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.

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.

Alloxan Disintegrates the Plant Cytoskeleton and Suppresses mlo-Mediated Powdery Mildew Resistance.

Recessively inherited mutant alleles of Mlo genes (mlo) confer broad-spectrum penetration resistance to powdery mildew pathogens in angiosperm plants. Although a few components are known to be required for mlo resistance, the detailed molecular mechanism underlying this type of immunity remains elusive. In this study, we identified alloxan (5,5-dihydroxyl pyrimidine-2,4,6-trione) and some of its structural analogs as chemical suppressors of mlo-mediated resistance in monocotyledonous barley (Hordeum vulgare) and dicotyledonous Arabidopsis thaliana. Apart from mlo resistance, alloxan impairs nonhost resistance in Arabidopsis. Histological analysis revealed that the chemical reduces callose deposition and hydrogen peroxide accumulation at attempted fungal penetration sites. Fluorescence microscopy revealed that alloxan interferes with the motility of cellular organelles (peroxisomes, endosomes and the endoplasmic reticulum) and the pathogen-triggered redistribution of the PEN1/SYP121 t-SNARE protein. These cellular defects are likely the consequence of disassembly of actin filaments and microtubules upon alloxan treatment. Similar to the situation in animal cells, alloxan elicited the temporary accumulation of reactive oxygen species (ROS) in cotyledons and rosette leaves of Arabidopsis plants. Our results suggest that alloxan may destabilize cytoskeletal architecture via induction of an early transient ROS burst, further leading to the failure of molecular and cellular processes that are critical for plant immunity.

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.

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.

Desmosome assembly and cell-cell adhesion are membrane raft-dependent processes.

The aim of our study was to investigate the association of desmosomal proteins with cholesterol-enriched membrane domains, commonly called membrane rafts, and the influence of cholesterol on desmosome assembly in epithelial Madin-Darby canine kidney cells (clone MDc-2). Biochemical analysis proved an association of desmosomal cadherin desmocollin 2 (Dsc2) in cholesterol-enriched fractions that contain membrane raft markers caveolin-1 and flotillin-1 and the novel raft marker ostreolysin. Cold detergent extraction of biotinylated plasma membranes revealed that ∼60% of Dsc2 associates with membrane rafts while the remainder is present in nonraft and cholesterol-poor membranes. The results of immunofluorescence microscopy confirmed colocalization of Dsc2 and ostreolysin. Partial depletion of cholesterol with methyl-ß-cyclodextrin disturbs desmosome assembly, as revealed by sequential recordings of live cells. Moreover, cholesterol depletion significantly reduces the strength of cell-cell junctions and partially releases Dsc2 from membrane rafts. Our data indicate that a pool of Dsc2 is associated with membrane rafts, particularly with the ostreolysin type of membrane raft, and that intact membrane rafts are necessary for desmosome assembly. Taken together, these data suggest cholesterol as a potential regulator that promotes desmosome assembly.

The keratin-filament cycle of assembly and disassembly.

Continuous and regulated remodelling of the cytoskeleton is crucial for many basic cell functions. In contrast to actin filaments and microtubules, it is not understood how this is accomplished for the third major cytoskeletal filament system, which consists of intermediate-filament polypeptides. Using time-lapse fluorescence microscopy of living interphase cells, in combination with photobleaching, photoactivation and quantitative fluorescence measurements, we observed that epithelial keratin intermediate filaments constantly release non-filamentous subunits, which are reused in the cell periphery for filament assembly. This cycle is independent of protein biosynthesis. The different stages of the cycle occur in defined cellular subdomains: assembly takes place in the cell periphery and newly formed filaments are constantly transported toward the perinuclear region while disassembly occurs, giving rise to diffusible subunits for another round of peripheral assembly. Remaining juxtanuclear filaments stabilize and encage the nucleus. Our data suggest that the keratin-filament cycle of assembly and disassembly is a major mechanism of intermediate-filament network plasticity, allowing rapid adaptation to specific requirements, notably in migrating cells.

Redistribution of adhering junctions in human endometrial epithelial cells during the implantation window of the menstrual cycle.

