Keratin filaments mediate the expansion of extra-embryonic membranes in the post-gastrulation mouse embryo.

Mesoderm arises at gastrulation and contributes to both the mouse embryo proper and its extra-embryonic membranes. Two-photon live imaging of embryos bearing a keratin reporter allowed recording filament nucleation and elongation in the extra-embryonic region. Upon separation of amniotic and exocoelomic cavities, keratin 8 formed apical cables co-aligned across multiple cells in the amnion, allantois, and blood islands. An influence of substrate rigidity and composition on cell behavior and keratin content was observed in mesoderm explants. Embryos lacking all keratin filaments displayed a deflated extra-embryonic cavity, a narrow thick amnion, and a short allantois. Single-cell RNA sequencing of sorted mesoderm cells and micro-dissected amnion, chorion, and allantois, provided an atlas of transcriptomes with germ layer and regional information. It defined the cytoskeleton and adhesion expression profile of mesoderm-derived keratin 8-enriched cells lining the exocoelomic cavity. Those findings indicate a novel role for keratin filaments in the expansion of extra-embryonic structures and suggest mechanisms of mesoderm adaptation to the environment.

Meeting report - Desmosome dysfunction and disease: Alpine desmosome disease meeting.

Desmosome diseases are caused by dysfunction of desmosomes, which anchor intermediate filaments (IFs) at sites of cell-cell adhesion. For many decades, the focus of attention has been on the role of actin filament-associated adherens junctions in development and disease, especially cancer. However, interference with the function of desmosomes, their molecular constituents or their attachments to IFs has now emerged as a major contributor to a variety of diseases affecting different tissues and organs including skin, heart and the digestive tract. The first Alpine desmosome disease meeting (ADDM) held in Grainau, Germany, in October 2022 brought together international researchers from the basic sciences with clinical experts from diverse fields to share and discuss their ideas and concepts on desmosome function and dysfunction in the different cell types involved in desmosome diseases. Besides the prototypic desmosomal diseases pemphigus and arrhythmogenic cardiomyopathy, the role of desmosome dysfunction in inflammatory bowel diseases and eosinophilic esophagitis was discussed.

An Assessment of the Mechanophysical and Hormonal Impact on Human Endometrial Epithelium Mechanics and Receptivity.

The endometrial epithelium and underlying stroma undergo profound changes to support and limit embryo adhesion and invasion, which occur in the secretory phase of the menstrual cycle during the window of implantation. This coincides with a peak in progesterone and estradiol production. We hypothesized that the interplay between hormone-induced changes in the mechanical properties of the endometrial epithelium and stroma supports this process. To study it, we used hormone-responsive endometrial adenocarcinoma-derived Ishikawa cells growing on substrates of different stiffness. We showed that Ishikawa monolayers on soft substrates are more tightly clustered and uniform than on stiff substrates. Probing for mechanical alterations, we found accelerated stress-relaxation after apical nanoindentation in hormone-stimulated monolayers on stiff substrates. Traction force microscopy furthermore revealed an increased number of foci with high traction in the presence of estradiol and progesterone on soft substrates. The detection of single cells and small cell clusters positive for the intermediate filament protein vimentin and the progesterone receptor further underscored monolayer heterogeneity. Finally, adhesion assays with trophoblast-derived AC-1M-88 spheroids were used to examine the effects of substrate stiffness and steroid hormones on endometrial receptivity. We conclude that the extracellular matrix and hormones act together to determine mechanical properties and, ultimately, embryo implantation.

The intestinal intermediate filament network responds to and protects against microbial insults and toxins.

The enrichment of intermediate filaments in the apical cytoplasm of intestinal cells is evolutionarily conserved, forming a sheath that is anchored to apical junctions and positioned below the microvillar brush border, which suggests a protective intracellular barrier function. To test this, we used Caenorhabditiselegans, the intestinal cells of which are endowed with a particularly dense intermediate filament-rich layer that is referred to as the endotube. We found alterations in endotube structure and intermediate filament expression upon infection with nematicidal B.thuringiensis or treatment with its major pore-forming toxin crystal protein Cry5B. Endotube impairment due to defined genetic mutations of intermediate filaments and their regulators results in increased Cry5B sensitivity as evidenced by elevated larval arrest, prolonged time of larval development and reduced survival. Phenotype severity reflects the extent of endotube alterations and correlates with reduced rescue upon toxin removal. The results provide in vivo evidence for a major protective role of a properly configured intermediate filament network as an intracellular barrier in intestinal cells. This notion is further supported by increased sensitivity of endotube mutants to oxidative and osmotic stress.

