino-terminal adhesive interface of desmoglein 3.
      ). In aggregate, these studies suggest that multiple mechanisms, perhaps acting synergistically, contribute to PV pathogenesis. However, previous studies have not directly compared the desmosomal alterations in the skin of patients with PV to those previously observed in cell culture models.
      A significant deficit in our understanding of PV pathomechanisms in patients is due to the difficulty in assessing the organization and localization of the desmosomal proteins in multiple patients at high levels of spatial resolution. Although electron microscopy provides high-resolution ultrastructural information, it is limited by difficult sample preparation, small sample size, and the challenges associated with quantitatively assessing the colocalization of various desmosomal proteins with other cellular antigens. In contrast, optical microscopy approaches such as wide-field and confocal immunofluorescence allow for high throughput of samples and detection of multiple proteins, but lack the spatial resolution sufficient to discern how desmosomal structures are altered in disease states such as PV.
      Here, we take advantage of recent advances in optical imaging approaches that bridge the resolution gap between electron microscopy and standard immunofluorescence imaging. Using structured illumination microscopy (SIM), a form of superresolution optical imaging, we assessed how desmosomes are altered in patients with PV. Further, we directly compared the desmosomal alterations observed in patients with PV with the PV IgG-induced alterations that occur in vitro. Our results indicate that desmosomal protein clustering and Dsg endocytosis occur in patient tissue and that these changes are associated with reduced desmosome size and desmosome splitting at the adhesive interface. We further demonstrate that desmosome splitting can be replicated in cultured keratinocytes by exposing PV IgG-treated cells to mechanical stress. These findings reveal new insights into the changes in desmosomal protein organization, trafficking, and function that occur in tissue of patients with PV, and serve to validate the use of purified PV IgG and cultured keratinocytes as an invaluable model system to understand PV pathomechanisms.

      Results and Discussion

      PV IgG cause desmosomal protein clustering in patient tissue and in cultured keratinocytes

      Desmosomal protein organization in the basal layer of the epidermis of normal human (NH, N1-3) and PV (P1-6) patient tissue was analyzed using SIM, a form of superresolution microscopy that doubles the resolution afforded by conventional light microscopy (Supplementary Figure S1 online). Patients with clinically active disease and high titers of Dsg3 autoantibodies (Dsg3 ELISA >100) were selected for analysis. Mucocutaneous tissue from patients with PV was analyzed using biopsies taken from both lip and skin (Supplementary Table S1 online).
      Biopsies from NH epidermis were negative for hIgG deposition (Figure 1a, top panel). SIM imaging of NH tissue revealed uniform organization of Dsg3, Dsg1, DP, and E-cadherin along cell borders (Figure 1a, top panel). In contrast, the skin of patients with PV exhibited hIgG deposition and disrupted Dsg3, Dsg1, and DP organization (Figure 1a, bottom panel; Supplementary Figure S3 online). Importantly, E-cadherin staining remained largely unchanged (Figure 1a, bottom panel). Alterations in junctional protein organization were quantified using a clustering index measuring the average distance between fluorescence puncta along cell borders (
      • Saito M.
      • Stahley S.N.
      • Caughman C.Y.
      • et al.
      Signaling dependent and independent mechanisms in pemphigus vulgaris blister formation.
      ; see the Materials and Methods section). Desmoglein and DP clustering were significantly increased in patients with PV, with Dsg3 and DP displaying the greatest alterations relative to controls (Figure 1b; clustering scores from individual patients are displayed in Supplementary Figure S2 online). E-cadherin distribution was also altered, although to a lesser degree than observed for desmosomal proteins. The small but statistically significant changes in E-cadherin organization are likely an indirect consequence of desmosome disruption in the skin of patients with PV, because alterations in adherens junctions also have been observed in desmoplakin knockout keratinocytes (
      • Vasioukhin V.
      • Bowers E.
      • Bauer C.
      • Degenstein L.
      • Fuchs E.
      Desmoplakin is essential in epidermal sheet formation.
      ). The organization of plakoglobin also displayed altered organization in tissue of patients with PV (Supplementary Figure S4 online). These data support previous observations that desmosomal proteins are clustered in biopsies from patients with pemphigus (
      • Oktarina D.A.
      • van der Wier G.
      • Diercks G.F.
      • Jonkman M.F.
      • Pas H.H.
      IgG-induced clustering of desmogleins 1 and 3 in skin of patients with pemphigus fits with the desmoglein nonassembly depletion hypothesis.
      ). Last, we verified that the IgG from each patient analyzed in Figure 1 caused similar alterations in Dsg3 organization in vitro. Similar to patient tissue, Dsg3 was clustered in response to IgG from all six patients with PV tested (Figure 1c). Collectively, these results demonstrate that clustering of desmosomal proteins is a hallmark feature of both PV IgG–treated cells and tissue of patients with PV.
      Figure 1
      Figure 1Desmosomal proteins are clustered in patients with PV as assessed using SIM and immunofluorescence analysis. (a) Top panel: normal human (NH) epidermal tissue is negative for IgG (hIgG) deposition, and junctional proteins desmoglein 3 (Dsg3), desmoglein 1 (Dsg1), desmoplakin (DP), and E-cadherin (Ecad) are uniformly distributed along cell borders. Bottom panel: epidermis of patient with PV is positive for hIgG deposition and border localization of Dsg3, Dsg1, and DP is disorganized and clustered. Ecad staining is largely unaltered. D, dermis. Solid line, epidermis-dermis interface. Images oriented dermis down. Images from N1-3 and P1-2. Scale bar = 5 μm. (b) Quantification of protein clustering. Means ± SEM; *P < 0.05. Mean clustering index for each junctional protein represents data derived from two to three NH biopsies and three to six biopsies from patients with PV and the analysis of 135–850 μm of cell border length per patient. (c) Quantification of average Dsg3 clustering using purified patient IgG (P1-6) added to cultured keratinocytes. Means ± SEM; *P < 0.05. Mean clustering index was derived from 765 to 1,830 μm of cell border length per IgG sample. IgG, antibodies; PV, pemphigus vulgaris; SIM, structured illumination microscopy.

