Keratin Intermediate Filaments Research Articles

Intermediate Filament Structure, Assembly and Nanomechanics

Intermediate filaments (IFs) consist of two-stranded coiled coils that form anti-parallel, half-staggered tetramers. By time-lapse electron microscopy, complemented with total internal reflection fluorescence (TIRF) microscopy, we have investigated the in vitro assembly of vimentin to define the assembly pathway for vertebrate cytoplasmic IFs. First, we have characterized the physical and structural state of the soluble vimentin subunits by analytical ultracentrifugation (AUC) and X-ray crystallography. Assembly is induced by a change in the ionic strength and starts with the lateral association of tetramers to full-width unit-length filaments (ULFs) driven by the interaction of the basic, non-structured head domains with the acidic coiled-coil rods. In a next step, ULFs longitudinally anneal by an end-on-addition mechanism to yield filaments. This mechanism is also exhibited by muscle desmin and the epithelial keratins, whereas the nuclear IF proteins, i.e. the lamins, do not assemble into ULFs. In a next step, the subunit composition of ULFs and IFs of different IF proteins was analyzed by scanning transmission electron microscopy (STEM) and cryo-electron tomography of native specimens. Depending on the ionic conditions used for assembly, on average keratin IFs harbor 8, vimentin IFs 16 and desmin IFs 24 coiled-coil dimers per filament cross-section. The formation of ULFs was investigated further by small-angle X-ray scattering (SAXS) and AUC, employing a mutant vimentin variant that is arrested in the ULF state. With these data at hand, we investigated the impact of human disease mutations found in desmin that cause myofibrillar myopathy. Last but not least, we explored the network formation of lamin A and some of its disease variants, which strongly deviates from that of cytoplasmic IFs. These data give a first mechanistic clue how the lamin network provides mechanical stability to the nuclear envelope and nuclear architecture.

Chapter Six - Beyond Expectations: Novel Insights into Epidermal Keratin Function and Regulation

The epidermis is a stratified epithelium that relies on its cytoskeleton and cell junctions to protect the body against mechanical injury, dehydration, and infections. Keratin intermediate filament proteins are involved in many of these functions by forming cell-specific cytoskeletal scaffolds crucial for the maintenance of cell and tissue integrity. In response to various stresses, the expression and organization of keratins are altered at transcriptional and posttranslational levels to restore tissue homeostasis. Failure to restore tissue homeostasis in the presence of keratin gene mutations results in acute and chronic skin disorders for which currently no rational therapies are available. Here, we review the recent progress on the role of keratins in cytoarchitecture, adhesion, signaling, and inflammation. By focusing on epidermal keratins, we illustrate the contribution of keratin isotypes to differentiated epithelial functions.

Transition from embryonic to adult epidermis in reptiles occurs by the production of corneous beta-proteins.

The adaptation of the epidermis in amniote vertebrates to life on land took place by a drastic change from an embryonic epidermis made of two-four periderm layers to a terrestrial-proof epidermis. This transition occurred by the increase in types and number of specialized corneous proteins coded by genes of the Epidermal Differentiation Complex. The prevalent types of corneous proteins produced in the reptilian epidermis contain a beta-sheet region of high amino acid homology which allows their polymerization into a meshwork of filaments forming the hard corneous material of scales and claws. The present immunogold ultrastructural study shows that this transition occurs with the synthesis of glycine-rich corneous beta-proteins (formerly indicated as beta-keratins) that are added to the initial framework of acidic intermediate filaments produced in the embryonic epidermis of lizards, snake, alligator and turtle. These corneous beta-proteins are accumulated in the transitional and definitive layers of reptilian epidermis formed underneath the transitory two-four layered embryonic epidermis. In the more specialized reptiles capable of shedding the epidermis as a single unit, such as lizards and snakes, special glycine-cysteine rich beta-proteins are initially produced in a single layer immediately formed beneath the embryonic epidermis, the oberhautchen. The latter layer allows the in ovo shedding of the embryonic epidermis in preparation for hatching, and in the following shedding cycles of the adult epidermis. The production of specialized corneous-specific beta-proteins in addition to intermediate filament keratins was probably an essential addition for terrestrial life during the evolution of reptiles into different lineages, including birds. The increase of glycine and cysteine in epidermal proteins enhanced the hydrophobicity, insolubility and mechanical strength of the stratum corneum in these amniotes.

