Biology subjects

Waschke, J.

Publications and source records attributed to Waschke, J..

12 recordsLinked to original sources

bioRxiv2025

STED/AFM as a tool to investigate mechanical and adhesive properties of migrating keratinocytes

E-cadherin is a central junctional molecule of adherens junctions that regulates epithelial integrity. In keratinocytes, wound healing critically depends on dynamic modulation of adhesion and cytoskeletal organization. Here, we applied wound healing assay and the combination of the stimulated emission depletion (STED) microscopy with the atomic force microscopy (AFM) (STED/AFM hybrid technique), to study E-cadherin binding properties during keratinocyte migration. Wound closure occurred within 6 h, an effect associated with E-cadherin accumulation at the leading edge. For location-specific binding measurements with the STED/AFM technique, we overexpressed E-cadherin in mouse keratinocytes and performed single molecule force spectroscopy measurements. Inhibition of actin polymerization abolished E-cadherin binding and further reduced overall cellular stiffness. To study adhesion mechanics during migration, we used the STED/AFM technique and performed single-molecule force spectroscopy at the leading edge of keratinocytes migrating into cell-free areas, generated by removal of two-well inserts. This approach enabled, for the first time, simultaneous measurements of single-molecule binding properties and cellular mechanistic properties in actively migrating keratinocytes. Our results revealed that E-Cad molecules present during migration exhibit binding properties comparable to those of E-Cad in stable AJs. This suggests that these molecules remain functional competent and may be readily available for rapid re-engagement in cell-cell adhesion when required. We introduce a powerful methodology to investigate single molecule binding properties in migrating cells, offering new opportunities to analyze epithelial repair at molecular resolution.

cell biology↗

bioRxiv2025

Autoantibodies in patients with arrhythmogenic cardiomyopathy activate GSK-3β resulting in a loss of cardiomyocyte cohesion

Arrhythmogenic cardiomyopathy (ACM) is an inherited cardiac desmosome disease, as more than 50% of affected patients carry pathogenic variants in desmosome protein-coding genes., In this study, we focused on the role and mechanisms of pathogenic and non-pathogenic autoantibodies against intercalated disc (ICD) proteins such as desmoglein2 (DSG2) in ACM patients, healthy relatives and murine ACM models. IgG fractions from ACM patients and healthy relatives, but not murine ACM model-derived or healthy control IgGs, revealed positive ICD staining. Antibodies reducing the loss of cardiomyocyte cohesion were found in three out of five ACM patients. Pathogenic autoantibodies, bound to DSG2 in hiPSC-CMs, cleaved DSG2 and reduced DSG2 interaction at the molecular level. We investigated GSK-3{beta} contribution to the loss of cardiomyocyte cohesion and observed that GSK-3{beta} reduced baseline cardiomyocyte cohesion in cultured cardiomyocytes and cardiac slices. Pathogenic ACM-IgGs activated GSK-3{beta} upstream of p38MAPK, leading to phosphorylation and junctional loss of {beta}-catenin. GSK-3{beta} inhibition rescued the loss of cell cohesion induced by ACM-IgGs in ACM hiPSC-CMs. Pathogenic autoantibodies targeting DSG2 are present in ACM patients and impair cardiomyocyte cohesion in a GSK-3{beta}-dependent manner. In contrast, autoantibodies are absent in murine ACM models and are non-pathogenic in some patients, healthy relatives.

cell biology↗

bioRxiv2025

Apremilast improves cardiomyocyte cohesion and arrhythmia in different models for arrhythmogenic cardiomyopathy

