Plectin.

Prechova et al. introduce the giant cytoskeletal crosslinker protein plectin.

Measurement of Liver Stiffness using Atomic Force Microscopy Coupled with Polarization Microscopy.

Matrix stiffening has been recognized as one of the key drivers of the progression of liver fibrosis. It has profound effects on various aspects of cell behavior such as cell function, differentiation, and motility. However, as these processes are not homogeneous throughout the whole organ, it has become increasingly important to understand changes in the mechanical properties of tissues on the cellular level. To be able to monitor the stiffening of collagen-rich areas within the liver lobes, this paper presents a protocol for measuring liver tissue elastic moduli with high spatial precision by atomic force microscopy (AFM). AFM is a sensitive method with the potential to characterize local mechanical properties, calculated as Young's (also referred to as elastic) modulus. AFM coupled with polarization microscopy can be used to specifically locate the areas of fibrosis development based on the birefringence of collagen fibers in tissues. Using the presented protocol, we characterized the stiffness of collagen-rich areas from fibrotic mouse livers and corresponding areas in the livers of control mice. A prominent increase in the stiffness of collagen-positive areas was observed with fibrosis development. The presented protocol allows for a highly reproducible method of AFM measurement, due to the use of mildly fixed liver tissue, that can be used to further the understanding of disease-initiated changes in local tissue mechanical properties and their effect on the fate of neighboring cells.

Plectin-mediated cytoskeletal crosstalk controls cell tension and cohesion in epithelial sheets.

The coordinated interplay of cytoskeletal networks critically determines tissue biomechanics and structural integrity. Here, we show that plectin, a major intermediate filament-based cytolinker protein, orchestrates cortical cytoskeletal networks in epithelial sheets to support intercellular junctions. By combining CRISPR/Cas9-based gene editing and pharmacological inhibition, we demonstrate that in an F-actin-dependent context, plectin is essential for the formation of the circumferential keratin rim, organization of radial keratin spokes, and desmosomal patterning. In the absence of plectin-mediated cytoskeletal cross-linking, the aberrant keratin-desmosome (DSM)-network feeds back to the actin cytoskeleton, which results in elevated actomyosin contractility. Also, by complementing a predictive mechanical model with Förster resonance energy transfer-based tension sensors, we provide evidence that in the absence of cytoskeletal cross-linking, major intercellular junctions (adherens junctions and DSMs) are under intrinsically generated tensile stress. Defective cytoarchitecture and tensional disequilibrium result in reduced intercellular cohesion, associated with general destabilization of plectin-deficient sheets upon mechanical stress.

Perceived discrimination among Syrian refugees in Germany and its associations with mental health.

Discrimination is a crucial post migratory stressor but there is little research on perceived discrimination by Syrian refugees. This study aims to assess self-reported discrimination by Syrian refugees with a residence permit in Germany, determine its forms, places, and predictors, and explore its possible relation with mental health. Sociodemographics, migration-specific characteristics, traumatic life events, quality of life, perceived discrimination, depression, generalised anxiety disorder, post-traumatic symptoms, and somatic distress were assessed among 116 participants. More than one-third of the participants perceived discrimination in low frequency, and in general, perceived discrimination was confined to treatment with less courtesy or respect. Unfair treatment was perceived mostly while searching for accommodation and in the neighbourhood. Refugees attributed their experiences to their lack of language skills prior to national, religious, and racial causes. Lower age, higher number of completed years of education, and symptoms of posttraumatic stress disorder were significantly and substantially associated with perceived discrimination. Anxiety symptoms, number of traumatic experiences and gender may also be regarded as relevant predictors of perceived discrimination. Health care professionals must be aware of the links between discrimination and symptoms of mental disorders. Policymakers should address discrimination as a key refugee issue and risk to mental health.

Phenotypic drug screening in a human fibrosis model identified a novel class of antifibrotic therapeutics.

Fibrogenic processes instigate fatal chronic diseases leading to organ failure and death. Underlying biological processes involve induced massive deposition of extracellular matrix (ECM) by aberrant fibroblasts. We subjected diseased primary human lung fibroblasts to an advanced three-dimensional phenotypic high-content assay and screened a repurposing drug library of small molecules for inhibiting ECM deposition. Fibrotic Pattern Detection by Artificial Intelligence identified tranilast as an effective inhibitor. Structure-activity relationship studies confirmed N-(2-butoxyphenyl)-3-(phenyl)acrylamides (N23Ps) as a novel and highly potent compound class. N23Ps suppressed myofibroblast transdifferentiation, ECM deposition, cellular contractility, and altered cell shapes, thus advocating a unique mode of action. Mechanistically, transcriptomics identified SMURF2 as a potential therapeutic target network. Antifibrotic activity of N23Ps was verified by proteomics in a human ex vivo tissue fibrosis disease model, suppressing profibrotic markers SERPINE1 and CXCL8. Conclusively, N23Ps are a novel class of highly potent compounds inhibiting organ fibrosis in patients.

pyTFM: A tool for traction force and monolayer stress microscopy.

