In tail skin tissue sections, the immunostaining for -catenin and for lamin A/C but not that for desmoglein 1 are markedly increased in Krt14 C373A mice relative to WT controls (Figure 6A,B). (H) Quantification of relative fluorescence intensity of data shown in frame g, normalized to WT. Thank you for submitting your article "Keratin 14-dependent disulfides regulate epidermal homeostasis and barrier function via 14-3-3 and YAP1" for consideration by eLife. How will keratins from different tissues, ie, different keratin subtypes, influence their function as biomaterials? Oligonucleotides were used to clone the target into pT7gRNA, and the plasmid was amplified and linearized prior to T7 transcription. The diverse keratin intermediate filament family evolved from a common ancestral gene and shares a common structural plan. First, the perinuclear enrichment of keratin IFs, which is promoted by K14-dependent disulfide bonding, is poised to increase the local concentration of binding sites for 14-3-3 and YAP near the nucleus (simple mass action law). 2010 Jul;163(1):197-200. doi: The molecular reagents and assays available to us at present do not allow more resolution in our mechanistic understanding of the interplay between K14, K14-dependent disulfide bonding, YAP1, mechanosensing and mechanotransduction. Together these observations link the anomalies observed in epidermal homeostasis and skin barrier to defects in terminal keratinocyte differentiation in Krt14 C373A mouse skin.

In contrast to tail and ear epidermis, several markers including K14, K10, loricrin and filaggrin appear normal in the thin epidermis of back skin (Figure 3figure supplement 1AC), consistent with the markedly lower yield of K14-dependent disulfide bonding in this body site (Figure 1).

Here, we report on studies involving a new mouse model that provides evidence that the stutter cysteine in K14 protein regulates entry into differentiation and thus the balance between proliferation and differentiation through regulated interactions with 14-3-3 adaptor proteins and YAP1, a terminal effector of Hippo signaling (Pocaterra et al., 2020). After understanding how keratin subtypes interact, deciphering the conditions needed to organize and assemble them in an orchestrated way will be key. Non-reducing lysates were prepared directed in LDS sample buffer. By western immunoblotting, the levels of endogenous YAP1 and Ser127-phosphorylated YAP1 are similar in WT and Krt14 C373A skin (Figure 4F,G). Keratins, represented by more than 50 genes and 20 known polypeptides, are amongst the most abundant proteins of lung epithelial cells. N=3 biological replicates. For morphological evaluation, CE isolates from dorsal ear and tail skin were seeded on glass slides at a concentration of 1.5 106 CEs and 6 106 CEs, respectively, covered with a thin cover glass, and then imaged. Loss of Claudin5 enhanced histamine-induced leakage in an organotypic and vessel type-specific manner in an inducible, EC-specific, knock-out mouse. Dominantly-acting missense alleles in either KRT5 or KRT14 underlie the vast majority of cases of epidermolysis bullosa simplex (EBS), a rare genetic skin disorder in which trivial trauma results in skin blistering secondary to the lysis of fragile basal keratinocytes (Bonifas et al., 1991; Coulombe et al., 1991b; Fuchs and Coulombe, 1992; Lane et al., 1992).

Students t test: *p<0.05; **p<0.01; ***p<0.005; n.s., no statistical difference. Our study establishes that residue cysteine 373 in mouse K14 partakes in regulating the balance between keratinocyte proliferation and differentiation in epidermis in vivo, and ultimately barrier function, in skin. These findings demonstrate the importance of both the head and tail of the motor in regulating the activity of KIF22 and offer insight into the cellular consequences of preventing KIF22 inactivation and disrupting force balance in anaphase. Genetically determined alterations in keratin-coding sequences underlie highly penetrant and rare disorders whose pathophysiology reflects cell fragility and/or altered tissue homeostasis. N=3 biological replicates per genotype. and de novo mutations in the KRT5 and KRT14 genes, phenotype/genotype Since then, more than a dozen of extraction strategies have been proposed. Since keratin fragments are released from tumors and in other pathological conditions affecting epithelial cells, they may also serve as disease markers. How K14 (and possibly K10), 14-3-3, YAP, and other crucial effectors bind each other as part of this newly defined signaling axis, its regulation, and its significance have now emerged as open issues of high significance for future studies.