BackgroundArrhythmogenic cardiomyopathy (ACM) is a genetically inherited desmosome heart disease leading to life-threatening arrhythmias and sudden cardiac death. Currently, ACM treatment paradigms are merely symptom targeting. Recently, apremilast was shown to stabilize keratinocyte adhesion in the desmosomal disease pemphigus vulgaris. Therefore, this study investigated whether apremilast can be a therapeutic option for ACM. MethodsHuman induced pluripotent stem cells from a healthy control (hiPSC) and an ACM index patient (ACM-hiPSC) carrying a heterozygous desmoplakin (DSP) gene mutation (c.2854G>T, p.Glu952Ter), confirmed by whole exome sequencing (WES), were established. Cyclic-AMP ELISA, dissociation assay, immunostaining, and Western blotting analyses were performed in human iPSC-derived cardiomyocytes (hiPSC-CMs), murine HL-1 cardiomyocytes, and cardiac slices derived from wild-type (WT) mice, plakoglobin (PG, Jup) knockout (Jup-/-) (murine ACM model) or PG Serine 665 phosphodeficient (JUP-S665A) mice. Microelectrode array (MEA) analyses in ventricular cardiac slices and Langendorff heart perfusion were performed to analyze heart rate variability and arrhythmia. ResultsACM-hiPSC derived cardiomyocytes (ACM-hiPSC-CMs) revealed a significant loss of cohesion, which was rescued by apremilast. Further, treatment with apremilast strengthened basal cardiomyocyte cohesion in HL-1 cells and WT murine cardiac slices, paralleled by phosphorylation of PG at Serine 665 in human and murine models. In HL-1 cells, apremilast in addition activated ERK1/2, inhibition of which abolished apremilast-enhanced cardiomyocyte cohesion. Further, dissociation assays in slice cultures from JUP-S665A and Jup-/- mice revealed that PG is crucial for apremilast-enhanced cardiomyocyte cohesion. In parallel to enhanced cell adhesion, MEA and Langendorff measurements from WT and Jup-/- mice demonstrated decreased heart rate variability and arrhythmia after apremilast treatment. ConclusionsApremilast improves loss of cardiomyocyte cohesion, enhances localization of DSG2, and reduces arrhythmia in human and murine models of ACM ex vivo and in vitro, providing a novel treatment strategy for ACM by preserving desmosome function. Translational perspectiveThe current therapeutic options for patients with arrhythmogenic cardiomyopathy (ACM) include lifestyle changes, treatment with anti-arrhythmic drugs, catheter ablation, implantable cardiac defibrillators, and ultimately, heart transplantation for patients who are having therapy refractory arrhythmia or developed heart failure. However, lifestyle changes, such as restraining from physical endurance activities and {beta}-blocker therapy, are most used in patients carrying genetic variants coding for proteins of the desmosomal complex. Recent advancements hint that strategies enhancing intracellular cAMP could be beneficial in treating desmosomal diseases and can be effective therapeutics, which would be highly relevant for ACM patients. In this study, we show that apremilast improves loss of cardiomyocyte cohesion, enhances localization of desmosomal proteins, and reduces arrhythmia in both human and murine models of ACM ex vivo and in vitro, providing a novel treatment strategy for ACM by preserving desmosome function.

cell biology↗

bioRxiv2024

Dsg2 truncation causes a lethal barrier breakdown in mice

Inflammatory bowel diseases (IBD) such as Crohns disease (CD) have a complex aetiology with alterations of both the intestinal epithelial barrier and the IL23/IL17 immune response. Here, we investigated the role of a novel mutation in the desmosomal cadherin desmoglein 2 gene (DSG2) in the pathogenesis of IBD. DSG2 is known to regulate intestinal epithelial barrier integrity. Genetic analysis of a CD patient revealed a novel likely pathogenic DSG2 mutation leading to a truncated protein lacking part of the intracellular domain. We generated an enterocyte-specific mouse model, recapitulating the human mutation to study how the cytoplasmic truncation of Dsg2 affects intestinal barrier properties systemically. Moreover, we analysed the intestinal genetic profile in these mice and compared it to IBD patients. We describe a first CD patient with a rare mutation in the DSG2 gene causing cytoplasmic truncation with affects Dsg2 mobility. Mice with enterocyte-specific Dsg2 truncation suffered from a lethal intestinal barrier defect and presented a skewed IL17 response similar to CD patients. We identified the desmosomal cadherin Dsg2 as a regulator of the skewed IL17 response. These data indicate that desmosomes regulate inflammation similar to psoriasis which explains why the same novel immune therapies are effective for both diseases.

cell biology↗

bioRxiv2023

Nfe2l1-mediated proteasome function controls muscle energy metabolism in obesity

Muscle function is an important denominator of energy metabolism and metabolic health. Adapting the myocyte proteome to energetic challenges, in response to diet or fasting, is facilitated by programs of proteostasis, but the adaptive role of the ubiquitin-proteasome system (UPS) in muscle remains unclear. Here, we show that myocyte Nuclear factor erythroid derived 2,-like 1 (Nfe2l1, also known as Nrf1) is a key regulator of skeletal muscle proteostasis and function. In mice and humans, Nfe2l1 is highly expressed in skeletal myocytes, and its loss diminishes proteasomal activity and leads to hyperubiquitylation. Mice lacking myocyte Nfe2l1 display muscle fiber type switching and insulin resistance when fed a high-fed diet. Nfe2l1 protects myocytes from ferroptosis, which is enhanced in the presence of excess lipids. In conclusion, we define a new adaptive role for the Nfe2l1-ubiquitin proteasome system in the control of skeletal muscle function and energy metabolism.