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.

An overview of chemical straightening of human hair: technical aspects, potential risks to hair fibre and health and legal issues

Personal image, as it relates to external beauty, has attracted much attention from the cosmetic industry, and capillary aesthetics is a leader in consumption in this area. There is a great diversity of products targeting both the treatment and beautification of hair. Among them, hair straighteners stand out with a high demand by costumers aiming at beauty, social acceptance and ease of daily hair maintenance. However, this kind of treatment affects the chemical structure of keratin and of the hair fibre, bringing up some safety concerns. Moreover, the development of hair is a dynamic and cyclic process, where the duration of growth cycles depends not only on where hair grows, but also on issues such as the individual's age, dietary habits and hormonal factors. Thus, although hair fibres are composed of dead epidermal cells, when they emerge from the scalp, there is a huge variation in natural wave and the response to hair cosmetics. Although it is possible to give the hair a cosmetically favourable appearance through the use of cosmetic products, for good results in any hair treatment, it is essential to understand the mechanisms of the process. Important information, such as the composition and structure of the hair fibres, and the composition of products and techniques available for hair straightening, must be taken into account so that the straightening process can be designed appropriately, avoiding undesirable side effects for hair fibre and for health. This review aims to address the morphology, chemical composition and molecular structure of hair fibres, as well as the products and techniques used for chemical hair relaxing, their potential risk to hair fibre and to health and the legal aspects of their use.

Radiographic findings regarding the toe crena

Radiographic findings regarding the toe crena

The Small Heat Shock Protein Hsp27 Affects Assembly Dynamics and Structure of Keratin Intermediate Filament Networks

The mechanical properties of living cells are essential for many processes. They are defined by the cytoskeleton, a composite network of protein fibers. Thus, the precise control of its architecture is of paramount importance. Our knowledge about the molecular and physical mechanisms defining the network structure remains scarce, especially for the intermediate filament cytoskeleton. Here, we investigate the effect of small heat shock proteins on the keratin 8/18 intermediate filament cytoskeleton using a well-controlled model system of reconstituted keratin networks. We demonstrate that Hsp27 severely alters the structure of such networks by changing their assembly dynamics. Furthermore, the C-terminal tail domain of keratin 8 is shown to be essential for this effect. Combining results from fluorescence and electron microscopy with data from analytical ultracentrifugation reveals the crucial role of kinetic trapping in keratin network formation.

Self-consistent field theory for the interactions between keratin intermediate filaments

BackgroundKeratins are important structural proteins found in skin, hair and nails. Keratin Intermediate Filaments are major components of corneocytes, nonviable horny cells of the Stratum Corneum, the outermost layer of skin. It is considered that interactions between unstructured domains of Keratin Intermediate Filaments are the key factor in maintaining the elasticity of the skin.ResultsWe have developed a model for the interactions between keratin intermediate filaments based on self-consistent field theory. The intermediate filaments are represented by charged surfaces, and the disordered terminal domains of the keratins are represented by charged heteropolymers grafted to these surfaces. We estimate the system is close to a charge compensation point where the heteropolymer grafting density is matched to the surface charge density. Using a protein model with amino acid resolution for the terminal domains, we find that the terminal chains can mediate a weak attraction between the keratin surfaces. The origin of the attraction is a combination of bridging and electrostatics. The attraction disappears when the system moves away from the charge compensation point, or when excess small ions and/or NMF-representing free amino acids are added.ConclusionsThese results are in concordance with experimental observations, and support the idea that the interaction between keratin filaments, and ultimately in part the elastic properties of the keratin-containing tissue, is controlled by a combination of the physico-chemical properties of the disordered terminal domains and the composition of the medium in the inter-filament region.

The cephalochordate Branchiostoma genome contains 26 intermediate filament (IF) genes: Implications for evolution of chordate IF proteins