The human uterine epithelium is characterised by remarkable plasticity with cyclic changes in differentiation that are controlled by ovarian steroid hormones to optimise conditions for embryo implantation. To understand whether and how cell-cell adhesion is affected, the localisation of junction proteins was studied throughout the menstrual cycle. Expression patterns were examined by immunofluorescence in 36 human endometrial specimens of different cycle stages. Antibodies against the desmosomal proteins desmoplakin 1/2 (Dp 1/2) and desmoglein 2 (Dsg 2), the adherens junction proteins E-cadherin and ß-catenin and also the common junctional linker protein plakoglobin showed a strong subapical staining during the proliferative phase until the early luteal phase (day 20). In the mid- to late luteal phase, however, these junctional proteins redistributed over the entire lateral plasma membranes. In contrast, tight junction proteins (ZO-1, claudin 4) remained at their characteristic subapical position throughout the menstrual cycle. mRNA levels of Dp 1/2, E-cadherin and ZO-1 obtained by real time RT-PCR were not significantly changed during the menstrual cycle. The observed redistribution of desmosomes and adherens junctions coincides with the onset of the so called implantation window of human endometrium. We propose that this change is controlled by ovarian steroids and prepares the endometrium for successful trophoblast invasion.

Placental vasculogenesis is regulated by keratin-mediated hyperoxia in murine decidual tissues.

The mammalian placenta represents the interface between maternal and embryonic tissues and provides nutrients and gas exchange during embryo growth. Recently, keratin intermediate filament proteins were found to regulate embryo growth upstream of the mammalian target of rapamycin pathway through glucose transporter relocalization and to contribute to yolk sac vasculogenesis through altered bone morphogenetic protein 4 signaling. Whether keratins have vital functions in extraembryonic tissues is not well understood. Here, we report that keratins are essential for placental function. In the absence of keratins, we find hyperoxia in the decidual tissue directly adjacent to the placenta, because of an increased maternal vasculature. Hyperoxia causes impaired vasculogenesis through defective hypoxia-inducible factor 1α and vascular endothelial growth factor signaling, resulting in invagination defects of fetal blood vessels into the chorion. In turn, the reduced labyrinth, together with impaired gas exchange between maternal and embryonic blood, led to increased hypoxia in keratin-deficient embryos. We provide evidence that keratin-positive trophoblast secretion of prolactin-like protein a (Prlpa) and placental growth factor (PlGF) during decidualization are altered in the absence of keratins, leading to increased infiltration of uterine natural killer cells into placental vicinity and increased vascularization of the maternal decidua. Our findings suggest that keratin mutations might mediate conditions leading to early pregnancy loss due to hyperoxia in the decidua.

p38 MAPK-dependent shaping of the keratin cytoskeleton in cultured cells.

Plasticity of the resilient keratin intermediate filament cytoskeleton is an important prerequisite for epithelial tissue homeostasis. Here, the contribution of stress-activated p38 MAPK to keratin network organization was examined in cultured cells. It was observed that phosphorylated p38 colocalized with keratin granules that were rapidly formed in response to orthovanadate. The same p38(p) recruitment was noted during mitosis, in various stress situations and in cells producing mutant keratins. In all these situations keratin 8 became phosphorylated on S73, a well-known p38 target site. To demonstrate that p38-dependent keratin phosphorylation determines keratin organization, p38 activity was pharmacologically and genetically modulated: up-regulation induced keratin granule formation, whereas down-regulation prevented keratin filament network disassembly. Furthermore, transient p38 inhibition also inhibited keratin filament precursor formation and mutant keratin granule dissolution. Collectively, the rapid and reversible effects of p38 activity on keratin phosphorylation and organization in diverse physiological, stress, and pathological situations identify p38-dependent signalling as a major intermediate filament-regulating pathway.

A Ca2+-Mediated Switch of Epiplakin from a Diffuse to Keratin-Bound State Affects Keratin Dynamics.

Keratins exert important structural but also cytoprotective functions. They have to be adaptable to support cellular homeostasis. Epiplakin (EPPK1) has been shown to decorate keratin filaments in epithelial cells and to play a protective role under stress, but the mechanism is still unclear. Using live-cell imaging of epithelial cells expressing fluorescently tagged EPPK1 and keratin, we report here an unexpected dynamic behavior of EPPK1 upon stress. EPPK1 was diffusely distributed throughout the cytoplasm and not associated with keratin filaments in living cells under standard culture conditions. However, ER-, oxidative and UV-stress, as well as cell fixation, induced a rapid association of EPPK1 with keratin filaments. This re-localization of EPPK1 was reversible and dependent on the elevation of cytoplasmic Ca2+ levels. Moreover, keratin filament association of EPPK1 led to significantly reduced keratin dynamics. Thus, we propose that EPPK1 stabilizes the keratin network in stress conditions, which involve increased cytoplasmic Ca2+.

Combining Image Restoration and Traction Force Microscopy to Study Extracellular Matrix-Dependent Keratin Filament Network Plasticity.

