Deficient plakophilin-1 expression due to a mutation in PKP1 causes ectodermal dysplasia-skin fragility syndrome in Chesapeake Bay retriever dogs

In humans, congenital and hereditary skin diseases associated with epidermal cell-cell separation (acantholysis) are very rare, and spontaneous animal models of these diseases are exceptional. Our objectives are to report a novel congenital acantholytic dermatosis that developed in Chesapeake Bay retriever dogs. Nine affected puppies in four different litters were born to eight closely related clinically normal dogs. The disease transmission was consistent with an autosomal recessive mode of inheritance. Clinical signs occurred immediately after birth with superficial epidermal layers sloughing upon pressure. At three month of age, dogs exhibited recurrent superficial skin sloughing and erosions at areas of friction and mucocutaneous junctions; their coat was also finer than normal and there were patches of partial hair loss. At birth, histopathology revealed severe suprabasal acantholysis, which became less severe with ageing. Electron microscopy demonstrated a reduced number of partially formed desmosomes with detached and aggregated keratin intermediate filaments. Immunostaining for desmosomal adhesion molecules revealed a complete lack of staining for plakophilin-1 and anomalies in the distribution of desmoplakin and keratins 10 and 14. Sequencing revealed a homozygous splice donor site mutation within the first intron of PKP1 resulting in a premature stop codon, thereby explaining the inability to detect plakophilin-1 in the skin. Altogether, the clinical and pathological findings, along with the PKP1 mutation, were consistent with the diagnosis of ectodermal dysplasia-skin fragility syndrome with plakophilin-1 deficiency. This is the first occurrence of ectodermal dysplasia-skin fragility syndrome in an animal species. Controlled mating of carrier dogs would yield puppies that could, in theory, be tested for gene therapy of this rare but severe skin disease of children.     

The desmoplakin carboxyl terminus coaligns with and specifically disrupts intermediate filament networks when expressed in cultured cells

Specific interactions between desmoplakins I and II (DP I and II) and other desmosomal or cytoskeletal molecules have been difficult to determine in part because of the complexity and insolubility of the desmosome and its constituents. We have used a molecular genetic approach to investigate the role that DP I and II may play in the association of the desmosomal plaque with cytoplasmic intermediate filaments (IF). A series of mammalian expression vectors encoding specific predicted domains of DP I were transiently expressed in cultured cells that form (COS-7) and do not form (NIH-3T3) desmosomes. Sequence encoding a small antigenic peptide was added to the 3' end of each mutant DP cDNA to facilitate immunolocalization of mutant DP protein. Light and electron microscopical observations revealed that DP polypeptides including the 90-kD carboxy-terminal globular domain of DP I specifically colocalized with and ultimately resulted in the complete disruption of IF in both cell lines. This effect was specific for IF as microtubule and microfilament networks were unaltered. This effect was also specific for the carboxyl terminus of DP, as the expression of the 95-kD rod domain of DP I did not visibly alter IF networks. Immunogold localization of COS-7 cells transfected with constructs including the carboxyl terminus of DP demonstrated an accumulation of mutant protein in perinuclear aggregates within which IF subunits were sequestered. These results suggest a role for the DP carboxyl terminus in the attachment of IF to the desmosome in either a direct or indirect manner.     

Rescuing desmoplakin function in extra-embryonic ectoderm reveals the importance of this protein in embryonic heart, neuroepithelium, skin and vasculature

