DPM1 modulates desmosomal adhesion and epidermal differentiation through SERPINB5.

Glycosylation is essential to facilitate cell-cell adhesion and differentiation. We determined the role of the dolichol phosphate mannosyltransferase (DPM) complex, a central regulator for glycosylation, for desmosomal adhesive function and epidermal differentiation. Deletion of the key molecule of the DPM complex, DPM1, in human keratinocytes resulted in weakened cell-cell adhesion, impaired localization of the desmosomal components desmoplakin and desmoglein-2, and led to cytoskeletal organization defects in human keratinocytes. In a 3D organotypic human epidermis model, loss of DPM1 caused impaired differentiation with abnormally increased cornification, reduced thickness of non-corneal layers, and formation of intercellular gaps in the epidermis. Using proteomic approaches, SERPINB5 was identified as a DPM1-dependent interaction partner of desmoplakin. Mechanistically, SERPINB5 reduced desmoplakin phosphorylation at serine 176, which was required for strong intercellular adhesion. These results uncover a novel role of the DPM complex in connecting desmosomal adhesion with epidermal differentiation.

Defective Desmosomal Adhesion Causes Arrhythmogenic Cardiomyopathy by Involving an Integrin-αVß6/TGF-ß Signaling Cascade.

BACKGROUND: Arrhythmogenic cardiomyopathy (ACM) is characterized by progressive loss of cardiomyocytes with fibrofatty tissue replacement, systolic dysfunction, and life-threatening arrhythmias. A substantial proportion of ACM is caused by mutations in genes of the desmosomal cell-cell adhesion complex, but the underlying mechanisms are not well understood. In the current study, we investigated the relevance of defective desmosomal adhesion for ACM development and progression. METHODS: We mutated the binding site of DSG2 (desmoglein-2), a crucial desmosomal adhesion molecule in cardiomyocytes. This DSG2-W2A mutation abrogates the tryptophan swap, a central interaction mechanism of DSG2 on the basis of structural data. Impaired adhesive function of DSG2-W2A was confirmed by cell-cell dissociation assays and force spectroscopy measurements by atomic force microscopy. The DSG2-W2A knock-in mouse model was analyzed by echocardiography, ECG, and histologic and biomolecular techniques including RNA sequencing and transmission electron and superresolution microscopy. The results were compared with ACM patient samples, and their relevance was confirmed in vivo and in cardiac slice cultures by inhibitor studies applying the small molecule EMD527040 or an inhibitory integrin-αVß6 antibody. RESULTS: The DSG2-W2A mutation impaired binding on molecular level and compromised intercellular adhesive function. Mice bearing this mutation develop a severe cardiac phenotype recalling the characteristics of ACM, including cardiac fibrosis, impaired systolic function, and arrhythmia. A comparison of the transcriptome of mutant mice with ACM patient data suggested deregulated integrin-αVß6 and subsequent transforming growth factor-ß signaling as driver of cardiac fibrosis. Blocking integrin-αVß6 led to reduced expression of profibrotic markers and reduced fibrosis formation in mutant animals in vivo. CONCLUSIONS: We show that disruption of desmosomal adhesion is sufficient to induce a phenotype that fulfils the clinical criteria to establish the diagnosis of ACM, confirming the dysfunctional adhesion hypothesis. Deregulation of integrin-αVß6 and transforming growth factor-ß signaling was identified as a central step toward fibrosis. A pilot in vivo drug test revealed this pathway as a promising target to ameliorate fibrosis. This highlights the value of this model to discern mechanisms of cardiac fibrosis and to identify and test novel treatment options for ACM.

The Actin-Binding Protein α-Adducin Modulates Desmosomal Turnover and Plasticity.

Intercellular adhesion is essential for tissue integrity and homeostasis. Desmosomes are abundant in the epidermis and the myocardium-tissues, which are under constantly changing mechanical stresses. Yet, it is largely unclear whether desmosomal adhesion can be rapidly adapted to changing demands, and the mechanisms underlying desmosome turnover are only partially understood. In this study we show that the loss of the actin-binding protein α-adducin resulted in reduced desmosome numbers and prevented the ability of cultured keratinocytes or murine epidermis to withstand mechanical stress. This effect was not primarily caused by decreased levels or impaired adhesive properties of desmosomal molecules but rather by altered desmosome turnover. Mechanistically, reduced cortical actin density in α-adducin knockout keratinocytes resulted in increased mobility of the desmosomal adhesion molecule desmoglein 3 and impaired interactions with E-cadherin, a crucial step in desmosome formation. Accordingly, the loss of α-adducin prevented increased membrane localization of desmoglein 3 in response to cyclic stretch or shear stress. Our data demonstrate the plasticity of desmosomal molecules in response to mechanical stimuli and unravel a mechanism of how the actin cytoskeleton indirectly shapes intercellular adhesion by restricting the membrane mobility of desmosomal molecules.

Clustering of desmosomal cadherins by desmoplakin is essential for cell-cell adhesion.