Dual proteotoxic stress accelerates liver injury via activation of p62-Nrf2.

Protein accumulation is the hallmark of various neuronal, muscular, and other human disorders. It is also often seen in the liver as a major protein-secretory organ. For example, aggregation of mutated alpha1-antitrypsin (AAT), referred to as PiZ, is a characteristic feature of AAT deficiency, whereas retention of hepatitis B surface protein (HBs) is found in chronic hepatitis B (CHB) infection. We investigated the interaction of both proteotoxic stresses in humans and mice. Animals overexpressing both PiZ and HBs (HBs-PiZ mice) had greater liver injury, steatosis, and fibrosis. Later they exhibited higher hepatocellular carcinoma load and a more aggressive tumor subtype. Although PiZ and HBs displayed differing solubility properties and distinct distribution patterns, HBs-PiZ animals manifested retention of AAT/HBs in the degradatory pathway and a marked accumulation of the autophagy adaptor p62. Isolation of p62-containing particles revealed retained HBs/AAT and the lipophagy adapter perilipin-2. p62 build-up led to activation of the p62-Nrf2 axis and emergence of reactive oxygen species. Our results demonstrate that the simultaneous presence of two prevalent proteotoxic stresses promotes the development of liver injury due to protein retention and activation of the p62-Nrf2 axis. In humans, the PiZ variant was over-represented in CHB patients with advanced liver fibrosis (unadjusted odds ratio = 9.92 [1.15-85.39]). Current siRNA approaches targeting HBs/AAT should be considered for these individuals. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

Desmoglein 2 mutation provokes skeletal muscle actin expression and accumulation at intercalated discs in murine hearts.

Arrhythmogenic cardiomyopathy (AC) is an incurable progressive disease that is linked to mutations in genes coding for components of desmosomal adhesions that are localized to the intercalated disc region, which electromechanically couples adjacent cardiomyocytes. To date, the underlying molecular dysfunctions are not well characterized. In two murine AC models, we find an upregulation of the skeletal muscle actin gene (Acta1), which is known to be a compensatory reaction to compromised heart function. Expression of this gene is elevated prior to visible morphological alterations and clinical symptoms, and persists throughout pathogenesis with an additional major rise during the chronic disease stage. We provide evidence that the increased Acta1 transcription is initiated through nuclear activation of the serum response transcription factor (SRF) by its transcriptional co-activator megakaryoblastic leukemia 1 protein (MKL1, also known as MRTFA). Our data further suggest that perturbed desmosomal adhesion causes Acta1 overexpression during the early stages of the disease, which is amplified by transforming growth factor ß (TGFß) release from fibrotic lesions and surrounding cardiomyocytes during later disease stages. These observations highlight a hitherto unknown molecular AC pathomechanism.

The keratin-desmosome scaffold: pivotal role of desmosomes for keratin network morphogenesis.

Desmosome-anchored keratin intermediate filaments (KFs) are essential for epithelial coherence. Yet, desmosomal KF attachment and network organization are still unexplored in vivo. We, therefore, monitored KF network morphogenesis in fluorescent keratin 8 knock-in murine embryos revealing keratin enrichment at newly formed desmosomes followed by KF formation, KF elongation and KF fusion. To examine details of this process and its coupling to desmosome formation, we studied fluorescent keratin and desmosomal protein reporter dynamics in the periphery of expanding HaCaT keratinocyte colonies. Less than 3 min after the start of desmosomal proteins clustering non-filamentous keratin enriched at these sites followed by KF formation and elongation. Subsequently, desmosome-anchored KFs merged into stable bundles generating a rim-and-spokes system consisting of subcortical KFs connecting desmosomes to each other and radial KFs connecting desmosomes to the cytoplasmic KF network. We conclude that desmosomes are organizing centers for the KF cytoskeleton with a hitherto unknown nucleation capacity.