      PV IgG associates with lipid raft markers in vivo and is trafficked to endosomes

      Previous studies have determined that desmosome disassembly and endocytosis occur in a lipid raft-dependent manner (
      • Delva E.
      • Jennings J.M.
      • Calkins C.C.
      • Kottke M.D.
      • Faundez V.
      • Kowalczyk A.P.
      Pemphigus vulgaris IgG-induced desmoglein-3 endocytosis and desmosomal disassembly are mediated by a clathrin- and dynamin-independent mechanism.
      ,
      • Stahley S.N.
      • Saito M.
      • Faundez V.
      • Koval M.
      • Mattheyses A.L.
      • Kowalczyk A.P.
      Desmosome assembly and disassembly are membrane raft-dependent.
      ). In vivo, patient IgG (hIgG) colocalized with raft markers CD59 and caveolin-1 both at cell borders and in vesicular-like puncta (Figure 2). A number of studies using cell culture models have found that the PV IgG-Dsg complex is internalized from the plasma membrane via lipid raft-mediated endocytosis, resulting in degradation of Dsg3 and reduced plasma membrane levels of the adhesive protein (
      • Calkins C.C.
      • Setzer S.V.
      • Jennings J.M.
      • et al.
      Desmoglein endocytosis and desmosome disassembly are coordinated responses to pemphigus autoantibodies.
      ,
      • Cirillo N.
      • Gombos F.
      • Lanza A.
      Changes in desmoglein 1 expression and subcellular localization in cultured keratinocytes subjected to anti-desmoglein 1 pemphigus autoimmunity.
      ,
      • Delva E.
      • Jennings J.M.
      • Calkins C.C.
      • Kottke M.D.
      • Faundez V.
      • Kowalczyk A.P.
      Pemphigus vulgaris IgG-induced desmoglein-3 endocytosis and desmosomal disassembly are mediated by a clathrin- and dynamin-independent mechanism.
      ,
      • Jolly P.S.
      • Berkowitz P.
      • Bektas M.
      • et al.
      p38MAPK signaling and desmoglein-3 internalization are linked events in pemphigus acantholysis.
      ,
      • Kitajima Y.
      150(th) anniversary series: desmosomes and autoimmune disease, perspective of dynamic desmosome remodeling and its impairments in pemphigus.
      ,
      • Mao X.
      • Sano Y.
      • Park J.M.
      • Payne A.S.
      p38 MAPK activation is downstream of the loss of intercellular adhesion in pemphigus vulgaris.
      ,
      • Mao X.
      • Li H.
      • Sano Y.
      • Gaestel M.
      • Mo Park J.
      • Payne A.S.
      MAPKAP kinase 2 (MK2)-dependent and -independent models of blister formation in pemphigus vulgaris.
      ,
      • Saito M.
      • Stahley S.N.
      • Caughman C.Y.
      • et al.
      Signaling dependent and independent mechanisms in pemphigus vulgaris blister formation.
      ,
      • Sato M.
      • Aoyama Y.
      • Kitajima Y.
      Assembly pathway of desmoglein 3 to desmosomes and its perturbation by pemphigus vulgaris-IgG in cultured keratinocytes, as revealed by time-lapsed labeling immunoelectron microscopy.
      ,
      • Schulze K.
      • Galichet A.
      • Sayar B.S.
      • et al.
      An adult passive transfer mouse model to study desmoglein 3 signaling in pemphigus vulgaris.
      ). To determine if PV IgG is internalized to endocytic compartments in patient tissue, biopsy sections were stained for hIgG and the early endosomal marker early endosomal antigen-1. Nearly every cell, particularly near sites of blister formation, displayed multiple puncta at or near the cell periphery containing hIgG and early endosomal antigen-1 (Figure 3a). Interestingly, steady-state Dsg3 levels were decreased in tissue from patients with PV compared with NH control tissue (Figure 3b; Supplementary Figure S5 online), further suggesting that PV IgG induces Dsg3 endocytosis and degradation in vivo.
      Figure 2
      Figure 2IgG of patients with PV colocalizes with lipid raft markers in vivo. (a) Colocalization of PV patient IgG (hIgG) with lipid raft markers CD59 (top panel) and caveolin-1 (middle panel), and non-raft marker clathrin (bottom panel) in biopsies from tissue of patients with PV. Colocalization is observed both at cell borders and in vesicular-like structures (asterisks). Note lack of clathrin colocalization in vesicular puncta. Images from P5. Scale bar = 5 μm. (b) Quantification of hIgG colocalization (Mander’s coefficient) with membrane markers at cell-cell borders. Means ± SEM, *P < 0.05; n = at least 54 cell borders from biopsies derived from 3 different patients. (c) Quantification of vesicular hIgG colocalization (Mander’s coefficient) with membrane markers. Means ± SEM; *P < 0.05; n = 30 vesicles analyzed from three patient biopsies. IgG, antibodies; PV, pemphigus vulgaris.
      Figure 3
      Figure 3IgG of patients with PV is internalized in vivo and Dsg3 levels are reduced. (a) PV IgG (hIgG) colocalizes with early endosomal marker EEA1. Numbered enlargements highlight vesicular colocalization. Images from P5. Scale bar = 2 μm. (b) Quantification of Dsg3 protein levels relative to adherens junction protein p120 in normal and PV patient tissue. Image J was used to analyze wide-field microscopy images of tissue. Following background subtraction, Dsg3 intensity was normalized to p120 levels using Image J. Means ± SEM; *P < 0.05. Data were collected from three NH biopsies and five biopsies from patients with PV. Average fluorescence intensity measurements were derived from the analysis of 6–10 images from each biopsy. Dsg3, desmoglein 3; EEA1, early endosomal antigen-1; IgG, antibodies; PV, pemphigus vulgaris; NH, normal human.