Keratin intermediate filaments are dynamic cytoskeletal components that are responsible for tuning the mechanical properties of epithelial tissues. Although it is known that keratin filaments (KFs) are able to sense and respond to changes in the physicochemical properties of the local niche, a direct correlation of the dynamic three-dimensional network structure at the single filament level with the microenvironment has not been possible. Using conventional approaches, we find that keratin flow rates are dependent on extracellular matrix (ECM) composition but are unable to resolve KF network organization at the single filament level in relation to force patterns. We therefore developed a novel method that combines a machine learning-based image restoration technique and traction force microscopy to decipher the fine details of KF network properties in living cells grown on defined ECM patterns. Our approach utilizes Content-Aware Image Restoration (CARE) to enhance the temporal resolution of confocal fluorescence microscopy by at least five fold while preserving the spatial resolution required for accurate extraction of KF network structure at the single KF/KF bundle level. The restored images are used to segment the KF network, allowing numerical analyses of its local properties. We show that these tools can be used to study the impact of ECM composition and local mechanical perturbations on KF network properties and corresponding traction force patterns in size-controlled keratinocyte assemblies. We were thus able to detect increased curvature but not length of KFs on laminin-322 versus fibronectin. Photoablation of single cells in microprinted circular quadruplets revealed surprisingly little but still significant changes in KF segment length and curvature that were paralleled by an overall reduction in traction forces without affecting global network orientation in the modified cell groups irrespective of the ECM coating. Single cell analyses furthermore revealed differential responses to the photoablation that were less pronounced on laminin-332 than on fibronectin. The obtained results illustrate the feasibility of combining multiple techniques for multimodal monitoring and thereby provide, for the first time, a direct comparison between the changes in KF network organization at the single filament level and local force distribution in defined paradigms.

Quantitative mapping of keratin networks in 3D.

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.

Desmoglein 2 mutant mice develop cardiac fibrosis and dilation.

Desmosomes are cell-cell adhesion sites and part of the intercalated discs, which couple adjacent cardiomyocytes. The connection is formed by the extracellular domains of desmosomal cadherins that are also linked to the cytoskeleton on the cytoplasmic side. To examine the contribution of the desmosomal cadherin desmoglein 2 to cardiomyocyte adhesion and cardiac function, mutant mice were prepared lacking a part of the extracellular adhesive domain of desmoglein 2. Most live born mutant mice presented normal overall cardiac morphology at 2 weeks. Some animals, however, displayed extensive fibrotic lesions. Later on, mutants developed ventricular dilation leading to cardiac insufficiency and eventually premature death. Upon histological examination, cardiomyocyte death by calcifying necrosis and replacement by fibrous tissue were observed. Fibrotic lesions were highly proliferative in 2-week-old mutants, whereas the fibrotic lesions of older mutants showed little proliferation indicating the completion of local muscle replacement by scar tissue. Disease progression correlated with increased mRNA expression of c-myc, ANF, BNF, CTGF and GDF15, which are markers for cardiac stress, remodeling and heart failure. Taken together, the desmoglein 2-mutant mice display features of dilative cardiomyopathy and arrhythmogenic right ventricular cardiomyopathy, an inherited human heart disease with pronounced fibrosis and ventricular arrhythmias that has been linked to mutations in desmosomal proteins including desmoglein 2.

Focal adhesions are hotspots for keratin filament precursor formation.

Recent studies showed that keratin filament (KF) formation originates primarily from sites close to the actin-rich cell cortex. To further characterize these sites, we performed multicolor fluorescence imaging of living cells and found drastically increased KF assembly in regions of elevated actin turnover, i.e., in lamellipodia. Abundant KF precursors (KFPs) appeared within these areas at the distal tips of actin stress fibers, moving alongside the stress fibers until their integration into the peripheral KF network. The earliest KFPs were detected next to actin-anchoring focal adhesions (FAs) and were only seen after the establishment of FAs in emerging lamellipodia. Tight spatiotemporal coupling of FAs and KFP formation were not restricted to epithelial cells, but also occurred in nonepithelial cells and cells producing mutant keratins. Finally, interference with FA formation by talin short hairpin RNA led to KFP depletion. Collectively, our results support a major regulatory function of FAs for KF assembly, thereby providing the basis for coordinated shaping of the entire cytoskeleton during cell relocation and rearrangement.

Requirements for leukocyte transmigration via the transmembrane chemokine CX3CL1.