Hemidesmosome-Related Keratin Filament Bundling and Nucleation.

The epithelial cytoskeleton encompasses actin filaments, microtubules, and keratin intermediate filaments. They are interconnected and attached to the extracellular matrix via focal adhesions and hemidesmosomes. To study their interplay, we inhibited actin and tubulin polymerization in the human keratinocyte cell line HaCaT by latrunculin B and nocodazole, respectively. Using immunocytochemistry and time-lapse imaging of living cells, we found that inhibition of actin and tubulin polymerization alone or in combination induced keratin network re-organization albeit differently in each situation. Keratin filament network retraction towards the nucleus and formation of bundled and radial keratin filaments was most pronounced in latrunculin-B treated cells but less in doubly-treated cells and not detectable in the presence of nocodazole alone. Hemidesmosomal keratin filament anchorage was maintained in each instance, whereas focal adhesions were disassembled in the absence of actin filaments. Simultaneous inhibition of actin and tubulin polymerization, therefore, allowed us to dissect hemidesmosome-specific functions for keratin network properties. These included not only anchorage of keratin filament bundles but also nucleation of keratin filaments, which was also observed in migrating cells. The findings highlight the fundamental role of hemidesmosomal adhesion for keratin network formation and organization independent of other cytoskeletal filaments pointing to a unique mechanobiological function.

Model for Bundling of Keratin Intermediate Filaments.

Keratin intermediate filaments form dynamic intracellular networks, which span the entire cytoplasm and provide mechanical strength to the cell. The mechanical resilience of the keratin intermediate filament network itself is determined by filament bundling. The bundling process can be reproduced in artificial conditions in the absence of any specific cross-linking proteins, which suggests that it is driven by generic physical forces acting between filaments. Here, we suggest a detailed model for bundling of keratin intermediate filaments based on interfilament electrostatic and hydrophobic interactions. It predicts that the process is limited by an optimal bundle thickness, which is determined by the electric charge of the filaments, the number of hydrophobic residues in the constituent keratin polypeptides, and the extent to which the electrolyte ions are excluded from the bundle interior. We evaluate the kinetics of the bundling process by considering the energy barrier a filament has to overcome for joining a bundle.

Inflammation shapes pathogenesis of murine arrhythmogenic cardiomyopathy.

Arrhythmogenic cardiomyopathy (AC) is an incurable genetic disease, whose pathogenesis is poorly understood. AC is characterized by arrhythmia, fibrosis, and cardiodilation that may lead to sudden cardiac death or heart failure. To elucidate AC pathogenesis and to design possible treatment strategies of AC, multiple murine models have been established. Among them, mice carrying desmoglein 2 mutations are particularly valuable given the identification of desmoglein 2 mutations in human AC and the detection of desmoglein 2 auto-antibodies in AC patients. Using two mouse strains producing either a mutant desmoglein 2 or lacking desmoglein 2 in cardiomyocytes, we test the hypothesis that inflammation is a major component of disease pathogenesis. We show that multifocal cardiomyocyte necrosis initiates a neutrophil-dominated inflammatory response, which also involves macrophages and T cells. Increased expression of Ccl2/Ccr2, Ccl3/Ccr5, and Cxcl5/Cxcr2 mRNA reflects the observed immune cell recruitment. During the ensuing acute disease phase, Mmp12+ and Spp1+ macrophages and T cells accumulate in scars, which mature from cell- to collagen-rich. The expression of Cx3cl1/Cx3cr1, Ccl2/Ccr2, and Cxcl10/Cxcr3 dominates this disease phase. We furthermore find that during chronic disease progression macrophages and T cells persist within mature scars and are present in expanding interstitial fibrosis. Ccl12 and Cx3cl1 are predominant chemokines in this disease phase. Together, our observations provide strong evidence that specific immune cell populations and chemokine expression profiles modulate inflammatory and repair processes throughout AC progression.

Identification of a Novel Link between the Intermediate Filament Organizer IFO-1 and Cholesterol Metabolism in the Caenorhabditis elegans Intestine.