physiology↗

bioRxiv2023

Catalytic antibodies in arrhythmogenic cardiomyopathy patients cleave desmoglein 2 and N-cadherin and impair cardiomyocyte cohesion

AimsArrhythmogenic cardiomyopathy (AC) is a severe heart disease predisposing to ventricular arrhythmias and sudden cardiac death caused by mutations affecting intercalated disc (ICD) proteins and aggravated by physical exercise. Recently, autoantibodies targeting ICD proteins, including the desmosomal cadherin desmoglein 2 (DSG2), were reported in AC patients and were considered relevant for disease development and progression, particularly in patients without underlying pathogenic mutations. However, it is unclear at present whether these autoantibodies are pathogenic and by which mechanisms show specificity for DSG2 and thus can be used as a diagnostic tool. Methods and ResultsIgG fractions were purified from 15 AC patients and 4 healthy controls. Immunostainings dissociation assays, atomic force microscopy (AFM), western blot analysis and Triton-X-100 assays were performed utilizing human heart left ventricle tissue, HL-1 cells, and murine cardiac slices. Immunostainings revealed that autoantibodies against ICD proteins are prevalent in AC and most autoantibody fractions have catalytic properties and cleave the ICD adhesion molecules DSG2 and N-cadherin, thereby reducing cadherin interactions as revealed by AFM. Furthermore, most of the AC-IgG fractions causing loss of cardiomyocyte cohesion activated p38MAPK, which is known to contribute to a loss of desmosomal adhesion in different cell types, including cardiomyocytes. In addition, p38MAPK inhibition rescued the loss of cardiomyocyte cohesion induced by AC-IgGs. ConclusionOur study demonstrates that catalytic autoantibodies play a pathogenic role by cleaving ICD cadherins and thereby reducing cardiomyocyte cohesion by a mechanism involving p38MAPK activation. Finally, we conclude that DSG2 cleavage by autoantibodies could be used as a diagnostic tool for AC.

cell biology↗

bioRxiv2022

Soluble VE-cadherin disrupts endothelial barrier function via VE-PTP/RhoA signalling

AimIncreased levels of soluble Vascular endothelial (VE)-cadherin fragments (sVE-cadherin) have previously been linked with inflammation-induced loss of endothelial barrier function. We tested whether sVE-cadherin is critically involved in the onset of endothelial barrier dysfunction. Methods and ResultsApplication of recombinant human sVE-cadherin (extracellular domains EC1-5) on human microvascular endothelial cells in vitro and in a rat model in vivo induced loss of endothelial barrier function and reduced microcirculatory flow. sVE-cadherinEC1-5 led to decreased localization of VE-cadherin at cell borders. Additionally, sVE-cadherinEC1-5 perturbed VE-protein tyrosine phosphatase (VE-PTP)/VE-cadherin interaction. VE-PTP inhibitor AKB9778 blunted all sVE-cadherinEC1-5-induced effects in vitro and in vivo. Downstream effects involve VE-PTP-dependent RhoA activation which was attenuated by AKB9778. Rho-kinase inhibitor Y27632 blocked sVE-cadherinEC1-5-induced loss of endothelial barrier function. ConclusionsVE-cadherin disrupts endothelial barrier function by dismantling the VE-cadherin complex at cell borders via VE-PTP-dependent RhoA activation. This uncovers a novel pathophysiological role of sVE-cadherin in the context of endothelial barrier dysfunction in inflammation.

physiology↗

bioRxiv2022

EGFR inhibition stabilizes cardiomyocyte cohesion in a murine arrhythmogenic cardiomyopathy model

Arrhythmogenic cardiomyopathy (AC) is a familial heart disease partly caused by impaired desmosome turnover. Thus, stabilization of desmosome integrity may provide potential new treatment options. Desmosomes, apart from cellular cohesion, provide the structural framework of a signaling hub. Here, we investigated the role of the epidermal growth factor receptor (EGFR) in cardiomyocyte cohesion. We inhibited EGFR under physiological and pathophysiological conditions using the murine plakoglobin knockout AC model, in which EGFR was upregulated. EGFR inhibition enhanced cardiomyocyte cohesion. Immunoprecipitation showed an interaction of EGFR and desmoglein 2 (DSG2). Immunostaining and AFM revealed enhanced DSG2 localization and binding at cell borders upon EGFR inhibition. Enhanced area composita length and desmosome assembly were observed upon EGFR inhibition, confirmed by enhanced DSG2 and desmoplakin (DP) recruitment to cell borders. Erlotinib, an EGFR inhibitor, activated ROCK. Erlotinib mediated desmosome assembly and cardiomyocyte cohesion were abolished upon ROCK inhibition. Thus, inhibiting EGFR, thereby stabilizing desmosome integrity, might provide new treatment options for AC. SummaryShoykhet et al. show that EGFR inhibition led ROCK activation enhances cardiomyocyte cohesion via enhanced desmosomal assembly which is evidenced by enhanced DP/DSG2 localization at cell borders. It is the first step towards a novel therapeutic approach for arrhythmogenic cardiomyopathy.

cell biology↗

bioRxiv2022

Apremilast prevents blistering in human epidermis by stabilization of keratinocyte adhesion in pemphigus.