The surface-expressed transmembrane CX3C chemokine ligand 1 (CX3CL1/fractalkine) induces firm adhesion of leukocytes expressing its receptor CX3CR1. After shedding by the disintegrins and metalloproteinases (ADAM) 10 and 17, CX3CL1 also acts as soluble leukocyte chemoattractant. Here, we demonstrate that transmembrane CX3CL1 expressed on both endothelial and epithelial cells induces leukocyte transmigration. To investigate the underlying mechanism, we generated CX3CR1 variants lacking the intracellular aspartate-arginine-tyrosine (DRY) motif or the intracellular C-terminus which led to a defect in intracellular calcium response and impaired ligand uptake, respectively. While both variants effectively mediated firm cell adhesion, they failed to induce transmigration and rather mediated retention of leukocytes on the CX3CL1-expressing cell layer. Targeting of ADAM10 led to increased adhesion but reduced transmigration in response to transmembrane CX3CL1, while transmigration towards soluble CX3CL1 was not affected. Thus, transmembrane CX3CL1 mediates leukocyte transmigration via the DRY motif and C-terminus of CX3CR1 and the activity of ADAM10.

Scratch-induced partial skin wounds re-epithelialize by sheets of independently migrating keratinocytes.

Re-epithelialization is a crucial process to reestablish the protective barrier upon wounding of the skin. Although this process is well described for wounds where the complete epidermis and dermis is damaged, little is known about the re-epithelialization strategy in more frequently occurring smaller scratch wounds in which structures such as the hair follicles and sweat glands stay intact. To study this, we established a scratch wound model to follow individual keratinocytes in all epidermal layers in the back skin of mice by intravital microscopy. We discover that keratinocytes adopt a re-epithelialization strategy that enables them to bypass immobile obstacles such as hair follicles. Wound-induced cell loss is replenished by proliferation in a distinct zone away from the wound and this proliferation does not affect overall migration pattern. Whereas suprabasal keratinocytes are rather passive, basal keratinocytes move as a sheet of independently migrating cells into the wound, thereby constantly changing their direct neighboring cells enabling them to bypass intact obstacles. This re-epithelialization strategy results in a fast re-establishment of the protective skin barrier upon wounding.

Actin-dependent dynamics of keratin filament precursors.

Actin filament and microtubule growth characteristics are defined by their different plus and minus ends. In contrast, intermediate filaments lack this type of polarity. Yet, intermediate filament network growth occurs by selective addition of newly formed and polymerizing keratin particles at peripheral network domains thereby allowing polarized network reorganization. To examine this process at high resolution in living cells, mammary epithelium-derived, immortalized EpH4-cells were infected with retroviral cDNA constructs coding for human keratin 18-fluorescent protein hybrids. Several stable cell lines were established presenting characteristic fluorescent keratin filament (KF) networks. These cells contain particularly large and abundant lamellipodia in which nascent keratin particle dynamics are easily detected by time-lapse fluorescence microscopy. These keratin particles originate close to the plasma membrane, translocate continuously toward the cell center, and integrate end-on into the peripheral KF network. We show that this inward-directed transport relies on intact actin filaments. After treatment with the actin filament-disrupting drug cytochalasin newly polymerizing keratin assemblies still appear in the peripheral cytoplasm but remain stationary. On the other hand, nocodazole-mediated disruption of microtubules does not affect the centripetal KF precursor transport. From these and other observations a model is deduced which postulates that focal adhesion-dependent keratin polymerization occurs in forming lamellipodia and that transport of newly formed keratin particles is mediated by actin filaments until network integration. This mechanism allows extension of the KF network toward the leading edge in migrating cells and may be of relevance for tissue development and regeneration.

Intermediate filaments in Caenorhabditis elegans.

Intermediate filaments (IFs) make up one of the three major fibrous cytoskeletal systems in metazoans. Numerous IF polypeptides are synthesized in cell type-specific combinations suggesting specialized functions. The review concentrates on IFs in the model organism Caenorhabditis elegans which carries great promise to elucidate the still unresolved mechanisms of IF assembly into complex networks and to determine IF function in a living organism. In contrast to Drosophila melanogaster, which lacks cytoplasmic IFs altogether, the nematode genome contains 11 genes coding for cytoplasmic IFs and only a single gene for a nuclear lamin. Its cytoplasmic IFs are expressed in developmentally and spatially defined patterns. As an example we present the case of the intestinal IFs which are abundant in the mechanically resilient endotube, a prominent feature of the C. elegans intestinal terminal web region. This IF-rich structure brings together all three cytoskeletal filaments that are integrated into a coherent entity by the C. elegans apical junction (CeAJ) thereby completely surrounding and stabilizing the intestinal lumen with its characteristic brush border. Concepts on the developmental establishment of the endotube in relation to polarization and its function for maintenance of epithelial integrity are discussed. Furthermore, possible connections of the cytoplasmic cytoskeleton to the nuclear lamin IFs and the importance of these links for nuclear positioning are summarized.