We analyzed the draft genome of the cephalochordate Branchiostoma floridae (B. floridae) for genes encoding intermediate filament (IF) proteins. From 26 identified IF genes 13 were not reported before. Four of the new IF genes belong to the previously established Branchiostoma IF group A, four to the Branchiostoma IF group B, one is homologous to the type II keratin E2 while the remaining four new IF sequences N1 to N4 could not be readily classified in any of the previously established Branchiostoma IF groups. All eleven identified A and B2-type IF genes are located on the same genomic scaffold and arose due to multiple cephalochordate-specific duplications. Another IF gene cluster, identified in the B. floridae genome, contains three keratins (E1, Y1, D1), two keratin-like IF genes (C2, X1), one new IF gene (N1) and one IF unrelated gene, but does not show any similarities to the well defined vertebrate type I or type II keratin gene clusters. In addition, some type III sequence features were documented in the new IF protein N2, which, however, seems to share a common ancestry with the Branchiostoma keratins D1 and two keratin-related genes C. Thus, a few type I and type II keratin genes existed in a common ancestor of cephalochordates and vertebrates, which after separation of these two lineages gave rise to the known complexities of the vertebrate cytoplasmic type I–IV IF proteins, as well as to the multiple keratin and related IF genes in cephalochordates, due to multiple gene duplications, deletions and sequence divergences.

The human squamous epithelial cell envelope: the structural model by Peter M Steinert

The human squamous epithelial cell envelope: the structural model by Peter M Steinert

Investigating the origins of nanostructural variations in differential ethnic hair types using X‐ray scattering techniques

Human hair is a major determinant of visual ethnic differentiation. Although hair types are celebrated as part of our ethnic diversity, the approach to hair care has made the assumption that hair types are structurally and chemically similar. Although this is clearly not the case at the macroscopic level, the intervention of many hair treatments is at the nanoscopic and molecular levels. The purpose of the work presented here is to identify the main nanoscopic and molecular hierarchical differences across five different ethnic hair types from hair fibres taken exclusively from the scalp. These are Afro (subdivided into elastic 'rubber' and softer non-elastic 'soft'), Chinese, European and Mullato (mixed race). Small angle X-Ray scattering (SAXS) is a technique capable of resolving nanostructural variations in complex materials. Individual hair fibres from different ethnic hair types were used to investigate structural features found in common and also specific to each type. Simultaneous wide angle X-Ray scattering (WAXS) was used to analyse the submolecular level structure of the fibrous keratin present. The data sets from both techniques were analysed with principal component analysis (PCA) to identify underlying variables. Principal component analysis of both SAXS and WAXS data was shown to discriminate the scattering signal between different hair types. The X-ray scattering results show a common underlying keratin intermediate filament (KIF) structure. However, distinct differences were observed in the preferential orientation and intensity signal from the lipid component of the hair. In addition, differences were observed in the intensity distribution of the very low-angle sample-dependent diffuse scatter surrounding the 'beamstop.' The results indicate that the fibrous keratin scaffold remains consistent between ethnic hair types. The hierarchies made by these may be modulated by variation in the content of keratin-associated proteins (KAPs) and lipids that alter the interfacial structures and lead to macroscopic differences in hair morphology.

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.

The effect of tension on the growth of keratinocytes in a skin equivalent culture model

The skin organotypic culture environment (Vaughan, et al 2009) was used to study the effect of tension forces on the fine structural features of the epithelium. Two different keratinocyte cell cultures were used in these experiments, Ker‐CT‐Ras and the HaCaT cell line. Both cell types grew to 7–15 layers of keratinocytes with a distinct basal layer present on a collagenous matrix composed of fibroblasts. The skin explants were harvested and prepared by standard methods for both light and transmission electron microscopy. Keratin intermediate filaments were not as dense and tonofibrils appeared reduced in the no‐tension model. In the tension model, desmosomes showed distinct features with evident attachment plaques and a dense radial band. Keratin filaments interacted with plaques of the desmosomes. In apical layers, early quantitative assessment showed 3 desmosomes/5μm of cell surface as compared to 8 desmosomes/5μm of cell surface in the no‐tension vs. tension model, respectively. Hopefully, these results will help to serve as a further means of understanding keratinocyte interaction and keratinocyte‐dermal interaction in wound healing, aging, and skin development.

Influence of microfluidic shear on keratin networks in living cells

Intermediate filaments play a key role in cell mechanics, providing cells with compliance to small deformations and reinforcing them when large forces are applied. Here, we present a study of networks of keratin intermediate filaments in living cells under the influence of external forces. We expose the cells to controlled shear forces applied by microflow and investigate the response of the keratin network in situ. Our results show that bundle dynamics are reduced upon the application of shear flow. It is likely that cytoskeletal cross-talk is involved in this shear stress response via actin–keratin coupling.