      Desmosomes are smaller and split in patients with PV

      Ultrastructural studies of desmosome morphology in patients with PV and mouse models have suggested that desmosomes either split at the adhesive interface or are reduced in size (
      • Shimizu A.
      • Ishiko A.
      • Ota T.
      • Tsunoda K.
      • Amagai M.
      • Nishikawa T.
      IgG binds to desmoglein 3 in desmosomes and causes a desmosomal split without keratin retraction in a pemphigus mouse model.
      ,
      • van der Wier G.
      • Pas H.H.
      • Kramer D.
      • Diercks G.F.
      • Jonkman M.F.
      Smaller desmosomes are seen in the skin of pemphigus patients with anti-desmoglein 1 antibodies but not in patients with anti-desmoglein 3 antibodies.
      ). We utilized SIM imaging to assess desmosome morphology in patient tissue and directly compared these changes with cultured keratinocytes exposed to patient IgG. We took advantage of information from previous immuno-gold EM studies defining the position of various desmosomal proteins (
      • North A.J.
      • Bardsley W.G.
      • Hyam J.
      • et al.
      Molecular map of the desmosomal plaque.
      ) and the subdiffraction limit resolution of SIM to identify bona fide desmosomes in tissues and cells by immunofluorescence localization. Using this approach, antibodies directed against the carboxyl terminal domain of DP result in a “railroad track” pattern of fluorescence that defines the localization of the inner desmosomal plaque, whereas antibodies against the Dsg extracellular domain identify the adhesive core (Figure 4a–c).
      Figure 4
      Figure 4Desmosomes in patients with PV are smaller and split. (a) Desmosome schematic depicting staining with N-terminal Dsg3 antibody (hIgG in patient tissue) and a C-terminal desmoplakin (DP) antibody. (b) SIM resolves the plaque-to-plaque distance and desmosomes appear as regions of parallel DP staining, or “railroad” tracks, along a cell border (1) or a sandwich of DP-Dsg3-DP staining (2). Split desmosomes appear as regions of green-red staining (3). (c) In vivo examples of (b). Scale bar = 0.3 μm. (d–f) Normal human (NH) biopsies and biopsies from patients with PV stained as detailed above and imaged by SIM. Railroad tracks were used to identify and measure desmosome size. (d) DP staining. Arrows indicating intercellular space highlight smaller desmosomes. Images from N3 and P1. Scale bar = 0.5 μm. (e) Many split (or half) desmosomes with IgG staining (asterisks) are observed adjacent to the blister space. Small desmosomes (arrowheads) were also observed. DP (green), hIgG (red). Images from P5. Scale bar = 0.5 μm. (f) Quantification of desmosome size. Means ± SEM; *P < 0.05 comparing PV-whole or PV-split to NH; measurements were obtained from 6 to 163 desmosomes per patient; data from three NH controls and six patients with PV. Dsg3, desmoglein 3; PV, pemphigus vulgaris.
      This pattern of DP railroad track staining was used to identify and measure desmosome size in human tissue. Desmosomes in NH samples averaged 0.43 μm in size, whereas desmosomes in patients with PV were significantly smaller, averaging 0.35 μm (Figure 4d and f). This observation is consistent with recent electron microscopy studies of patients with PV (
      • van der Wier G.
      • Pas H.H.
      • Kramer D.
      • Diercks G.F.
      • Jonkman M.F.
      Smaller desmosomes are seen in the skin of pemphigus patients with anti-desmoglein 1 antibodies but not in patients with anti-desmoglein 3 antibodies.
      ) and analysis of desmosomes in cultured keratinocytes treated with PV IgG (
      • Saito M.
      • Stahley S.N.
      • Caughman C.Y.
      • et al.
      Signaling dependent and independent mechanisms in pemphigus vulgaris blister formation.
      ,
      • Tucker D.K.
      • Stahley S.N.
      • Kowalczyk A.P.
      Plakophilin-1 protects keratinocytes from pemphigus vulgaris IgG by forming calcium-independent desmosomes.
      ). Interestingly, SIM revealed three types of fluorescence staining patterns in patient tissue (Figure 4b–c and e). First, when DP railroad track staining was intact, we often observed hIgG deposition in the desmosome core (red) that was bracketed by DP staining on opposing keratinocyte membranes (green). Second, a few instances of DP railroad track staining devoid of hIgG deposition in the desmosomal core were also observed, possibly indicating Dsg-depleted desmosomes (Supplementary Figure S6 online). Third, we observed numerous split desmosomes in which hIgG was adjacent to only one “rail” of DP (Figure 4e, asterisks), indicating desmosome splitting. The presence of split desmosomes in patients and mouse models of PV has been attributed to pathogenic antibodies physically interfering with, or sterically hindering, desmosomal cadherin adhesive interactions (
      • Amagai M.
      • Karpati S.
      • Prussick R.
      • Klaus-Kovtun V.
      • Stanley J.R.
      Autoantibodies against the amino-terminal cadherin-like binding domain of pemphigus vulgaris antigen are pathogenic.
      ,
      • Sharma P.
      • Mao X.
      • Payne A.S.
      Beyond steric hindrance: the role of adhesion signaling pathways in the pathogenesis of pemphigus.
      ,
      • Stahley S.N.
      • Kowalczyk A.P.
      Desmosomes in acquired disease.
      ,
      • Tsunoda K.
      • Ota T.
      • Aoki M.
      • et al.
      Induction of pemphigus phenotype by a mouse monoclonal antibody against the amino-terminal adhesive interface of desmoglein 3.
      ). If steric hindrance were the sole mechanism for loss of adhesion, we would predict that desmosomes would be split but remain otherwise unchanged morphologically. Interestingly, the majority of split desmosomes in tissue of patients with PV were also found to be smaller than intact desmosomes in NH tissue (Figure 4f). The observation that desmosomes were often found to be smaller and occasionally depleted of Dsg supports a pathomechanism model for PV that includes altered desmosome assembly or disassembly dynamics.