Mutation analysis of the entire keratin 5 and 14 genes in patients with We next assessed markers relevant to keratinocyte proliferation to identify possible causes for the defective barrier of Krt14 C373A skin relative to WT. 2016 Oct 13]. Keratins constitute two classes: designated as type I (acidic) keratin and type II (basic) keratin. Verma S, Pasternack SM, Rtten A, Ruzicka T, Betz RC, Hanneken S. The First Keratins modulate cell functions including receptor signaling, protein targeting, proliferation, migration, differentiation and inflammatory and immune responses. Krt14-/- skin keratinocytes (Feng and Coulombe, 2015a; Feng and Coulombe, 2015b) were cultured in FAD medium. Mice were anesthetized using isoflurane (delivered by inhalation) during TEWL measurements. We also showed that loss of the stutter cysteine alters K14s ability to become part of the dense meshwork of keratin filaments that occurs in the perinuclear space of early differentiating keratinocytes (Lee et al., 2012; Feng and Coulombe, 2015a; Feng and Coulombe, 2015b). Keratin genes expressed in differentiating keratinocytes of epidermis, including the type I Krt10 and type Krt1 and Krt2, do not feature proximal TEAD binding sites (Figure 7figure supplement 1; Supplementary file 2) and thus are not likely to be transcribed in a TEAD/YAP-dependent fashion in epidermis. Outside the cell, it provides nature with the building blocks for making hard and strong appendages such as hair, horns, nails and hooves. Four lines of evidence support the contention that K10, in particular, is a strong candidate for K14-like regulation of YAP1 subcellular partitioning and Hippo signaling in the epidermis. The human keratin family c. 2011. Keratins are among the most abundant proteins in epithelial cells, in which they occur as a cytoplasmic network of 10-nm-wide intermediate filaments. 2014 Further, they link this modification to the YAP binding partner 14-3-3 which impacts YAP signaling.

N=3 biological replicates. In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses. Eur J Cell Biol. Cells or minced tissue were lysed in cold urea lysis buffer (pH 7.0, 6.5M urea, 50 mM Tris-HCl, 150 mM sodium chloride, 5 mM ethylenediaminetetraacetic acid (EDTA), 0.1% Triton X-100, 50 M N-ethylmaleimide, 1 mM phenylmethanesulfonyl fluoride (PMSF), 1 g/mL each of cymostatin, leupeptin, and pepstatin, 10 g/mL each of aprotinin and benzamidine, 2 g/mL antipain, and 50 mM sodium fluoride).

2003 Apr;21(4):447. Review. While these molecules are designed by Nature to self-assemble into filaments, they contain ample chemical modalities to allow for alternative arrangements such that in vitro assembly is not limited to filaments.

Potential transgenic founders were screened using restriction digestion of PCR product extending beyond the repair template oligonucleotide and findings were confirmed by direct DNA sequencing (data not shown). The mechanisms through which new progenitor cells are produced at the base of this stratified epithelium, pace themselves through differentiation, and maintain tissue architecture and function in spite of a high rate of cell loss at the skin surface are only partially understood (Wells and Watt, 2018). Approx. This chapter focuses on keratins that are expressed in skin epithelia, and details a number of basic protocols and assays that have proven useful for analyses being carried out in skin. (F)same as E, except that F-actin (via phalloidin) and Ser19-phosphorylated myosin light chain pMLC (Ser19) are stained. Mechanically, the high sulfur (high cysteine) proteins make up the matrix of these tissues that are reinforced by low sulfur keratins that take a more fibrous structure. Co-immunoprecipitation assays confirmed that transfected, HA tagged-14-3-3 physically interact with endogenous WT K14 and Krt14373A mutant protein in mouse keratinocytes in primary culture (Figure 4B and data not shown).

This study demonstrates that tracking the dynamic behavior of a GPCR is an efficient way to link GPCR conformations to their functions, therefore improving the development of drugs targeting specific receptor conformations. Keratins play an important role in biological functioning of cells. K14-dependent disulfide bonding is low in the absence of calcium and rises of the course of days after adding calcium to primary cultures of WT mouse keratinocytes (Figure 4figure supplement 1A,B). Across tissues, Claudin5 exhibited diminishing expression along the arteriovenous axis, correlating with EC barrier integrity.

As highlighted by the color code used in frames (a) and (b), individual type I and type II keratin genes belonging to the same subgroup, based on the primary structure of their protein products, tend to be clustered in the genome. This screen identified 14-3-3 and other 14-4-3 isoforms as major interacting partners for K14 in WT cell cultures (Figure 4A and Supplementary file 1). Keratin fragments are released in pathological conditions, serving as disease markers, and keratins assist in the differential diagnosis of lung neoplasms. The tools to induce MDBs, ways of their visualization and quantification, as well as the possibilities to detect SEK variants in humans are summarized. Available from ScienceDirect is a registered trademark of Elsevier B.V. ScienceDirect is a registered trademark of Elsevier B.V. Ocular Surface Disease: Cornea, Conjunctiva and Tear Film, Encyclopedia of Respiratory Medicine(Second Edition), Fibrous protein-based biomaterials (silk, keratin, elastin, and resilin proteins) for tissue regeneration and repair, Peptides and Proteins as Biomaterials for Tissue Regeneration and Repair, Biologically Inspired and Biomolecular Materials, Tissues & Organs | Keratins and the Skin, Encyclopedia of Biological Chemistry (Third Edition), Physiology of the Gastrointestinal Tract (Sixth Edition), Encyclopedia of Biological Chemistry (Second Edition). From an environmental point of view, the success of a top-down approach of recycling keratinous biowastes from both human and animal sources, through the creation of high value keratin based products, is a highly green and sustainable strategy that will be welcome by society and consumers.