Cellular force generation and force transmission are of fundamental importance for numerous biological processes and can be studied with the methods of Traction Force Microscopy (TFM) and Monolayer Stress Microscopy. Traction Force Microscopy and Monolayer Stress Microscopy solve the inverse problem of reconstructing cell-matrix tractions and inter- and intra-cellular stresses from the measured cell force-induced deformations of an adhesive substrate with known elasticity. Although several laboratories have developed software for Traction Force Microscopy and Monolayer Stress Microscopy computations, there is currently no software package available that allows non-expert users to perform a full evaluation of such experiments. Here we present pyTFM, a tool to perform Traction Force Microscopy and Monolayer Stress Microscopy on cell patches and cell layers grown in a 2-dimensional environment. pyTFM was optimized for ease-of-use; it is open-source and well documented (hosted at https://pytfm.readthedocs.io/) including usage examples and explanations of the theoretical background. pyTFM can be used as a standalone Python package or as an add-on to the image annotation tool ClickPoints. In combination with the ClickPoints environment, pyTFM allows the user to set all necessary analysis parameters, select regions of interest, examine the input data and intermediary results, and calculate a wide range of parameters describing forces, stresses, and their distribution. In this work, we also thoroughly analyze the accuracy and performance of the Traction Force Microscopy and Monolayer Stress Microscopy algorithms of pyTFM using synthetic and experimental data from epithelial cell patches.

ADAM10 and ADAM17 regulate EGFR, c-Met and TNF RI signalling in liver regeneration and fibrosis.

ADAM10 and ADAM17 are proteases that affect multiple signalling pathways by releasing molecules from the cell surface. As their substrate specificities partially overlaps, we investigated their concurrent role in liver regeneration and fibrosis, using three liver-specific deficient mouse lines: ADAM10- and ADAM17-deficient lines, and a line deficient for both proteases. In the model of partial hepatectomy, double deficient mice exhibited decreased AKT phosphorylation, decreased release of EGFR activating factors and lower shedding of HGF receptor c-Met. Thus, simultaneous ablation of ADAM10 and ADAM17 resulted in inhibited EGFR signalling, while HGF/c-Met signalling pathway was enhanced. In contrast, antagonistic effects of ADAM10 and ADAM17 were observed in the model of chronic CCl4 intoxication. While ADAM10-deficient mice develop more severe fibrosis manifested by high ALT, AST, ALP and higher collagen deposition, combined deficiency of ADAM10 and ADAM17 surprisingly results in comparable degree of liver damage as in control littermates. Therefore, ADAM17 deficiency is not protective in fibrosis development per se, but can ameliorate the damaging effect of ADAM10 deficiency on liver fibrosis development. Furthermore, we show that while ablation of ADAM17 resulted in decreased shedding of TNF RI, ADAM10 deficiency leads to increased levels of soluble TNF RI in serum. In conclusion, hepatocyte-derived ADAM10 and ADAM17 are important regulators of growth receptor signalling and TNF RI release, and pathological roles of these proteases are dependent on the cellular context.

Somatic distress among Syrian refugees with residence permission in Germany: analysis of a cross-sectional register-based study.

BACKGROUND: Previous studies have already proven high rates of common mental disorders in Syrian refugees. Nevertheless, little is known about the patterns of somatic distress among this refugee population. For this reason, we aimed to examine the prevalence, co-occurrence, and risk factors of somatic distress among Syrian refugees in Germany. METHODS: This study analyzes the second measurement point (N = 116) of a prospective register-based survey among 200 adult Syrian refugees with residence permission in Germany. The survey consisted of information on sociodemographic and migration-specific characteristics, health care utilization, traumatic life events, acculturative stress (Barcelona Immigration Stress Scale (BISS); subscales: perceived discrimination, intercultural contact stress, homesickness, and general psychosocial stress), and self-reported outcomes of somatic distress (Patient Health Questionnaire (PHQ-15)), depression (PHQ-9), generalized anxiety disorder (GAD-7), and post-traumatic symptoms (Essen Trauma Inventory (ETI)). RESULTS: Almost half of the respondents (49.1%) were identified as being at risk of somatic distress (PHQ-15 score ≥ 6), and even 24.1% being bothered by moderate-to-severe levels of somatic distress (PHQ-15 score ≥ 10). The most robust associations with somatic distress were found for female gender, the amount of health care utilization, multiple trauma exposures, general psychosocial stress, and self-reported depression and anxiety symptoms. High comorbidities with somatic distress were shown for all of the common mental disorders studied. CONCLUSIONS: The presented study reveals a significant risk of somatic distress among this displaced population and highlights implications for policy and health care providers.