      Mechanical stress causes desmosome splitting

      Our analysis of patient tissue using SIM revealed striking similarities in desmosomal protein organization and trafficking in cultured keratinocyte models and tissue of patients with PV. However, split desmosomes have not been reported in cell culture models of disease, possibly because unlike patient tissue, cultured keratinocytes are not exposed to mechanical stress. To test the hypothesis that mechanical stress causes desmosome splitting, cultured keratinocyte monolayers were subjected to a dispase cell fragmentation assay (
      • Ishii K.
      • Harada R.
      • Matsuo I.
      • Shirakata Y.
      • Hashimoto K.
      • Amagai M.
      In vitro keratinocyte dissociation assay for evaluation of the pathogenicity of anti-desmoglein 3 IgG autoantibodies in pemphigus vulgaris.
      ) and then processed for immunofluorescence analysis by SIM. Similar to NH tissue, the keratinocyte cell sheet exposed to NH IgG remained adhesive and numerous intact desmosomes were observed (Figure 5). As expected, keratinocytes exposed to PV IgG dissociated on exposure to mechanical stress. SIM analysis of the free edge of the fragmented cell sheet exposed to PV IgG revealed extensive desmosome splitting (Figure 5) that was strikingly similar to that observed at the blister edge in biopsies from patients with PV (Figure 4e). These results indicate that the application of mechanical stress causes desmosomes that are weakened by PV IgG to split at the adhesive interface.
      Figure 5
      Figure 5Mechanical stress causes desmosome splitting in cultured keratinocytes exposed to PV IgG. Keratinocyte monolayers exposed to either normal human (NH) or PV IgG were subjected to mechanical stress by repeated pipetting and then processed for SIM. The keratinocyte sheet exposed to NH IgG displays intact desmosomes at cell borders indicated by the green-red-green “railroad” track staining pattern (top panel). The free edge of the fragmented sheet exposed to PV IgG exhibits multiple split desmosomes (bottom panel) indicated by only a green-red staining. IgG, antibodies; PV, pemphigus vulgaris; SIM, structured illumination microscopy. Scale bar = 0.5 μm.