The intestine is an organ essential to organismal nutrient absorption, metabolic control, barrier function and immunoprotection. The Caenorhabditis elegans intestine consists of 20 cells harboring a dense intermediate filament network positioned below the apical plasma membrane that forms a junction-anchored sheath around the intestinal lumen. This evolutionarily conserved arrangement provides mechanical and overall stress-protection, and it serves as an important model for deciphering the role of intestinal architecture in metazoan biology. We recently reported that the loss-of-function mutation of the intestinal intermediate filament organizer IFO-1 perturbs this architecture, leading to reduced body size and reproduction. Here, we demonstrate that the IFO-1 mutation dramatically affects cholesterol metabolism. Mutants showed an increased sensitivity to cholesterol depletion, reduced cholesterol uptake, and cholesterol transfer to the gonads, which is also observed in worms completely lacking an intermediate filament network. Accordingly, we found striking similarities to transcriptome and lipidome profiles of a nuclear hormone receptor (NHR)-8 mutant. NHR-8 is homologous to mammalian LXR (liver X receptor) that serves as a sterol sensor and transcriptional regulator of lipid metabolism. Remarkably, increasing exogenous cholesterol partially rescues the developmental retardation in IFO-1 mutants. Our results uncover a novel link of the intestinal intermediate filament cytoskeleton to cholesterol metabolism that contributes to compromised growth and reproduction.

A rim-and-spoke hypothesis to explain the biomechanical roles for cytoplasmic intermediate filament networks.

Textbook images of keratin intermediate filament (IF) networks in epithelial cells and the functional compromization of the epidermis by keratin mutations promulgate a mechanical role for this important cytoskeletal component. In stratified epithelia, keratin filaments form prominent radial spokes that are focused onto cell-cell contact sites, i.e. the desmosomes. In this Hypothesis, we draw attention to a subset of keratin filaments that are apposed to the plasma membrane. They form a rim of filaments interconnecting the desmosomes in a circumferential network. We hypothesize that they are part of a rim-and-spoke arrangement of IFs in epithelia. From our review of the literature, we extend this functional role for the subplasmalemmal rim of IFs to any cell, in which plasma membrane support is required, provided these filaments connect directly or indirectly to the plasma membrane. Furthermore, cytoplasmic IF networks physically link the outer nuclear and plasma membranes, but their participation in mechanotransduction processes remain largely unconsidered. Therefore, we also discuss the potential biomechanical and mechanosensory role(s) of the cytoplasmic IF network in terms of such a rim (i.e. subplasmalemmal)-and-spoke arrangement for cytoplasmic IF networks.

Cardiomyocyte Hypertrophy in Arrhythmogenic Cardiomyopathy.

Arrhythmogenic cardiomyopathy (AC) is a hereditary disease leading to sudden cardiac death or heart failure. AC pathology is characterized by cardiomyocyte loss and replacement fibrosis. Our goal was to determine whether cardiomyocytes respond to AC progression by pathological hypertrophy. To this end, we examined tissue samples from AC patients with end-stage heart failure and tissue samples that were collected at different disease stages from desmoglein 2-mutant mice, a well characterized AC model. We find that cardiomyocyte diameters are significantly increased in right ventricles of AC patients. Increased mRNA expression of the cardiac stress marker natriuretic peptide B is also observed in the right ventricle of AC patients. Elevated myosin heavy chain 7 mRNA expression is detected in left ventricles. In desmoglein 2-mutant mice, cardiomyocyte diameters are normal during the concealed disease phase but increase significantly after acute disease onset on cardiomyocyte death and fibrotic myocardial remodeling. Hypertrophy progresses further during the chronic disease stage. In parallel, mRNA expression of myosin heavy chain 7 and natriuretic peptide B is up-regulated in both ventricles with right ventricular preference. Calcineurin/nuclear factor of activated T cells (Nfat) signaling, which is linked to pathological hypertrophy, is observed during AC progression, as evidenced by Nfatc2 and Nfatc3 mRNA in cardiomyocytes and increased mRNA of the Nfat target regulator of calcineurin 1. Taken together, we demonstrate that pathological hypertrophy occurs in AC and is secondary to cardiomyocyte loss and cardiac remodeling.