Pemphigus vulgaris (PV) is a life-threatening blistering skin disease caused by autoantibodies (PV-IgG) destabilizing desmosomal adhesion. Current therapies focus on suppression of autoantibody formation and thus treatments directly stabilizing keratinocyte adhesion would fulfill an unmet medical need. We here demonstrate that apremilast, a phosphodiesterase 4 inhibitor used e.g. in psoriasis, prevents blistering in PV. Apremilast abrogated PV-IgG-induced loss of keratinocyte cohesion in ex-vivo epidermis and in vitro. This was paralleled by inhibition of keratin retraction and desmosome splitting but affected neither desmoglein (Dsg) depletion nor Dsg3 binding properties. Apremilast induced phosphorylation of plakoglobin at serine 665 - a mechanisms which is known to stabilize cardiomyocyte cohesion. Interestingly, keratinocytes phospho-deficient at this side showed altered organization of Dsg1, Dsg3 and keratin filaments and impaired adhesion, which was not rescued by apremilast. These data identified a new mechanism of desmosome regulation and propose that apremilast is protective in pemphigus by stabilizing keratinocyte cohesion.

cell biology↗

bioRxiv2020

linus: Conveniently explore, share and present large-scale biological trajectory data from a web browser.

In biology, we are often confronted with information-rich, large-scale trajectory data, but exploring and communicating patterns in such data is often a cumbersome task. Ideally, the data should be wrapped with an interactive visualisation in one concise package that makes it straightforward to create and test hypotheses collaboratively. To address these challenges, we have developed a tool, linus, which makes the process of exploring and sharing 3D trajectories as easy as browsing a website. We provide a python script that reads trajectory data and enriches them with additional features, such as edge bundling or custom axes and generates an interactive web-based visualisation that can be shared offline and online. The goal of linus is to facilitate the collaborative discovery of patterns in complex trajectory data.

bioinformatics↗

bioRxiv2020

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

Desmoplakin (Dp) localizes to desmosomes, linking clusters of desmosomal adhesion molecules to the intermediate filament cytoskeleton. Here, we generated Dp knockout (ko) cell lines of human keratinocytes to study the impact on desmosomal adhesion molecules and desmosome turnover using atomic force microscopy and superresolution imaging. In comparison to ko of another desmosomal component, plakoglobin (Pg), loss of Dp resulted in absence of desmosomes and drastically impaired cell cohesion. In Dp ko, the desmosomal adhesion molecules desmoglein 2 (Dsg2) and desmocollin 3 (Dsc3) were redistributed into small clusters in the cell membrane with no further increase in loss of intercellular adhesion by silencing of Dsg2. This suggests that extradesmosomal cadherins do not significantly contribute to cell cohesion but rather localization within desmosomes is required. Our data outline a crucial role of Dp for both desmosomal molecule clustering and mature desmosome formation and provide novel insights into the regulation of intercellular adhesion.

cell biology↗

bioRxiv2019

The actin binding protein α-adducin modulates desmosomal turnover and plasticity

Intercellular adhesion is essential for tissue integrity and homeostasis. Desmosomes are especially 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. We here show that loss of the actin-binding protein -adducin prevented the ability of cultured keratinocytes or murine epidermis to withstand mechanical stress paralleled with reduced desmosome number. This effect was not caused by decreased levels or impaired adhesive properties of desmosomal molecules but rather by altered desmosome turnover. Mechanistically, reduced cortical actin density in -adducin knockout keratinocytes resulted in increased mobility of the desmosomal adhesion molecule desmoglein 3 (Dsg3) and impaired interactions with E-cadherin, a crucial step in desmosome formation. Accordingly, loss of -adducin prevented increased membrane localization of Dsg3 in response to cyclic stretch or shear stress. Our data demonstrate plasticity of desmosomal molecules in response to mechanical stimuli and unravel a mechanism how the actin cytoskeleton indirectly shapes intercellular adhesion by restricting the mobility of desmosomal molecules.

cell biology↗