AIM: Desmoplakin (Dp) is a crucial component of the desmosome, a supramolecular cell junction complex anchoring intermediate filaments. The mechanisms how Dp modulates cell-cell adhesion are only partially understood. Here, we studied the impact of Dp on the function of desmosomal adhesion molecules, desmosome turnover and intercellular adhesion. METHODS: CRISPR/Cas9 was used for gene editing of human keratinocytes which were characterized by Western blot and immunostaining. Desmosomal ultrastructure and function were assessed by electron microscopy and cell adhesion assays. Single molecule binding properties and localization of desmosomal cadherins were studied by atomic force microscopy and super-resolution imaging. RESULTS: Knockout (ko) of Dp impaired cell cohesion to drastically higher extents as ko of another desmosomal protein, plakoglobin (Pg). In contrast to Pg ko, desmosomes were completely absent in Dp ko. Binding properties of the desmosomal adhesion molecules desmocollin (Dsc) 3 and desmoglein (Dsg) 3 remained unaltered under loss of Dp. Dp was required for assembling desmosomal cadherins into large clusters, as Dsg2 and Dsc3, adhesion molecules primarily localized within desmosomes, were redistributed into small puncta in the cell membrane of Dp ko cells. Additional silencing of desmosomal cadherins in Dp ko did not further increase loss of intercellular adhesion. CONCLUSION: Our data demonstrate that Dp is essential for desmosome formation but does not influence intercellular adhesion on the level of individual cadherin binding properties. Rather, macro-clustering of desmosomal adhesion molecules through Dp is crucial. These results may help to better understand severe diseases which are caused by Dp dysfunction.

Role of Dsg1- and Dsg3-Mediated Signaling in Pemphigus Autoantibody-Induced Loss of Keratinocyte Cohesion.

Pemphigus is an autoimmune dermatosis in which mucocutaneous blisters are induced primarily by autoantibodies against Desmoglein (Dsg) 1 and 3. Pemphigus vulgaris (PV) usually is associated with autoantibodies against Dsg3 whereas pemphigus foliaceus (PF) patients present autoantibodies against Dsg1. Several signaling pathways were proposed to cause loss of keratinocyte adhesion. However, relevance of different signaling pathways and role of Dsg1 and 3 to trigger signaling are not fully understood. Here, we show that Ca2+ chelation reduced PV-IgG- and PF-IgG-mediated loss of HaCaT keratinocyte cohesion whereas EGFR inhibition did not inhibit effects of PF-IgG. PV-IgG activated EGFR in a Src-dependent manner whereas both PV-IgG and PF-IgG caused Ca2+ influx independent of EGFR. ERK activation was Src-dependent in response to PV-IgG but not PF-IgG. To delineate the roles of Dsg isoforms to trigger signaling pathways, Dsg3- and Dsg2-deficient HaCaT keratinocyte cell lines were generated using CRISPR/Cas9. Dsg3- but not Dsg2-deficient cells were protected against PV-IgG-induced loss of cell adhesion. Ca2+ influx and ERK activation in response to PF-IgG were preserved in both cell lines.

Keratins Regulate the Adhesive Properties of Desmosomal Cadherins through Signaling.

Tightly controlled intercellular adhesion is crucial for the integrity and function of the epidermis. The keratin filament cytoskeleton anchors desmosomes, supramolecular complexes required for strong intercellular adhesion. We tested whether keratin filaments control cell adhesion by regulating the adhesive properties of desmosomal cadherins such as desmoglein (Dsg) 3. Atomic force microscopy and fluorescence recovery after photobleaching experiments showed reduced Dsg3 adhesive forces and membrane stability in murine keratinocytes lacking all keratin filaments. Impairment of the actin cytoskeleton also resulted in decreased Dsg3 immobilization but did not affect Dsg3 binding properties, indicating that the latter are exclusively controlled by keratins. Reduced binding forces were dependent on p38 mitogen-activated protein kinase activity, which was deregulated in keratin-deficient cells. In contrast, inhibition of protein kinase C signaling, which is known to be controlled by keratins, promoted and spatially stabilized Dsg3-mediated interactions in the membrane. These results show a previously unreported mechanism for how keratins stabilize intercellular adhesion on the level of single desmosomal adhesion molecules.

Caenorhabditis elegans star formation and negative chemotaxis induced by infection with corynebacteria.

Caenorhabditis elegans is one of the major model systems in biology based on advantageous properties such as short life span, transparency, genetic tractability and ease of culture using an Escherichia coli diet. In its natural habitat, compost and rotting plant material, this nematode lives on bacteria. However, C. elegans is a predator of bacteria, but can also be infected by nematopathogenic coryneform bacteria such Microbacterium and Leucobacter species, which display intriguing and diverse modes of pathogenicity. Depending on the nematode pathogen, aggregates of worms, termed worm-stars, can be formed, or severe rectal swelling, so-called Dar formation, can be induced. Using the human and animal pathogens Corynebacterium diphtheriae and Corynebacterium ulcerans as well as the non-pathogenic species Corynebacterium glutamicum, we show that these coryneform bacteria can also induce star formation slowly in worms, as well as a severe tail-swelling phenotype. While C. glutamicum had a significant, but minor influence on survival of C. elegans, nematodes were killed after infection with C. diphtheriae and C. ulcerans. The two pathogenic species were avoided by the nematodes and induced aversive learning in C. elegans.