Desmosomes mediate intercellular adhesion through desmosomal cadherins, which interface with plakoglobin (PG) and desmoplakin (DP) to associate with the intermediate filament (IF) cytoskeleton. Desmosomes first assemble in the E3.5 mouse trophectoderm, concomitant with establishment of epithelial polarity and appearance of a blastocoel cavity. Increasing in size and number, desmosomes continue their prominence in extra-embryonic tissues, but as development proceeds, they also become abundant in a number of embryonic tissues, including heart muscle, epidermis and neuroepithelium. Previously, we explored the functional importance of desmosomes by ablating the Dsp gene. Homozygous Dsp mutant embryos progressed through implantation, but did not survive beyond E6.5, owing to a loss or instability of desmosomes and tissue integrity. We have now rescued the extra-embryonic tissues by aggregation of tetraploid (wild-type) and diploid (Dsp mutant) morulae. These animals survive several days longer, but die shortly after gastrulation, with major defects in the heart muscle, neuroepithelium and skin epithelium, all of which possess desmosomes, as well as the microvasculature, which does not. Interestingly, although wild-type endothelial cells of capillaries do not form desmosomes, they possess unusual intercellular junctions composed of DP, PG and VE-cadherin. The severity in phenotype and the breadth of defects in the Dsp mutant embryo is greater than PG mutant embryos, substantiating redundancy between PG and other armadillo proteins (e.g. beta-catenin). The timing of lethality is similar to that of the VE-cadherin null embryo, suggesting that a participating cause of death may be a defect in vasculature, not reported for PG null embryos.     

Carboxyl terminus of Plakophilin-1 recruits it to plasma membrane, whereas amino terminus recruits desmoplakin and promotes desmosome assembly

Plakophilins are armadillo repeat-containing proteins, initially identified as desmosomal plaque proteins that have subsequently been shown to also localize to the nucleus. Loss of plakophilin-1 is the underlying cause of ectodermal dysplasia/skin fragility syndrome, and skin from these patients exhibits desmosomes that are reduced in size and number. Thus, it has been suggested that plakophilin-1 plays an important role in desmosome stability and/or assembly. In this study, we used a cell culture system (A431DE cells) that expresses all of the proteins necessary to assemble a desmosome, except plakophilin-1. Using this cell line, we sought to determine the role of plakophilin-1 in de novo desmosome assembly. When exogenous plakophilin-1 was expressed in these cells, desmosomes were assembled, as assessed by electron microscopy and immunofluorescence localization of desmoplakin, into punctate structures. Deletion mutagenesis experiments revealed that amino acids 686-726 in the carboxyl terminus of plakophilin-1 are required for its localization to the plasma membrane. In addition, we showed that amino acids 1-34 in the amino terminus were necessary for subsequent recruitment of desmoplakin to the membrane and desmosome assembly.     

Mice expressing a mutant desmosomal cadherin exhibit abnormalities in desmosomes, proliferation, and epidermal differentiation

Desmogleins are members of the cadherin superfamily which form the core of desmosomes. In vitro studies indicate that the cytoplasmic domain of desmogleins associates with plakoglobin; however, little is known about the role of this domain in desmosome recognition or assembly in vivo, or about the possible relation of desmoglein mutations to epidermal differentiation and disease. To address these questions we used transgenic mouse technology to produce an NH2-terminally truncated desmoglein (Pemphigus Vulgaris Antigen or Dsg3) in cells known to express its wild-type counterpart. Within 2 d, newborn transgenic animals displayed swelling of their paws, flakiness on their back, and blackening of the tail tip. When analyzed histologically and ultrastructurally, widening of intercellular spaces and disruption of desmosomes were especially striking in the paws and tail. Desmosomes were reduced dramatically in number and were smaller and often peculiar in structure. Immunofluorescence and immunoelectron microscopy revealed no major abnormalities in localization of hemidesmosomal components, but desmosomal components organized aberrantly, resulting in a loss of ultrastructure within the plaque. In regions where desmosome loss was prevalent but where some adhesive structures persisted, the epidermis was thickened, with a marked increase in spinous and stratum corneum layers, variability in granular layer thickness, and parakeratosis in some regions. Intriguingly, a dramatic increase in cell proliferation was also observed concomitant with biochemical changes, including alterations in integrin expression, known to be associated with hyperproliferation. An inflammatory response was also detected in some skin regions. Collectively, these findings demonstrate that a mutation in a desmoglein can perturb epidermal cell-cell adhesion, triggering a cascade of changes in the skin.     