Ultrastructural changes in endometrial desmosomes of desmoglein 2 mutant mice.

The intercellular binding of desmosomal junctions is mediated by cadherins of the desmoglein (Dsg) and desmocollin (Dsc) type. Dsg2 mutant mice with deletion of a substantial segment of the extracellular EC1-EC2 domain, which is believed to participate in homo- and heterophilic desmosomal cadherin interactions, develop cardiac fibrosis and ventricular dilation. Widening of the intercellular cleft and complete intercalated disc ruptures can be observed in the hearts of these mice. Since a reduced litter size of homozygous Dsg2 mutant mice was noted and a functional correlation between desmosomes and embryo implantation has been deduced from animal studies, we looked for an alteration of desmosomes in uterine endometrial epithelium. Shape and number of desmosomes as well as the expression of Dsg2 and the desmosomal plaque protein desmoplakin (Dsp) were investigated by electron microscopy and immunohistochemistry in 12 oestrous-dated mice (7 wild type and 5 homozygous Dsg2 mutant mice) at the age of 9-17 weeks. The immunohistochemical detection of Dsg2 was diminished in the mutants and the number of desmosomes was significantly reduced as revealed by electron microscopy. In addition, the intercellular desmosomal space measured in electron micrographs was considerably widened in the Dsg2 mutants. The increased intercellular spacing can be explained by the partial deletion of the extracellular EC1-EC2 domain of Dsg2. Whether these changes explain the reduced number of offspring of homozygous Dsg2 mutant mice remains to be further investigated.

Measuring the regulation of keratin filament network dynamics.

The organization of the keratin intermediate filament cytoskeleton is closely linked to epithelial function. To study keratin network plasticity and its regulation at different levels, tools are needed to localize and measure local network dynamics. In this paper, we present image analysis methods designed to determine the speed and direction of keratin filament motion and to identify locations of keratin filament polymerization and depolymerization at subcellular resolution. Using these methods, we have analyzed time-lapse fluorescence recordings of fluorescent keratin 13 in human vulva carcinoma-derived A431 cells. The fluorescent keratins integrated into the endogenous keratin cytoskeleton, and thereby served as reliable markers of keratin dynamics. We found that increased times after seeding correlated with down-regulation of inward-directed keratin filament movement. Bulk flow analyses further revealed that keratin filament polymerization in the cell periphery and keratin depolymerization in the more central cytoplasm were both reduced. Treating these cells and other human keratinocyte-derived cells with EGF reversed all these processes within a few minutes, coinciding with increased keratin phosphorylation. These results highlight the value of the newly developed tools for identifying modulators of keratin filament network dynamics and characterizing their mode of action, which, in turn, contributes to understanding the close link between keratin filament network plasticity and epithelial physiology.

Keratins as the main component for the mechanical integrity of keratinocytes.

Keratins are major components of the epithelial cytoskeleton and are believed to play a vital role for mechanical integrity at the cellular and tissue level. Keratinocytes as the main cell type of the epidermis express a differentiation-specific set of type I and type II keratins forming a stable network and are major contributors of keratinocyte mechanical properties. However, owing to compensatory keratin expression, the overall contribution of keratins to cell mechanics was difficult to examine in vivo on deletion of single keratin genes. To overcome this problem, we used keratinocytes lacking all keratins. The mechanical properties of these cells were analyzed by atomic force microscopy (AFM) and magnetic tweezers experiments. We found a strong and highly significant softening of keratin-deficient keratinocytes when analyzed by AFM on the cell body and above the nucleus. Magnetic tweezers experiments fully confirmed these results showing, in addition, high viscous contributions to magnetic bead displacement in keratin-lacking cells. Keratin loss neither affected actin or microtubule networks nor their overall protein concentration. Furthermore, depolymerization of actin preserves cell softening in the absence of keratin. On reexpression of the sole basal epidermal keratin pair K5/14, the keratin filament network was reestablished, and mechanical properties were restored almost to WT levels in both experimental setups. The data presented here demonstrate the importance of keratin filaments for mechanical resilience of keratinocytes and indicate that expression of a single keratin pair is sufficient for almost complete reconstitution of their mechanical properties.

