Filaggrin and Atopic Dermatitis: Clinical and Pathogenetic Parallels and Therapeutic Possibilities

Atopic dermatitis (AtD) is multifactorial inflammatory skin disease, one of the aspects of its pathogenesis is epidermal barrier dysfunction. Early development of AtD is associated with filaggrin dysfunction. Filaggrin is a protein involved in aggregation of keratin filaments in the upper layers of epidermis and the retention of lipids and proteins between corneocytes. Frequently, filaggrin dysfunction can be accompanied with secondary infection and high risk of other allergic diseases development. This can happen due to disturbance in terminal differentiation of epidermal cells and, as consequence, malfunction of epidermal barrier. Thus, the long regular use of emollients is the basis of AtD therapy. New class of emollients (“emollents plus”) allowed us to achieve more significant treatment results in patients with AtD. These emollients reduce inflammatory process activity in the skin by replacing structural components of abnormal epidermal barrier.

Structural heterogeneity of cellular K5/K14 filaments as revealed by cryo-electron microscopy

Keratin intermediate filaments are an essential and major component of the cytoskeleton in epithelial cells. They form a stable yet dynamic filamentous network extending from the nucleus to the cell periphery, which provides resistance to mechanical stresses. Mutations in keratin genes are related to a variety of epithelial tissue diseases. Despite their importance, the molecular structure of keratin filaments remains largely unknown. In this study, we analyzed the structure of keratin 5/keratin 14 filaments within ghost mouse keratinocytes by cryo-electron microscopy and cryo-electron tomography. By averaging a large number of keratin segments, we have gained insights into the helical architecture of the filaments. Two-dimensional classification revealed profound variations in the diameter of keratin filaments and their subunit organization. Computational reconstitution of filaments of substantial length uncovered a high degree of internal heterogeneity along single filaments, which can contain regions of helical symmetry, regions with less symmetry and regions with significant diameter fluctuations. Cross section views of filaments revealed that keratins form hollow cylinders consisting of multiple protofilaments, with an electron dense core located in the center of the filament. These findings shed light on the complex and remarkable heterogenic architecture of keratin filaments, suggesting that they are highly flexible, dynamic cytoskeletal structures.

Roles for Ndel1 in keratin organization and desmosome function

Keratin intermediate filaments form dynamic polymer networks that organize in specific ways dependent on the cell type, the stage of the cell cycle, and the state of the cell. In differentiated cells of the epidermis, they are organized by desmosomes, cell-cell adhesion complexes which provide essential mechanical integrity to this tissue. Despite this, we know little about how keratin organization is controlled and whether desmosomes locally regulate keratin dynamics in addition to binding pre-assembled filaments. Ndel1 is a desmosome-associated protein in the differentiated epidermis, though its function at these structures has not been examined. Here, we show that Ndel1 binds directly to keratin subunits through a motif conserved in all intermediate filament proteins. Further, Ndel1 was necessary for robust desmosome-keratin association and sufficient to reorganize keratins at distinct cellular sites. Lis1, a Ndel1 binding protein, was required for desmosomal localization of Ndel1, but not for its effects on keratin filaments. Finally, we use mouse genetics to demonstrate that loss of Ndel1 results in desmosome defects in the epidermis. Our data thus identifies Ndel1 as a desmosome-associated protein which promotes local assembly/reorganization of keratin filaments and is essential for robust desmosome formation.

Post Zygotic, Somatic, Deletion in KERATIN 1 V1 Domain Generates Structural Alteration of the K1/K10 Dimer, Producing a Monolateral Palmar Epidermolytic Nevus