Decreased extracellular deposition of fibrillin and decorin in neonatal Marfan syndrome fibroblasts

Abnormalities of the microfibrillar protein fibrillin (Fib) have been reported in Marfan syndrome (MFS). The so-called neonatal Marfan syndrome (nMFS) is a lethal phenotype displaying features that are not seen in classical MFS. We have therefore studied the biosynthesis and extracellular deposition of Fib and decorin in fibroblasts from a patient with nMFS and controls. Immunofluorescence of the patient's cell cultures showed an almost complete absence of Fib and a marked reduction of decorin in the extracellular matrix (ECM). The nMFS skin revealed Fib on subbasal microfibrillar bundles in the papillary dermis, and Fib associated with elastic fibers in the reticular dermis; the bundles and fibers were fragmented and thinner than normal. Pulse-chase labeling of cells with [³⁵S]Met/Cys revealed moderately reduced secretion, but a diminished deposition of Fib in the ECM; this was more apparent at a longer chase time. Fib mRNA and synthesis appeared to be normal, where-as both decorin mRNA and biosynthesis were reduced. We therefore assume a structural Fib defect in this patient causing reduced deposition into and/or enhanced removal from the ECM, whereas the reduced decorin biosynthesis may be a secondary regulatory phenomenon. The clinical relevance of this remains unclear. Our findings imply that Fib defects may be responsible for the severe, complex phenotype of nMFS.     

Extracellularly truncated desmoglein 1 compromises desmosomes in MDCK cells

The formation and stability of epithelial tissue involves cell adhesion and the connection of the intermediate filaments of contiguous cells, mediated by desmosomes. The cadherin family members Desmocollins (Dsc) and Desmogleins (Dsg) mediate desmosome extracellular adhesion. The main intracellular molecules identified linking Dscs and Dsgs with the intermediate filament network are Plakoglobin (PG), Plakophilins (PPs) and Desmoplakin (DP). Previous studies on desmosome-mediated adhesion have focused on the intracellular domains of Dsc and Dsg because of their capacity to interact with PG, PPs and DP. This study examines the role of the extracellular domain of Dsg1 upon desmosome stability in MDCK cells. Dsg1 was constructed containing an extracellular deletion (Dsg delta 1EC) and was expressed in MDCK cells. A high expressor Dsg delta 1EC/MDCK clone was obtained and analysed for its capacity to form desmosomes in cell monolayers and when growing under mechanical stress in three-dimensional collagen cultures. Phenotypic changes associated with the ectopic expression of Dsg1 delta EC in MDCK cells were: disturbance of the cytokeratin network, a change in the quality and number of desmosomes and impairment of the formation of cysts in suspension cultures. Interestingly, Dsg1 delta EC was not localized in desmosomes, but was still able to maintain its intracytoplasmic interaction with PG, suggesting that the disruptive effects were largely due to PG and/or PP sequestration.     

Assessment of splice variant-specific functions of desmocollin 1 in the skin

Desmocollin 1 (Dsc1) is part of a desmosomal cell adhesion receptor formed in terminally differentiating keratinocytes of stratified epithelia. The dsc1 gene encodes two proteins (Dsc1a and Dsc1b) that differ only with respect to their COOH-terminal cytoplasmic amino acid sequences. On the basis of in vitro experiments, it is thought that the Dsc1a variant is essential for assembly of the desmosomal plaque, a structure that connects desmosomes to the intermediate filament cytoskeleton of epithelial cells. We have generated mice that synthesize a truncated Dsc1 receptor that lacks both the Dsc1a- and Dsc1b-specific COOH-terminal domains. This mutant transmembrane receptor, which does not bind the common desmosomal plaque proteins plakoglobin and plakophilin 1, is integrated into functional desmosomes. Interestingly, our mutant mice did not show the epidermal fragility previously observed in dsc1-null mice. This suggests that neither the Dsc1a- nor the Dsc1b-specific COOH-terminal cytoplasmic domain is required for establishing and maintaining desmosomal adhesion. However, a comparison of our mutants with dsc1-null mice suggests that the Dsc1 extracellular domain is necessary to maintain structural integrity of the skin.     