The novel intestinal filament organizer IFO-1 contributes to epithelial integrity in concert with ERM-1 and DLG-1.

The nematode Caenorhabditis elegans is an excellent model system in which to study in vivo organization and function of the intermediate filament (IF) system for epithelial development and function. Using a transgenic ifb-2::cfp reporter strain, a mutagenesis screen was performed to identify mutants with aberrant expression patterns of the IF protein IFB-2, which is expressed in a dense network at the subapical endotube just below the microvillar brush border of intestinal cells. Two of the isolated alleles (kc2 and kc3) were mapped to the same gene, which we refer to as ifo-1 (intestinal filament organizer). The encoded polypeptide colocalizes with IF proteins and F-actin in the intestine. The apical localization of IFO-1 does not rely on IFB-2 but is dependent on LET-413, a basolateral protein involved in apical junction assembly and maintenance of cell polarity. In mutant worms, IFB-2 and IFC-2 are mislocalized in cytoplasmic granules and accumulate in large aggregates at the C. elegans apical junction (CeAJ) in a DLG-1-dependent fashion. Electron microscopy reveals loss of the prominent endotube and disordered but still intact microvilli. Semiquantitative fluorescence microscopy revealed a significant decrease of F-actin, suggesting a general role of IFO-1 in cytoskeletal organization. Furthermore, downregulation of the cytoskeletal organizer ERM-1 and the adherens junction component DLG-1, each of which leads to F-actin reduction on its own, induces a novel synthetic phenotype in ifo-1 mutants resulting in disruption of the lumen. We conclude that IFO-1 is a multipurpose linker between different cytoskeletal components of the C. elegans intestinal terminal web and contributes to proper epithelial tube formation.

Endurance Training Provokes Arrhythmogenic Right Ventricular Cardiomyopathy Phenotype in Heterozygous Desmoglein-2 Mutants: Alleviation by Preload Reduction.

Desmoglein-2 mutations are detected in 5-10% of patients with arrhythmogenic right ventricular cardiomyopathy (ARVC). Endurance training accelerates the development of the ARVC phenotype, leading to earlier arrhythmic events. Homozygous Dsg2 mutant mice develop a severe ARVC-like phenotype. The phenotype of heterozygous mutant (Dsg2mt/wt) or haploinsufficient (Dsg20/wt) mice is still not well understood. To assess the effects of age and endurance swim training, we studied cardiac morphology and function in sedentary one-year-old Dsg2mt/wt and Dsg20/wt mice and in young Dsg2mt/wt mice exposed to endurance swim training. Cardiac structure was only occasionally affected in aged Dsg20/wt and Dsg2mt/wt mice manifesting as small fibrotic foci and displacement of Connexin 43. Endurance swim training increased the right ventricular (RV) diameter and decreased RV function in Dsg2mt/wt mice but not in wild types. Dsg2mt/wt hearts showed increased ventricular activation times and pacing-induced ventricular arrhythmia without obvious fibrosis or inflammation. Preload-reducing therapy during training prevented RV enlargement and alleviated the electrophysiological phenotype. Taken together, endurance swim training induced features of ARVC in young adult Dsg2mt/wt mice. Prolonged ventricular activation times in the hearts of trained Dsg2mt/wt mice are therefore a potential mechanism for increased arrhythmia risk. Preload-reducing therapy prevented training-induced ARVC phenotype pointing to beneficial treatment options in human patients.

Plasticity of cytoplasmic intermediate filament architecture determines cellular functions.

Cytoplasmic intermediate filaments endow cells with mechanical stability. They are subject to changes in morphology and composition if needed. This remodeling encompasses entire cells but can also be restricted to specific intracellular regions. Intermediate filaments thereby support spatially and temporally defined cell type-specific functions. This review focuses on recent advances in our understanding of how intermediate filament dynamics affect the underlying regulatory pathways. We will elaborate on the role of intermediate filaments for the formation and maintenance of surface specializations, cell migration, contractility, organelle positioning, nucleus protection, stress responses and axonal conduction velocity. Together, the selected examples highlight the modulatory role of intermediate filament plasticity for multiple cellular functions.