      Conclusions and implications

      In this study, superresolution immunofluorescence microscopy revealed changes in desmosomal protein distribution and trafficking that occur in epidermis of patients with PV. We compared these changes in patient tissue with those that occur in cultured keratinocytes exposed to PV IgG, and have identified hallmark features of the disease that occur both in vivo and in vitro. In both cases, desmosomal proteins become clustered, enter lipid raft–enriched linear arrays, and are internalized to endosomes. These organizational changes are accompanied by a reduction in steady-state Dsg3 levels as well as a reduction in desmosome size. Superresolution imaging also revealed examples of individual desmosomes in patient tissue that were depleted of desmogleins. It is likely that these changes, along with the ability of PV IgG to sterically interfere with desmosomal cadherin adhesion, compromise desmosome function, resulting in mechanical failure on exposure to mechanical stress. Indeed, desmosome splitting was recapitulated in vitro by exposing PV IgG-treated keratinocytes to physical forces. Altogether, these results provide further support for a multifactorial model in which PV IgG weaken cell adhesion by altering desmosomal protein distribution, by perturbing the dynamics of desmosome assembly and/or disassembly, and by sterically interfering with desmosome assembly and adhesion (
      • Kitajima Y.
      New insights into desmosome regulation and pemphigus blistering as a desmosome-remodeling disease.
      ,
      • Kitajima Y.
      150(th) anniversary series: desmosomes and autoimmune disease, perspective of dynamic desmosome remodeling and its impairments in pemphigus.
      ,
      • Stahley S.N.
      • Kowalczyk A.P.
      Desmosomes in acquired disease.
      ). Finally, this study provides a foundation for using advanced optical imaging techniques to investigate alterations in adhesion structures in a variety of epidermal diseases, and for the development of new optical imaging-based diagnostic metrics for pemphigus and related disorders.

      Materials and Methods

      Human subjects statement

      The use of human IgG and skin biopsies was approved by the Institutional Review Board at Emory University. Guidelines set forth in the Declaration of Helsinki were adhered to, and written informed consent was obtained from all participants.

      Antibodies

      The following antibodies were used in this study: mouse anti-Dsg3 antibody AK15 (
      • Tsunoda K.
      • Ota T.
      • Aoki M.
      • et al.
      Induction of pemphigus phenotype by a mouse monoclonal antibody against the amino-terminal adhesive interface of desmoglein 3.
      ) was a kind gift from Dr Masayuki Amagai (Keio University, Tokyo); rabbit anti-desmoplakin antibody NW6 was a kind gift from Dr Kathleen Green (Northwestern University); mouse anti-Dsg1 antibody P124 (Progen Biotechnik GmbH, Heidelberg); mouse anti-desmoplakin I/II antibody (Fitzgerald, Acton, MA); rabbit anti-γ-catenin (plakoglobin, H-80) and rabbit anti-p120 antibodies (Santa Cruz Biotechnology, Santa Cruz, CA); mouse anti-E-cadherin (HECD-1, Abcam, Cambridge, MA); mouse anti-CD59-FITC conjugated antibody (Invitrogen, Grand Island, NY); rabbit anti-caveolin-1 antibody (BD Biosciences, San Jose, CA); rabbit anti-early endosomal antigen-1 antibody (Thermo Scientific, Waltham, MA). Secondary antibodies conjugated to Alexa Fluors were purchased from Invitrogen. PV sera (used in Figure 5) was a generous gift from Dr M. Amagai. Sera of patients with PV used in all other figures were obtained from patients seen at Emory University, Department of Dermatology. IgG was purified from PV sera according to the manufacturer’s protocol using Melon Gel IgG Purification Resins and Kits (Thermo Fisher Scientific, Rockford, IL).

      Human tissue biopsy processing

      Perilesional biopsies (mucosa lip or skin) from six patients with mucocutaneous PV seen at the Emory Clinic Dermatology Department were collected and stored at −80 °C. Sections (5 μm) from the biopsies were mounted onto glass slides and processed for immunostaining as described below.

      Cells and culture conditions

      Primary human keratinocytes (HKs, passage 2 or 4) were isolated as previously described (
      • Calkins C.C.
      • Setzer S.V.
      • Jennings J.M.
      • et al.
      Desmoglein endocytosis and desmosome disassembly are coordinated responses to pemphigus autoantibodies.
      ) and cultured in the KBM-Gold basal medium (100 μM calcium) supplemented with a KGM-Gold Single-Quot Kit (Lonza, Walkersville, MD). For Figure 1, HKs were cultured to 70% confluence on glass coverslips and switched to 550 μM calcium for 16–18 hours to induce junction assembly. HKs were exposed to NH IgG or IgG from patients with PV for 6 hours at 37 °C, processed for wide-field immunofluorescence and then analyzed for clustering as described below. For the dispase assay in Figure 5, HKs were cultured to 100% confluence in four-well tissue culture plates and switched to 50 μM calcium to prevent any junction assembly for 16–18 hours before switching to 550 μM calcium for 3 hours to allow for junction assembly. HKs were exposed to NH or PV IgG for 3 hours at 37 °C and processed for a dispase fragmentation assay followed by SIM, as described below.