Plectin ensures intestinal epithelial integrity and protects colon against colitis.

Plectin, a highly versatile cytolinker protein, provides tissues with mechanical stability through the integration of intermediate filaments (IFs) with cell junctions. Here, we hypothesize that plectin-controlled cytoarchitecture is a critical determinant of the intestinal barrier function and homeostasis. Mice lacking plectin in an intestinal epithelial cell (IEC; PleΔIEC) spontaneously developed colitis characterized by extensive detachment of IECs from the basement membrane (BM), increased intestinal permeability, and inflammatory lesions. Moreover, plectin expression was reduced in the colons of ulcerative colitis (UC) patients and negatively correlated with the severity of colitis. Mechanistically, plectin deficiency in IECs led to aberrant keratin filament (KF) network organization and the formation of dysfunctional hemidesmosomes (HDs) and intercellular junctions. In addition, the hemidesmosomal α6ß4 integrin (Itg) receptor showed attenuated association with KFs, and protein profiling revealed prominent downregulation of junctional constituents. Consistent with the effects of plectin loss in the intestinal epithelium, plectin-deficient IECs exhibited remarkably reduced mechanical stability and limited adhesion capacity in vitro. Feeding mice with a low-residue liquid diet that reduced mechanical stress and antibiotic treatment successfully mitigated epithelial damage in the PleΔIEC colon.

A low-cost uniaxial cell stretcher for six parallel wells.

Cells in the lungs, the heart, and numerous other organs, are constantly exposed to dynamic forces and deformations. To mimic these dynamic mechanical loading conditions and to study the resulting cellular responses such as morphological changes or the activation of biochemical signaling pathways, cells are typically seeded on flexible 2D substrates that are uniaxially or biaxially stretched. Here, we present an open-source cell stretcher built from parts of an Anet A8 3D printer. The cell stretcher is controlled by a fully programmable open-source software using GCode and Python. Up to six flexible optically clear substrates can be stretched simultaneously, allowing for comparative multi-batch biological studies including microscopic image analysis. The cell yield from the cell culture area of 4 cm2 per substrate is sufficient for Western-blot protein analysis. As a proof-of-concept, we study the activation of the Yes-associated protein (YAP) mechanotransduction pathway in response to increased cytoskeletal tension induced by uniaxial stretching of epithelial cells. Our data support the previously observed activation of the YAP transcription pathway by stretch-induced increase in cytoskeletal tension and demonstrate the suitability of the cell stretcher to study complex mechano-biological processes.

The prevalence and risk factors for mental distress among Syrian refugees in Germany: a register-based follow-up study.

BACKGROUND: Mental disorders among refugees as well as their risk factors are already well documented in cross-sectional reports. However, longitudinal follow-up designs are widely lacking. Therefore, the aim of this study was to examine the change of the prevalence of mental disorders among Syrian refugees with German residence permission, taking into account their increasing length of stay in Germany, and to uncover the change in their relationship to pre- and post-migration risk factors. METHODS: This study formed part of a register-based follow-up study with two measurement points in Erlangen (Germany). At the first time of recruitment in 2017, 200 of the 518 Syrian refugees with residence permission living in Erlangen took part. During the second survey timeframe 1.5 years later, in 2019, 108 of the former 200 Syrian refugees participated again and formed the total sample for this follow-up study. The survey instruments included demographics, migration-related variables and symptoms of post-traumatic stress (Essen Trauma Inventory, ETI), depression (Patient Health Questionnaire - PHQ-9) and generalized anxiety disorder (GAD-7). RESULTS: At the time of the first survey, 26.9% of the participants exceeded the cut-off for a clinically relevant depression diagnosis, 16.7% for an anxiety disorder and 13.9% for a PTSD diagnosis. At the second measurement point, it was 30.6% for depression, 15.7% for an anxiety disorder and 13.0% for PTSD. No significant changes between the measurement points were found for any of the disorders. In multiple linear regression analyses, higher perceived discrimination, a higher number of traumatic experiences and a shorter duration of residence permission were shown to be the most important pre- and post-migration predictors of psychological stress independent of the time of measurement. CONCLUSIONS: There is strong empirical evidence that the prevalence rates of mental distress among refugees are significantly higher compared to the overall population. However, it has not yet become clear how these prevalence rates change with an increasing length of stay in the host countries. The results of our study indicate that the psychological burden on this refugee population remains consistently high over time, despite partly improved living conditions, and confirm the importance of therapeutic interventions.