Defining the Interactions Between Intermediate Filaments and Desmosomes

Desmoplakin (DP), plakoglobin (PG), and plakophilin 1 (PP1) are desmosomal components lacking a transmembrane domain, thus making them candidate linker proteins for connecting intermediate filaments and desmosomes. Using deletion and site-directed mutagenesis, we show that remarkably, removal of ~1% of DP's sequence obliterates its ability to associate with desmosomes. Conversely, when linked to a foreign protein, as few as 86 NH₂-terminal DP residues are sufficient to target to desmosomes efficiently. In in vitro overlay assays, the DP head specifically associates with itself and with desmocollin 1a (Dsc1a). In similar overlay assays, PP1 binds to DP and Dsc1a, and to a lesser extent, desmoglein 1 (Dsg1), while PG binds to Dsg1 and more weakly to Dsc1a and DP. Interestingly, like DP, PG and PP1 associate with epidermal keratins, although PG is considerably weaker in its ability to do so. As judged by overlay assays, the amino terminal head domain of type II keratins appears to have a special importance in establishing these connections. Taken together, our findings provide new insights into the complexities of the links between desmosomes and intermediate filaments (IFs). Our results suggest a model whereby at desmosome sites within dividing epidermal cells, DP and PG anchor to desmosomal cadherins and to each other, forming an ordered array of nontransmembrane proteins that then bind to keratin IFs. As epidermal cells differentiate, PP1 is added as a molecular reinforcement to the plaque, enhancing anchorage to IFs and accounting at least partially for the increase in numbers and stability of desmosomes in suprabasal cells.     

Single-cell sequencing reveals the new existence form of dermal papilla cells in the hair follicle regeneration of cashmere goats

The problem of human hair loss has caused widespread concern, however, such research is difficult because the periodicity is not obvious and the deeper levels knowledge of dermal papilla (DP) stem cells' differentiation are limited. Here, cashmere goats which have obvious periodicity of hair follicles were used, based on unbiased scRNA sequencing, we constructed DP cell lineage differentiation trajectory and revealed the key genes, signals and functions involved in cell fate decisions. And then we revealed the molecular landscape of hair follicle on regeneration. Revealed that DP cells differentiate into four intermediate cell states at different periodicity: Intermediate-cell-10 showed important functions in the growth and maintenance of cashmere; intermediate-cell-1 acting on apoptosis and cashmere shedding; intermediate-cell-0 initiated new follicular cycles, the migration of hair follicles and the occurrence of cashmere; and intermediate-cell-15 are suggested to be DP progenitor cells. In general, we provide new insights for hair regrowth. At the same time, it provides a new research ideas, directions and molecular landscape for the mechanism of dermal papilla cells.     

Making a connection: direct binding between keratin intermediate filaments and desmosomal proteins

In epidermal cells, keratin intermediate filaments connect with desmosomes to form extensive cadherin-mediated cytoskeletal architectures. Desmoplakin (DPI), a desmosomal component lacking a transmembrane domain, has been implicated in this interaction, although most studies have been conducted with cells that contain few or no desmosomes, and efforts to demonstrate direct interactions between desmoplakin and intermediate filaments have not been successful. In this report, we explore the biochemical nature of the connections between keratin filaments and desmosomes in epidermal keratinocytes. We show that the carboxy terminal "tail" of DPI associates directly with the amino terminal "head" of type II epidermal keratins, including K1, K2, K5, and K6. We have engineered and purified recombinant K5 head and DPI tail, and we demonstrate direct interaction in vitro by solution- binding assays and by ligand blot assays. This marked association is not seen with simple epithelial type II keratins, vimentin, or with type I keratins, providing a possible explanation for the greater stability of the epidermal keratin filament architecture over that of other cell types. We have identified an 18-amino acid residue stretch in the K5 head that is conserved only among type II epidermal keratins and that appears to play some role in DPI tail binding. This finding might have important implications for understanding a recent point mutation found within this binding site in a family with a blistering skin disorder.     