      Immunofluorescence

      Patient tissue slices were allowed to come to room temperature and immunostained with primary and secondary antibodies for 1 hour each at room temperature with triple PBS+ washes between antibody incubations. HKs in Figure 1 were fixed in methanol and processed for immunofluorescence. Primary antibodies described above and patient IgG present in tissues were detected with Alexa Fluor-conjugated secondary antibodies. Widefield fluorescence microscopy was performed as previously described (
      • Stahley S.N.
      • Saito M.
      • Faundez V.
      • Koval M.
      • Mattheyses A.L.
      • Kowalczyk A.P.
      Desmosome assembly and disassembly are membrane raft-dependent.
      ). Superresolution SIM was performed using the N-SIM system equipped with a ×100/1.49 NA oil immersion objective and 488- and 561-nm solid-state lasers. Three-dimensional SIM images were captured with an EM charge-coupled device camera (DU-897, Andor Technology, Belfast, UK) and reconstructed using the NIS-Elements software with the N-SIM module (version 3.22, Nikon, Melville, NY). Colocalization analysis via Mander’s coefficient was performed using ImageJ Fiji and plugin JACoP (
      • Bolte S.
      • Cordelieres F.P.
      A guided tour into subcellular colocalization analysis in light microscopy.
      ).

      Clustering analysis

      Protein clustering was measured as previously described (
      • Saito M.
      • Stahley S.N.
      • Caughman C.Y.
      • et al.
      Signaling dependent and independent mechanisms in pemphigus vulgaris blister formation.
      ). Briefly, lines were drawn along cell borders to measure fluorescence intensity using ImageJ/Fiji (NIH, Bethesda, MD). A custom-designed MATLAB program identified all local maxima (peaks) with an intensity at least half the maximum intensity. The clustering index is defined as 1 over the number of peaks per 5 μm distance.

      Dispase-based fragmentation assay with immunofluorescence

      After PV IgG treatment, cells were subjected to a dispase fragmentation assay as previously described (
      • Saito M.
      • Stahley S.N.
      • Caughman C.Y.
      • et al.
      Signaling dependent and independent mechanisms in pemphigus vulgaris blister formation.
      ). In parallel, cell sheets and/or fragments were fixed in paraformaldehyde, permeablized in Triton X-100 and incubated with primary and secondary antibodies in the tissue culture wells. Gentle washes with PBS+ were carried out between Triton and antibody incubations. Cell sheets and/or fragments were then mounted in ProLong Gold (Molecular Probes, Eugene, OR) and then imaged by SIM.

      Statistics

      Statistical analysis comparing the PV group to NH was performed using a t-test, assuming unequal variances with a significance level of α = 0.05. For Figure 2b–c, pairwise multiple comparisons were performed via the Holm-Sidak method with a significance level of α = 0.05.

      Conflict of Interest

      The authors state no conflict of interest.

      Acknowledgments

      We acknowledge members of the Kowalczyk Laboratory for their insightful advice and discussions, and especially Susan Summers for keratinocyte isolations. We greatly appreciate the assistance of Bridget Bradley (Emory Dermatology) and Sue Manos (Emory Pathology) in obtaining and processing pemphigus patient samples. This work was conducted using funding and instrumentation made available through the Integrated Cellular Imaging Core (ICI) of Emory University. This work was supported by NIH R01AR048266 to APK and R21AR066920 to ALM. SNS was supported by NIH T32GM008367. MFW was supported by NIH UL1TR000454.

