Keratin are among the most abundant proteins in epithelial cells. Functions of the keratin network in cells are shaped by their dynamical organization. Using a collection of experimentally-driven ...mathematical models, different hypotheses for the turnover and transport of the keratin material in epithelial cells are tested. The interplay between turnover and transport and their effects on the keratin organization in cells are hence investigated by combining mathematical modeling and experimental data. Amongst the collection of mathematical models considered, a best model strongly supported by experimental data is identified. Fundamental to this approach is the fact that optimal parameter values associated with the best fit for each model are established. The best candidate among the best fits is characterized by the disassembly of the assembled keratin material in the perinuclear region and an active transport of the assembled keratin. Our study shows that an active transport of the assembled keratin is required to explain the experimentally observed keratin organization.
The desmosomal cadherin desmoglein 2 (Dsg2) localizes to the intercalated disc coupling adjacent cardiomyocytes. Desmoglein 2 gene (DSG2) mutations cause arrhythmogenic cardiomyopathy (AC) in human ...and transgenic mice. AC is characterized by arrhythmia, cardiodilation, cardiomyocyte necrosis with replacement fibrosis, interstitial fibrosis, and intercalated disc dissociation. The genetic DSG2 constellations encountered are compatible with loss of adhesion and altered signaling. To further elucidate pathomechanisms, we examined whether heart-specific Dsg2 depletion triggers cardiomyopathy.
Because DSG2 knockouts die during early embryogenesis, mice were prepared with cardiomyocyte-specific DSG2 ablation. Healthy transgenic animals were born with a functional heart presenting intercalated discs with incorporated desmosomal proteins. Dsg2 protein expression was reduced below 3% in the heart. All animals developed AC during postnatal growth with pronounced chamber dilation, calcifying cardiomyocyte necrosis, aseptic inflammation, interstitial and focal replacement fibrosis, and conduction defects with altered connexin 43 distribution. Electron microscopy revealed absence of desmosome-like structures and regional loss of intercalated disc adhesion. Mice carrying 2 mutant DSG2 alleles coding for Dsg2 lacking part of the adhesive EC1-EC2 domains present an indistinguishable phenotype, which is similar to that observed in human AC patients.
The observations show that the presence of Dsg2 is not essential for late heart morphogenesis and for cardiac contractility to support postnatal life. On increasing mechanical demands, heart function is severely compromised as evidenced by the onset of cardiomyopathy with pronounced morphological alterations. We propose that loss of Dsg2 compromises adhesion, and that this is a major pathogenic mechanism in DSG2-related and probably other desmosome-related ACs.
A characteristic feature of the skin blistering disease epidermolysis bullosa simplex is keratin filament (KF) network collapse caused by aggregation of the basal epidermal keratin type II (KtyII) K5 ...and its type I partner keratin 14 (K14). Here, we examine the role of keratin phosphorylation in KF network rearrangement and cellular functions. We detect phosphorylation of the K5 head domain residue T150 in cytoplasmic epidermolysis bullosa simplex granules containing R125C K14 mutants. Expression of phosphomimetic T150D K5 mutants results in impaired KF formation in keratinocytes. The phenotype is enhanced upon combination with other phosphomimetic K5 head domain mutations. Remarkably, introduction of T150D K5 mutants into KtyII-lacking (KtyII–/–) keratinocytes prevents keratin network formation altogether. In contrast, phosphorylation-deficient T150A K5 leads to KFs with reduced branching and turnover. Assembly of T150D K5 is arrested at the heterotetramer stage coinciding with increased heat shock protein association. Finally, reduced cell viability and elevated response to stressors is noted in T150 mutant cells. Taken together, our findings identify T150 K5 phosphorylation as an important determinant of KF network formation and function with a possible role in epidermolysis bullosa simplex pathogenesis.
Mechanical stability is a fundamental and essential property of epithelial cell sheets. It is in large part determined by cell-cell adhesion sites that are tightly integrated by the cortical ...cytoskeleton. An intimate crosstalk between the adherens junction-associated contractile actomyosin system and the desmosome-anchored keratin intermediate filament system is decisive for dynamic regulation of epithelial mechanics. A major question in the field is whether and in which way mechanical stress affects junctional plasticity. This is especially true for the desmosome-keratin scaffold whose role in force-sensing is virtually unknown. To examine this question, we inactivated the actomyosin system in human keratinocytes (HaCaT) and canine kidney cells (MDCK) and monitored changes in desmosomal protein turnover.
Partial inhibition of myosin II by para-nitro-blebbistatin led to a decrease of the cells' elastic modulus and to reduced desmosomal protein turnover in regions where nascent desmosomes are formed and, to a lower degree, in regions where larger, more mature desmosomes are present. Interestingly, desmosomal proteins are affected differently: a significant decrease in turnover was observed for the desmosomal plaque protein desmoplakin I (DspI), which links keratin filaments to the desmosomal core, and the transmembrane cadherin desmoglein 2 (Dsg2). On the other hand, the turnover of another type of desmosomal cadherin, desmocollin 2 (Dsc2), was not significantly altered under the tested conditions. Similarly, the turnover of the adherens junction-associated E-cadherin was not affected by the low doses of para-nitro-blebbistatin. Inhibition of actin polymerization by low dose latrunculin B treatment and of ROCK-driven actomyosin contractility by Y-27632 treatment also induced a significant decrease in desmosomal DspI turnover. Taken together, we conclude that changes in the cortical force balance affect desmosome formation and growth. Furthermore, they differentially modulate desmosomal protein turnover resulting in changes of desmosome composition. We take the observations as evidence for a hitherto unknown desmosomal mechanosensing and mechanoresponse pathway responding to an altered force balance.