Dysregulated NADPH Oxidase Promotes Bone Damage in Murine Model of Autoinflammatory Osteomyelitis.

Autoinflammatory diseases are characterized by dysregulation of the innate immune system, leading to spontaneous inflammation. Pstpip2cmo mouse strain is a well-characterized model of this class of disorders. Because of the mutation leading to the lack of adaptor protein PSTPIP2, these animals suffer from autoinflammatory chronic multifocal osteomyelitis similar to several human syndromes. Current evidence suggests that it is driven by hyperproduction of IL-1ß by neutrophil granulocytes. In this study, we show that in addition to IL-1ß, PSTPIP2 also negatively regulates pathways governing reactive oxygen species generation by neutrophil NOX2 NADPH oxidase. Pstpip2cmo neutrophils display highly elevated superoxide production in response to a range of stimuli. Inactivation of NOX2 NADPH oxidase in Pstpip2cmo mice did not affect IL-1ß levels, and the autoinflammatory process was initiated with similar kinetics. However, the bone destruction was almost completely alleviated, suggesting that dysregulated NADPH oxidase activity is a key factor promoting autoinflammatory bone damage in Pstpip2cmo mice.

Does the separation from marital partners of Syrian refugees with a residence permit in Germany have an impact on their quality of life?

OBJECTIVE: Around 700,000 Syrian refugees live in Germany, most of them having come alone since 2015 as asylum seekers and waiting in Germany for family reunification. This study focused on separation from marital partner and its impact on quality of life among Syrian refugees with a residence permit. METHODS: For the present investigation, we included only married participants of a larger registry-based study. Therefore, we analyzed 119 participants; 93 of them were married, and their partner accompanied them in Germany at the time of the investigation (partner+), while a further 26 were married but separated from their partner (partner-). The respondents were investigated for mental stress, quality of life and protective factors. RESULTS: The partner- group reported significantly lower quality of life in the domains of psychological and social health in comparison to the partner+ group. Higher general quality of life was associated with higher social support, higher sense of coherence and fewer symptoms of depression. Predictors for a higher quality of life were male gender, fewer symptoms of depression, a higher sense of coherence, higher perceived social support and living together with the marital partner. CONCLUSION: Family separation of Syrian refugees with a residence permit in Germany can have an impact on their quality of life. Longitudinal studies are needed to confirm our results and determine the long-term effects of family separation.

Vimentin Intermediate Filaments as Potential Target for Cancer Treatment.

Intermediate filaments constitute the third component of the cellular skeleton. Unlike actin and microtubule cytoskeletons, the intermediate filaments are composed of a wide variety of structurally related proteins showing distinct expression patterns in tissues and cell types. Changes in the expression patterns of intermediate filaments are often associated with cancer progression; in particular with phenotypes leading to increased cellular migration and invasion. In this review we will describe the role of vimentin intermediate filaments in cancer cell migration, cell adhesion structures, and metastasis formation. The potential for targeting vimentin in cancer treatment and the development of drugs targeting vimentin will be reviewed.

Isolation and 3D Collagen Sandwich Culture of Primary Mouse Hepatocytes to Study the Role of Cytoskeleton in Bile Canalicular Formation In Vitro.

Hepatocytes are the central cells of the liver responsible for its metabolic function. As such, they form a uniquely polarized epithelium, in which two or more hepatocytes contribute apical membranes to form a bile canalicular network through which bile is secreted. Hepatocyte polarization is essential for correct canalicular formation and depends on interactions between the hepatocyte cytoskeleton, cell-cell contacts, and the extracellular matrix. In vitro studies of hepatocyte cytoskeleton involvement in canaliculi formation and its response to pathological situations are handicapped by the lack of cell culture, which would closely resemble the canaliculi network structure in vivo. Described here is a protocol for the isolation of mouse hepatocytes from the adult mouse liver using a modified collagenase perfusion technique. Also described is the production of culture in a 3D collagen sandwich setting, which is used for immunolabeling of cytoskeletal components to study bile canalicular formation and its response to treatments in vitro. It is shown that hepatocyte 3D collagen sandwich cultures respond to treatments with toxins (ethanol) or actin cytoskeleton altering drugs (e.g., blebbistatin) and serve as a valuable tool for in vitro studies of bile canaliculi formation and function.