      Supplementary Material

      References

        • Al-Jassar C.
        • Bikker H.
        • Overduin M.
        • et al.
        Mechanistic basis of desmosome-targeted diseases.
        J Mol Biol. 2013; 425: 4006-4022
        • Amagai M.
        The molecular logic of pemphigus and impetigo: the desmoglein story.
        Vet Dermatol. 2009; 20: 308-312
        • Amagai M.
        Autoimmune and infectious skin diseases that target desmogleins.
        Proc Jpn Acad Ser B Phys Biol Sci. 2010; 86: 524-537
        • Amagai M.
        • Karpati S.
        • Prussick R.
        • Klaus-Kovtun V.
        • Stanley J.R.
        Autoantibodies against the amino-terminal cadherin-like binding domain of pemphigus vulgaris antigen are pathogenic.
        J Clin Invest. 1992; 90: 919-926
        • Berika M.
        • Garrod D.
        Desmosomal adhesion in vivo.
        Cell Commun Adhes. 2014; 21: 65-75
        • Bolte S.
        • Cordelieres F.P.
        A guided tour into subcellular colocalization analysis in light microscopy.
        J Microsc. 2006; 224: 213-232
        • Calkins C.C.
        • Setzer S.V.
        • Jennings J.M.
        • et al.
        Desmoglein endocytosis and desmosome disassembly are coordinated responses to pemphigus autoantibodies.
        J Biol Chem. 2006; 281: 7623-7634
        • Cirillo N.
        150th anniversary series: desmosomes in physiology and disease.
        Cell Commun Adhes. 2014; 21: 85-88
        • Cirillo N.
        • Gombos F.
        • Lanza A.
        Changes in desmoglein 1 expression and subcellular localization in cultured keratinocytes subjected to anti-desmoglein 1 pemphigus autoimmunity.
        J Cell Physiol. 2007; 210: 411-416
        • Delva E.
        • Jennings J.M.
        • Calkins C.C.
        • Kottke M.D.
        • Faundez V.
        • Kowalczyk A.P.
        Pemphigus vulgaris IgG-induced desmoglein-3 endocytosis and desmosomal disassembly are mediated by a clathrin- and dynamin-independent mechanism.
        J Biol Chem. 2008; 283: 18303-18313
        • Desai B.V.
        • Harmon R.M.
        • Green K.J.
        Desmosomes at a glance.
        J Cell Sci. 2009; 122: 4401-4407
        • Di Zenzo G.
        • Di Lullo G.
        • Corti D.
        • et al.
        Pemphigus autoantibodies generated through somatic mutations target the desmoglein-3 cis-interface.
        J Clin Invest. 2012; 122: 3781-3790
        • Getsios S.
        • Huen A.C.
        • Green K.J.
        Working out the strength and flexibility of desmosomes.
        Nature Rev Mol Cell Biol. 2004; 5: 271-281
        • Ishii K.
        • Harada R.
        • Matsuo I.
        • Shirakata Y.
        • Hashimoto K.
        • Amagai M.
        In vitro keratinocyte dissociation assay for evaluation of the pathogenicity of anti-desmoglein 3 IgG autoantibodies in pemphigus vulgaris.
        J Invest Dermatol. 2005; 124: 939-946
        • Iwatsuki K.
        • Han G.W.
        • Fukuti R.
        • et al.
        Internalization of constitutive desmogleins with the subsequent induction of desmoglein 2 in pemphigus lesions.
        Br J Dermatol. 1999; 140: 35-43
        • Iwatsuki K.
        • Takigawa M.
        • Imaizumi S.
        • Yamada M.
        In vivo binding site of pemphigus vulgaris antibodies and their fate during acantholysis.
        J Am Acad Dermatol. 1989; 20: 578-582
        • Jennings J.M.
        • Tucker D.K.
        • Kottke M.D.
        • et al.
        Desmosome disassembly in response to pemphigus vulgaris IgG occurs in distinct phases and can be reversed by expression of exogenous Dsg3.
        J Invest Dermatol. 2011; 131: 706-718
        • Jolly P.S.
        • Berkowitz P.
        • Bektas M.
        • et al.
        p38MAPK signaling and desmoglein-3 internalization are linked events in pemphigus acantholysis.
        J Biol Chem. 2010; 285: 8936-8941
        • Kitajima Y.
        New insights into desmosome regulation and pemphigus blistering as a desmosome-remodeling disease.
        Kaohsiung J Med Sci. 2013; 29: 1-13
        • Kitajima Y.
        150(th) anniversary series: desmosomes and autoimmune disease, perspective of dynamic desmosome remodeling and its impairments in pemphigus.
        Cell Commun Adhes. 2014; 21: 269-280
        • Kneisel A.
        • Hertl M.
        Autoimmune bullous skin diseases. Part 1: Clinical manifestations.
        J Dtsch Dermatol Ges. 2011; 9: 844-856
        • Koga H.
        • Tsuruta D.
        • Ohyama B.
        • et al.
        Desmoglein 3, its pathogenecity and a possibility for therapeutic target in pemphigus vulgaris.
        Expert Opin Ther Targets. 2013; 17: 293-306
        • Kottke M.D.
        • Delva E.
        • Kowalczyk A.P.
        The desmosome: cell science lessons from human diseases.
        J Cell Sci. 2006; 119: 797-806
        • Kowalczyk A.P.
        • Green K.J.
        Structure, function, and regulation of desmosomes.
        Prog Mol Biol Transl Sci. 2013; 116: 95-118
        • Mao X.
        • Li H.
        • Sano Y.
        • Gaestel M.
        • Mo Park J.
        • Payne A.S.