Keratin 8 and 18 Loss in Epithelial Cancer Cells Increases Collective Cell Migration and Cisplatin Sensitivity through Claudin1 Up-regulation

Keratins 8 and 18 (K8/18) are simple epithelial cell-specific intermediate filament proteins. Keratins are essential for tissue integrity and are involved in intracellular signaling pathways that regulate cell response to injuries, cell growth, and death. K8/18 expression is maintained during tumorigenesis; hence, they are used as a diagnostic marker in tumor pathology. In recent years, studies have provided evidence that keratins should be considered not only as markers but also as regulators of cancer cell signaling. The loss of K8/18 expression during epithelial-mesenchymal transition (EMT) is associated with metastasis and chemoresistance. In the present study, we investigated whether K8/18 expression plays an active role in EMT. We show that K8/18 stable knockdown using shRNA increased collective migration and invasiveness of epithelial cancer cells without modulating EMT markers. K8/18-depleted cells showed PI3K/Akt/NF-κB hyperactivation and increased MMP2 and MMP9 expression. K8/18 deletion also increased cisplatin-induced apoptosis. Increased Fas receptor membrane targeting suggests that apoptosis is enhanced via the extrinsic pathway. Interestingly, we identified the tight junction protein claudin1 as a regulator of these processes. This is the first indication that modulation of K8/18 expression can influence the phenotype of epithelial cancer cells at a transcriptional level and supports the hypothesis that keratins play an active role in cancer progression.

A Pathophysiologic Role for Epidermal Growth Factor Receptor in Pemphigus Acantholysis

The pemphigus family of autoimmune bullous disorders is characterized by autoantibody binding to desmoglein 1 and/or 3 (dsg1/dsg3). In this study we show that EGF receptor (EGFR) is activated following pemphigus vulgaris (PV) IgG treatment of primary human keratinocytes and that EGFR activation is downstream of p38 mitogen-activated protein kinase (p38). Inhibition of EGFR blocked PV IgG-triggered dsg3 endocytosis, keratin intermediate filament retraction, and loss of cell-cell adhesion in vitro. Significantly, inhibiting EGFR prevented PV IgG-induced blister formation in the passive transfer mouse model of pemphigus. These data demonstrate cross-talk between dsg3 and EGFR, that this cross-talk is regulated by p38, and that EGFR is a potential therapeutic target for pemphigus. Small-molecule inhibitors and monoclonal antibodies directed against EGFR are currently used to treat several types of solid tumors. This study provides the experimental rationale for investigating the use of EGFR inhibitors in pemphigus.

Keratinocyte cytoskeletal roles in cell sheet engineering

BackgroundThere is an increasing need to understand cell-cell interactions for cell and tissue engineering purposes, such as optimizing cell sheet constructs, as well as for examining adhesion defect diseases. For cell-sheet engineering, one major obstacle to sheet function is that cell sheets in suspension are fragile and, over time, will contract. While the role of the cytoskeleton in maintaining the structure and adhesion of cells cultured on a rigid substrate is well-characterized, a systematic examination of the role played by different components of the cytoskeleton in regulating cell sheet contraction and cohesion in the absence of a substrate has been lacking.ResultsIn this study, keratinocytes were cultured until confluent and cell sheets were generated using dispase to remove the influence of the substrate. The effects of disrupting actin, microtubules or intermediate filaments on cell-cell interactions were assessed by measuring cell sheet cohesion and contraction. Keratin intermediate filament disruption caused comparable effects on cell sheet cohesion and contraction, when compared to actin or microtubule disruption. Interfering with actomyosin contraction demonstrated that interfering with cell contraction can also diminish cell cohesion.ConclusionsAll components of the cytoskeleton are involved in maintaining cell sheet cohesion and contraction, although not to the same extent. These findings demonstrate that substrate-free cell sheet biomechanical properties are dependent on the integrity of the cytoskeleton network.