Regulation of intestinal epithelial intercellular adhesion and barrier function by desmosomal cadherin desmocollin-2

The role of desmosomal cadherin desmocollin-2 (Dsc2) in regulating barrier function in intestinal epithelial cells (IECs) is not well understood. Here, we report the consequences of silencing Dsc2 on IEC barrier function in vivo using mice with inducible intestinal–epithelial-specific Dsc2 knockdown (KD) (Dsc2^ERΔIEC). While the small intestinal gross architecture was maintained, loss of epithelial Dsc2 influenced desmosomal plaque structure, which was smaller in size and had increased intermembrane space between adjacent epithelial cells. Functional analysis revealed that loss of Dsc2 increased intestinal permeability in vivo, supporting a role for Dsc2 in the regulation of intestinal epithelial barrier function. These results were corroborated in model human IECs in which Dsc2 KD resulted in decreased cell–cell adhesion and impaired barrier function. It is noteworthy that Dsc2 KD cells exhibited delayed recruitment of desmoglein-2 (Dsg2) to the plasma membrane after calcium switch-induced intercellular junction reassembly, while E-cadherin accumulation was unaffected. Mechanistically, loss of Dsc2 increased desmoplakin (DP I/II) protein expression and promoted intermediate filament interaction with DP I/II and was associated with enhanced tension on desmosomes as measured by a Dsg2-tension sensor. In conclusion, we provide new insights on Dsc2 regulation of mechanical tension, adhesion, and barrier function in IECs.     

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

The desmosomal plaque protein desmoplakin (DP), located at the juncture between the intermediate filament (IF) network and the cytoplasmic tails of the transmembrane desmosomal cadherins, has been proposed to link IF to the desmosomal plaque. Consistent with this hypothesis, previous studies of individual DP domains indicated that the DP COOH terminus associates with IF networks whereas NH2-terminal sequences govern the association of DP with the desmosomal plaque. Nevertheless, it had not yet been demonstrated that DP is required for attaching IF to the desmosome. To test this proposal directly, we generated A431 cell lines stably expressing DP NH2-terminal polypeptides, which were expected to compete with endogenous DP during desmosome assembly. As these polypeptides lacked the COOH-terminal IF-binding domain, this competition should result in the loss of IF anchorage if DP is required for linking IF to the desmosomal plaque. In such cells, a 70-kD DP NH2- terminal polypeptide (DP-NTP) colocalized at cell-cell interfaces with desmosomal proteins. As predicted, the distribution of endogenous DP was severely perturbed. At cell-cell borders where endogenous DP was undetectable by immunofluorescence, there was a striking absence of attached tonofibrils (IF bundles). Furthermore, DP-NTP assembled into ultrastructurally identifiable junctional structures lacking associated IF bundles. Surprisingly, immunofluorescence and immunogold electron microscopy indicated that adherens junction components were coassembled into these structures along with desmosomal components and DP-NTP. These results indicate that DP is required for anchoring IF networks to desmosomes and furthermore suggest that the DP-IF complex is important for governing the normal spatial segregation of adhesive junction components during their assembly into distinct structures.     

Plakophilin 2: a critical scaffold for PKC alpha that regulates intercellular junction assembly

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.     