Palmoplantar keratodermas (PPKs) are characterized by thickness of stratum corneum and epidermal hyperkeratosis localized in palms and soles. PPKs can be epidermolytic (EPPK) or non epidermolytic (NEPPK). Specific mutations of keratin 16 (K16) and keratin 1 (K1) have been associated to EPPK, and NEPPK. Cases of mosaicism in PPKs due to somatic keratin mutations have also been described in scientific literature. We evaluated a patient presenting hyperkeratosis localized monolaterally in the right palmar area, characterized by linear yellowish hyperkeratotic lesions following the Blaschko lines. No other relatives of the patient showed any dermatological disease. Light and confocal histological analysis confirmed the presence of epidermolityic hyperkeratosis. Genetic analysis performed demonstrates the heterozygous deletion NM_006121.4:r.274_472del for a total of 198 nucleotides, in KRT1 cDNA obtained by a palmar lesional skin biopsy, corresponding to the protein mutation NP_006112.3:p.Gly71_Gly137del. DNA extracted from peripheral blood lymphocytes did not display the presence of the mutation. These results suggest a somatic mutation causing an alteration in K1 N-terminal variable domain (V1). The deleted sequence involves the ISIS subdomain, containing a lysine residue already described as fundamental for epidermal transglutaminases in the crosslinking of IF cytoskeleton. Moreover, a computational analysis of the wild-type and V1-mutated K1/K10 keratin dimers, suggests an unusual interaction between these keratin filaments. The mutation taster in silico analysis also returned a high probability for a deleterious mutation. These data demonstrate once again the importance of the head domain (V1) of K1 in the formation of a functional keratinocyte cytoskeleton. Moreover, this is a further demonstration of the presence of somatic mutations arising in later stages of the embryogenesis, generating a mosaic phenotype.

Structures of the ß-Keratin Filaments and Keratin Intermediate Filaments in the Epidermal Appendages of Birds and Reptiles (Sauropsids)

The epidermal appendages of birds and reptiles (the sauropsids) include claws, scales, and feathers. Each has specialized physical properties that facilitate movement, thermal insulation, defence mechanisms, and/or the catching of prey. The mechanical attributes of each of these appendages originate from its fibril-matrix texture, where the two filamentous structures present, i.e., the corneous ß-proteins (CBP or ß-keratins) that form 3.4 nm diameter filaments and the α-fibrous molecules that form the 7–10 nm diameter keratin intermediate filaments (KIF), provide much of the required tensile properties. The matrix, which is composed of the terminal domains of the KIF molecules and the proteins of the epidermal differentiation complex (EDC) (and which include the terminal domains of the CBP), provides the appendages, with their ability to resist compression and torsion. Only by knowing the detailed structures of the individual components and the manner in which they interact with one another will a full understanding be gained of the physical properties of the tissues as a whole. Towards that end, newly-derived aspects of the detailed conformations of the two filamentous structures will be discussed and then placed in the context of former knowledge.

Keratin dynamics govern the establishment of the maternal-fetal interface

SUMMARYAfter implantation, the mouse embryo undergoes gastrulation and forms mesoderm and endoderm. Mesoderm participates in embryonic and extra-embryonic tissues including the amnion, yolk sac, chorion and allantois, the umbilical cord precursor.Extra-embryonic mesoderm is rich in intermediate filaments. Two-photon live imaging of keratin 8-eYFP knock-in embryos allowed recording nucleation and elongation of keratin filaments, which formed apical cables coordinated across multiple cells in amnion, allantois, and blood islands. Embryos lacking all keratins displayed a deflated exocoelomic cavity, a narrow thick amnion, and a short allantois, indicating a hitherto unknown role for keratin filaments in post-implantation extra-embryonic membranes morphogenesis.Single-cell RNA sequencing of mesoderm cells, microdissected amnion, chorion, and allantois provided an interactive atlas of transcriptomes with germ layer and regional information. Keratin 8highmesenchymal cells in contact with the exocoelom shared a cytoskeleton and adhesion expression profile that might explain the adaptation of extra-embryonic structures to the increasing mechanical pressure.GRAPHICAL ABSTRACT

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.

Keratins and plakin family cytolinker proteins control the length of epithelial microridge protrusions

Actin filaments and microtubules create diverse cellular protrusions, but intermediate filaments, the strongest and most stable cytoskeletal elements, are not known to directly participate in the formation of protrusions. Here we show that keratin intermediate filaments directly regulate the morphogenesis of microridges, elongated protrusions arranged in elaborate maze-like patterns on the surface of mucosal epithelial cells. We found that microridges on zebrafish skin cells contained both actin and keratin filaments. Keratin filaments stabilized microridges, and overexpressing keratins lengthened them. Envoplakin and periplakin, plakin family cytolinkers that bind F-actin and keratins, localized to microridges, and were required for their morphogenesis. Strikingly, plakin protein levels directly dictate microridge length. An actin-binding domain of periplakin was required to initiate microridge morphogenesis, whereas periplakin-keratin binding was required to elongate microridges. These findings separate microridge morphogenesis into distinct steps, expand our understanding of intermediate filament functions, and identify microridges as protrusions that integrate actin and intermediate filaments.