        MAPKAP kinase 2 (MK2)-dependent and -independent models of blister formation in pemphigus vulgaris.
        J Invest Dermatol. 2014; 134: 68-76
        • Mao X.
        • Sano Y.
        • Park J.M.
        • Payne A.S.
        p38 MAPK activation is downstream of the loss of intercellular adhesion in pemphigus vulgaris.
        J Biol Chem. 2011; 286: 1283-1291
        • Nekrasova O.
        • Green K.J.
        Desmosome assembly and dynamics.
        Trends Cell Biol. 2013; 23: 537-546
        • North A.J.
        • Bardsley W.G.
        • Hyam J.
        • et al.
        Molecular map of the desmosomal plaque.
        J Cell Sci. 1999; 112: 4325-4336
        • Oktarina D.A.
        • van der Wier G.
        • Diercks G.F.
        • Jonkman M.F.
        • Pas H.H.
        IgG-induced clustering of desmogleins 1 and 3 in skin of patients with pemphigus fits with the desmoglein nonassembly depletion hypothesis.
        Br J Dermatol. 2011; 165: 552-562
        • Patel H.P.
        • Diaz L.A.
        • Anhalt G.J.
        • Labib R.S.
        • Takahashi Y.
        Demonstration of pemphigus antibodies on the cell surface of murine epidermal cell monolayers and their internalization.
        J Invest Dermatol. 1984; 83: 409-415
        • Patel S.D.
        • Chen C.P.
        • Bahna F.
        • Honig B.
        • Shapiro L.
        Cadherin-mediated cell-cell adhesion: sticking together as a family.
        Curr Opin Struct Biol. 2003; 13: 690-698
        • Payne A.S.
        • Ishii K.
        • Kacir S.
        • et al.
        Genetic and functional characterization of human pemphigus vulgaris monoclonal autoantibodies isolated by phage display.
        J Clin Invest. 2005; 115: 888-899
        • Saito M.
        • Stahley S.N.
        • Caughman C.Y.
        • et al.
        Signaling dependent and independent mechanisms in pemphigus vulgaris blister formation.
        PloS One. 2012; 7: e50696
        • Sato M.
        • Aoyama Y.
        • Kitajima Y.
        Assembly pathway of desmoglein 3 to desmosomes and its perturbation by pemphigus vulgaris-IgG in cultured keratinocytes, as revealed by time-lapsed labeling immunoelectron microscopy.
        Lab Invest. 2000; 80: 1583-1592
        • Schulze K.
        • Galichet A.
        • Sayar B.S.
        • et al.
        An adult passive transfer mouse model to study desmoglein 3 signaling in pemphigus vulgaris.
        J Invest Dermatol. 2012; 132: 346-355
        • Sekiguchi M.
        • Futei Y.
        • Fujii Y.
        • Iwasaki T.
        • Nishikawa T.
        • Amagai M.
        Dominant autoimmune epitopes recognized by pemphigus antibodies map to the N-terminal adhesive region of desmogleins.
        J Immunol. 2001; 167: 5439-5448
        • Shapiro L.
        • Weis W.I.
        Structure and biochemistry of cadherins and catenins.
        Cold Spring Harb Perspect Biol. 2009; 1: a003053
        • Sharma P.
        • Mao X.
        • Payne A.S.
        Beyond steric hindrance: the role of adhesion signaling pathways in the pathogenesis of pemphigus.
        J Dermatol Sci. 2007; 48: 1-14
        • Shimizu A.
        • Ishiko A.
        • Ota T.
        • Tsunoda K.
        • Amagai M.
        • Nishikawa T.
        IgG binds to desmoglein 3 in desmosomes and causes a desmosomal split without keratin retraction in a pemphigus mouse model.
        J Invest Dermatol. 2004; 122: 1145-1153
        • Stahley S.N.
        • Kowalczyk A.P.
        Desmosomes in acquired disease.
        Cell Tissue Res. 2015; 360: 439-456
        • Stahley S.N.
        • Saito M.
        • Faundez V.
        • Koval M.
        • Mattheyses A.L.
        • Kowalczyk A.P.
        Desmosome assembly and disassembly are membrane raft-dependent.
        PloS One. 2014; 9: e87809
        • Thomason H.A.
        • Scothern A.
        • McHarg S.
        • Garrod D.R.
        Desmosomes: adhesive strength and signalling in health and disease.
        Biochem J. 2010; 429: 419-433
        • Tsunoda K.
        • Ota T.
        • Aoki M.
        • et al.
        Induction of pemphigus phenotype by a mouse monoclonal antibody against the amino-terminal adhesive interface of desmoglein 3.
        J Immunol. 2003; 170: 2170-2178
        • Tucker D.K.
        • Stahley S.N.
        • Kowalczyk A.P.
        Plakophilin-1 protects keratinocytes from pemphigus vulgaris IgG by forming calcium-independent desmosomes.
        J Invest Dermatol. 2014; 134: 1033-1043
        • van der Wier G.
        • Pas H.H.
        • Kramer D.
        • Diercks G.F.
        • Jonkman M.F.
        Smaller desmosomes are seen in the skin of pemphigus patients with anti-desmoglein 1 antibodies but not in patients with anti-desmoglein 3 antibodies.
        J Invest Dermatol. 2014; 134: 2287-2290
        • Vasioukhin V.
        • Bowers E.
        • Bauer C.
        • Degenstein L.
        • Fuchs E.
        Desmoplakin is essential in epidermal sheet formation.
        Nature Cell Biol. 2001; 3: 1076-1085
        • Waschke J.
        The desmosome and pemphigus.
        Histochem Cell Biol. 2008; 130: 21-54