An immunoelectron microscopic comparison of desmosomal constituents and hemidesmosomal ones originating from the same tissue of the same animal

The molecular constituents of desmosomes and hemidesmosomes were compared by examining bovine muzzle epidermis under immunoelectron microscopy using a postembedding method, first with antibodies prepared to four desmosomal antigens (DP1/2, DP3, DG1, DG2/3), followed by protein A-gold (PAG) complexes. The four antibodies showed almost negative labeling at hemidesmosomes as compared with the labeling observed at the desmosomes in the same tissue. By counting the number of PAG particles/200 millimicrons at hemidesmosomes and desmosomes, the above qualitative observation was confirmed quantitatively. These results support a new concept which has recently been proposed by several researchers that hemidesmosomes and desmosomes are immunochemically distinct.     

Desmoplakin Is Required Early in Development for Assembly of Desmosomes and Cytoskeletal Linkage

Desmosomes first assemble in the E3.5 mouse trophectoderm, concomitant with establishment of epithelial polarity and appearance of a blastocoel cavity. Throughout development, they increase in size and number and are especially abundant in epidermis and heart muscle. Desmosomes mediate cell–cell adhesion through desmosomal cadherins, which differ from classical cadherins in their attachments to intermediate filaments (IFs), rather than actin filaments. Of the proteins implicated in making this IF connection, only desmoplakin (DP) is both exclusive to and ubiquitous among desmosomes. To explore its function and importance to tissue integrity, we ablated the desmoplakin gene. Homozygous −/− mutant embryos proceeded through implantation, but did not survive beyond E6.5. Mutant embryos proceeded through implantation, but did not survive beyond E6.5. Surprisingly, analysis of these embryos revealed a critical role for desmoplakin not only in anchoring IFs to desmosomes, but also in desmosome assembly and/or stabilization. This finding not only unveiled a new function for desmoplakin, but also provided the first opportunity to explore desmosome function during embryogenesis. While a blastocoel cavity formed and epithelial cell polarity was at least partially established in the DP (−/−) embryos, the paucity of desmosomal cell–cell junctions severely affected the modeling of tissue architecture and shaping of the early embryo.     

Desmoplakin expression and distribution in cultured rat bladder epithelial cells of varying tumorigenic potential

The expression and distribution of the desmosomal plaque proteins, desmoplakins (DPs) I and II, were studied in nontumorigenic (RBE-8) and a series of tumorigenic (AY34, R-4909, SS-24B, RBTCC-8, and 804G) rat bladder epithelial cell lines. These cell lines ranged from slow-growing papillary transitional cells (AY34) to rapidly metastatic carcinoma cells (RBTCC-8). DPs I and II were shown by immunoblotting and Northern analysis to be present in nontumorigenic RBE-8 cells as well as in all of the tumorigenic cell lines, albeit in differing amounts. Immunofluorescence microscopy revealed striking differences in DP distribution, corresponding in general with increases in tumorigenic potential. Whereas DPs of normal RBE-8 cells and less tumorigenic AY34 cells were localized predominantly at cell interfaces, the more tumorigenic lines exhibited a high proportion of DP in the form of cytoplasmic dots, a distribution reminiscent of that seen in epithelial cells maintained in low levels of extracellular calcium. In 804G cells, which represented the most extreme example of this phenomenon, the majority of DPs were organized as cytoplasmic dots. Electron microscopy revealed intermediate filament (IF)-associated spots in the cytoplasm as well as an elaborate array of IF-associated plaques at the cell-substratum interface. The IF-associated spots in the cytoplasm reacted with anti-DP antibody in immunogold labeling experiments while those at the cell-substratum did not react. In more dense cultures of 804G cells, certain cells stratified and expressed increased amounts of DP followed by the induction of new keratins including those of the skin type. Decreasing extracellular calcium resulted in a rearrangement of DP in each cell line; staining at cell-cell interfaces disappeared and was replaced with a pattern of cytoplasmic dots. These results demonstrate a possible relationship between desmosome assembly and/or maintenance and tumorigenic potential.