Keratins perform major structural and regulatory functions in epithelia. Owing to redundancy, their respective contribution to epidermal integrity, adhesion, and cell junction formation has not been ...addressed in full. Unexpectedly, the constitutive deletion of type II keratins in mice was embryonic lethal ∼E9.5 without extensive tissue damage. This prompted us to analyze keratin functions in skin where keratins are best characterized. Here, we compare the mosaic and complete deletion of all type II keratins in mouse skin, with distinct consequences on epidermal integrity, adhesion, and organismal survival. Mosaic knockout (KO) mice survived ∼12 days while global KO mice died perinatally because of extensive epidermal damage. Coinciding with absence of keratins, epidermal fragility, inflammation, increased epidermal thickness, and increased proliferation were noted in both strains of mice, accompanied by significantly smaller desmosomes. Decreased desmosome size was due to accumulation of desmosomal proteins in the cytoplasm, causing intercellular adhesion defects resulting in intercellular splits. Mixing different ratios of wild-type and KO keratinocytes revealed that ∼60% of keratin-expressing cells were sufficient to maintain epithelial sheets under stress. Our data reveal a major contribution of keratins to the maintenance of desmosomal adhesion and epidermal integrity with relevance for the treatment of epidermolysis bullosa simplex and other keratinopathies.
Mechanobiology requires precise quantitative information on processes taking place in specific 3D microenvironments. Connecting the abundance of microscopical, molecular, biochemical, and cell ...mechanical data with defined topologies has turned out to be extremely difficult. Establishing such structural and functional 3D maps needed for biophysical modeling is a particular challenge for the cytoskeleton, which consists of long and interwoven filamentous polymers coordinating subcellular processes and interactions of cells with their environment. To date, useful tools are available for the segmentation and modeling of actin filaments and microtubules but comprehensive tools for the mapping of intermediate filament organization are still lacking. In this work, we describe a workflow to model and examine the complete 3D arrangement of the keratin intermediate filament cytoskeleton in canine, murine, and human epithelial cells both, in vitro and in vivo. Numerical models are derived from confocal airyscan high-resolution 3D imaging of fluorescence-tagged keratin filaments. They are interrogated and annotated at different length scales using different modes of visualization including immersive virtual reality. In this way, information is provided on network organization at the subcellular level including mesh arrangement, density and isotropic configuration as well as details on filament morphology such as bundling, curvature, and orientation. We show that the comparison of these parameters helps to identify, in quantitative terms, similarities and differences of keratin network organization in epithelial cell types defining subcellular domains, notably basal, apical, lateral, and perinuclear systems. The described approach and the presented data are pivotal for generating mechanobiological models that can be experimentally tested.
The abundance and diversity of intermediate filaments (IFs) in the C. elegans intestine indicate important contributions to intestinal function and organismal wellbeing. Fluorescent IF reporters ...localize below the actin-rich brush border and are highly enriched in the lumen-enveloping endotube, which is attached to the C. elegans apical junction. Mapping intestinal viscoelasticity by contact-free Brillouin microscopy reveals that the IF-rich endotube is positioned at the interface between the stiff brush border and soft cytoplasm suggesting a mechanical buffering function to deal with the frequent luminal distortions occurring during food intake and movement. In accordance, depletion of IFB-2, IFC-2 and IFD-2 leads to intestinal lumen dilation although depletion of IFC-1, IFD-1 and IFP-1 do not. Ultrastructural analyses of loss of function mutants further show that IFC-2 mutants have a rarefied endotube and IFB-2 mutants lack an endotube altogether. Remarkably, almost all IFB-2- and IFC-2-deficient animals develop to fertile adults. But developmental retardation, reduced brood size, altered survival and increased sensitivity to microbial toxin, osmotic and oxidative stress are seen in both mutants albeit to different degrees. Taken together, we propose that individual intestinal IF polypeptides contribute in different ways to endotube morphogenesis and cooperate to cope with changing environments.
The integrity of intercellular junctions that form the "terminal bar" in intestinal epithelium is crucial for sealing the intestinal barrier. Whereas specific roles of tight and adherens junctions ...are well known, the contribution of desmosomal adhesion for maintaining the intestinal epithelial barrier has not been specifically addressed. For the present study, we generated a desmoglein 2 antibody directed against the extracellular domain (Dsg2 ED) to test whether impaired Dsg2-mediated adhesion affects intestinal epithelial barrier functions in vitro. This antibody was able to specifically block Dsg2 interaction in cell-free atomic-force microscopy experiments. For in vitro studies of the intestinal barrier we used Caco2 cells following differentiation into tight enterocyte-like epithelial monolayers. Application of Dsg2 ED to Caco2 monolayers resulted in increased cell dissociation compared with controls in a dispase-based enterocyte dissociation assay. Under similar conditions, Dsg2 antibody significantly decreased transepithelial electrical resistance and increased FITC-dextran flux, indicating that Dsg2 interaction is critically involved in the maintenance of epithelial intestinal barrier functions. As revealed by immunostaining, this was due to Dsg2 ED antibody-induced rupture of tight junctions because tight junction proteins claudins 1, 4, and 5, occludin, and tight junction-associated protein zonula occludens-1 were partially removed from cell borders by Dsg2 ED treatment. Similar results were obtained by application of a commercial monoclonal antibody directed against the ED of Dsg2. Antibody-induced effects were blocked by absorption experiments using Dsg2-Fc-coated beads. Our data indicate that Dsg2-mediated adhesion affects tight junction integrity and is required to maintain intestinal epithelial barrier properties.
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