Alteration in composition of keratin intermediate filaments in a model of breast cancer progression and the potential to reverse hallmarks of metastasis

In breast cancer the development of metastasis is a major turning point in the treatment and outcome of the disease. Throughout tumour development, and especially in the development of metastasis, epithelial mesenchymal transition takes place. During this transformation into a mesenchymal phenotype, the tumour cells undergo a series of structural changes. The loss of structural integrity and adoption of mesenchymal filaments enables cells to detach from the epithelial cell layer and metastasise. Keratins form the intermediate filaments of the cytoskeleton and provide scaffold structures within cells. During cancer progression the intermediate filaments are reorganised, and dramatic changes are seen in their protein components. Keratins K8, K18, K19 and vimentin are intermediate filament proteins with altered expression profiles during tumour development. We have used in vivo and in vitro models to analyse changes in intermediate filament proteins. Antibody-based methods were used to study K8 levels and proteomic analysis to profile the protein content of metastatic breast cancer cell variants. K8 expression declines as human breast tumours progress into an invasive phenotype. Analysis of IF proteins indicated altered expression profiles of K8, K18, K19 and vimentin, with K8, K18, K19 expressed in high levels in the T47D and MCF-7 cell lines, whereas the highly metastatic cell lines expressed lower levels of K8 and K18 and no detectable K19. Vimentin showed reverse expression profile with T47D and MCF-7 cells having no detectable vimentin expression whereas the highly metastatic MDA-MB-231 and MDA-MB-436 showed high levels. Analysis of acetylation status using specific antibodies suggested acetylation occurred within the central coiled domain in the MCF-7 and T47D cells. Inhibition of tumour growth by tissue factor (TF) shRNA resulted in a dramatic re-elevation of expression of K8 in xenographs of the highly metastatic MDA-MB-436 line. Intermediate filament expression alters during epithelial mesenchymal transition. Identified post translational modifications may play a role in alterations seen in the organisation, solubility and stability of these filaments. Epithelial mesenchymal transition can be reversed and an epithelial phenotype re-established.

A severe familial phenotype of Ichthyosis Curth-Macklin caused by a novel mutation in theKRT1gene

Funding sources: This study was supported by the Universidad del Rosario, Grant CS/Genetics 2012. Conflicts of interest: none declared. Madam, Ichthyosis Curth–Macklin (ICM; Mendelian Inheritance in Man database #146590) is a rare genetic disorder that is clinically characterized by severe palmoplantar keratoderma. The skin of patients with ICM is characterized by extensive dark spiky or verrucous plaques that affect the large joints and the trunk. In some cases these lesions can cover the entire body. Differentiating keratinocytes of ICM tissues display ultrastructural anomalies such as keratin intermediate filaments (KIF), aggregation into peripheral shells, perinuclear vacuolization and binucleated cell formation.1 ICM, as well as other keratinopathic ichthyoses, is caused by mutations of the keratin 1 gene (KRT1) that lead to an impairment of the KIF network assembly in the cytoplasm of keratinocytes.2 At the protein level, these mutations are located in the variable (V2) domain of the protein.1, 3, 4 In this study, we report two related women of Colombian origin, affected by a severe ICM phenotype, who present a novel KRT1 mutation. These patients attended the Dermatology Unit of the Carlos Ardila Lulle Clinic of Bucaramanga (Colombia). This study has been approved by the Ethical Committee at Universidad del Rosario and was conducted according to the Declaration of Helsinki principles. The index case (IC) provided a consent form to participate in the study and signed it on behalf of her daughter.

Depolymerization of Cytokeratin Intermediate Filaments Facilitates Intracellular Infection of HeLa Cells by Bartonella henselae

Bartonella henselae is capable of invading epithelial and endothelial cells by modulating the function of actin-dependent cytoskeleton proteins. Although understanding of the pathogenesis has been increased by the development of an in vitro infection model involving endothelial cells, little is known about the mechanism of interaction between B. henselae and epithelial cells. This study aims to identify the binding candidates of B. henselae in epithelial cells and explores their effect on B. henselae infection. Pull-down assays and mass spectrometry analysis confirmed that some of the binding proteins (keratin 14, keratin 6, and F-actin) are cytoskeleton associated. B. henselae infection significantly induces the expression of the cytokeratin genes. Chemical disruption of the keratin network by using ethylene glycol tetraacetic acid promotes the intracellular persistence of B. henselae in HeLa cells. However, cytochalasin B and phalloidin treatment inhibits B. henselae invasion. Immunofluorescent staining demonstrates that B. henselae infection induces an F-actin-dependent rearrangement of the cytoskeleton. However, we demonstrated via immunofluorescent staining and whole-mount cell electron microscopy that keratin intermediate filaments are depolymerized by B. henselae. The results indicate that B. henselae achieves an intracellular persistence in epithelial cells through the depolymerization of cytokeratin intermediate filaments that are protective against B. henselae invasion.