Plectin controls biliary tree architecture and stability in cholestasis.

BACKGROUND & AIMS: Plectin, a highly versatile cytolinker protein, controls intermediate filament cytoarchitecture and cellular stress response. In the present study, we investigate the role of plectin in the liver under basal conditions and in experimental cholestasis. METHODS: We generated liver-specific plectin knockout (PleΔalb) mice and analyzed them using two cholestatic liver injury models: bile duct ligation (BDL) and 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) feeding. Primary hepatocytes and a cholangiocyte cell line were used to address the impact of plectin on keratin filament organization and stability in vitro. RESULTS: Plectin deficiency in hepatocytes and biliary epithelial cells led to aberrant keratin filament network organization, biliary tree malformations, and collapse of bile ducts and ductules. Further, plectin ablation significantly aggravated biliary damage upon cholestatic challenge. Coincidently, we observed a significant expansion of A6-positive progenitor cells in PleΔalb livers. After BDL, plectin-deficient bile ducts were prominently dilated with more frequent ruptures corresponding to an increased number of bile infarcts. In addition, more abundant keratin aggregates indicated less stable keratin filaments in PleΔalb hepatocytes. A transmission electron microscopy analysis revealed a compromised tight junction formation in plectin-deficient biliary epithelial cells. In addition, protein profiling showed increased expression of the adherens junction protein E-Cadherin, and inefficient upregulation of the desmosomal protein desmoplakin in response to BDL. In vitro analyses revealed a higher susceptibility of plectin-deficient keratin networks to stress-induced collapse, paralleled by elevated activation of p38 MAP kinase. CONCLUSION: Our study shows that by maintaining proper keratin network cytoarchitecture and biliary epithelial stability, plectin plays a critical role in protecting the liver from stress elicited by cholestasis. LAY SUMMARY: Plectin is a cytolinker protein capable of interconnecting all three cytoskeletal filament systems and linking them to plasma membrane-bound junctional complexes. In liver, the plectin-controlled cytoskeleton mechanically stabilizes epithelial cells and provides them with the capacity to adapt to increased bile pressure under cholestasis.

Structural insights into Ca2+-calmodulin regulation of Plectin 1a-integrin ß4 interaction in hemidesmosomes.

The mechanical stability of epithelial cells, which protect organisms from harmful external factors, is maintained by hemidesmosomes via the interaction between plectin 1a (P1a) and integrin α6ß4. Binding of calcium-calmodulin (Ca(2+)-CaM) to P1a together with phosphorylation of integrin ß4 disrupts this complex, resulting in disassembly of hemidesmosomes. We present structures of the P1a actin binding domain either in complex with the N-ter lobe of Ca(2+)-CaM or with the first pair of integrin ß4 fibronectin domains. Ca(2+)-CaM binds to the N-ter isoform-specific tail of P1a in a unique manner, via its N-ter lobe in an extended conformation. Structural, cell biology, and biochemical studies suggest the following model: binding of Ca(2+)-CaM to an intrinsically disordered N-ter segment of plectin converts it to an α helix, which repositions calmodulin to displace integrin ß4 by steric repulsion. This model could serve as a blueprint for studies aimed at understanding how Ca(2+)-CaM or EF-hand motifs regulate F-actin-based cytoskeleton.

Mechanosensing through focal adhesion-anchored intermediate filaments.

Integrin-based mechanotransduction involves a complex focal adhesion (FA)-associated machinery that is able to detect and respond to forces exerted either through components of the extracellular matrix or the intracellular contractile actomyosin network. Here, we show a hitherto unrecognized regulatory role of vimentin intermediate filaments (IFs) in this process. By studying fibroblasts in which vimentin IFs were decoupled from FAs, either because of vimentin deficiency (V0) or loss of vimentin network anchorage due to deficiency in the cytolinker protein plectin (P0), we demonstrate attenuated activation of the major mechanosensor molecule FAK and its downstream targets Src, ERK1/2, and p38, as well as an up-regulation of the compensatory feedback loop acting on RhoA and myosin light chain. In line with these findings, we show strongly reduced FA turnover rates in P0 fibroblasts combined with impaired directional migration, formation of protrusions, and up-regulation of "stretched" high-affinity integrin complexes. By exploiting tension-independent conditions, we were able to mechanistically link these defects to diminished cytoskeletal tension in both P0 and V0 cells. Our data provide important new insights into molecular mechanisms underlying cytoskeleton-regulated mechanosensing, a feature that is fundamental for controlled cell movement and tumor progression.