N=3 replicates for each sample. For ATAC-seq data, alignment files were loaded into the Galaxy web platform (Afgan et al., 2016) using the public server at usegalaxy.org. Krt14 C373A mice were generated using the RNA-guided CRISPR-Cas9 system as described (Wang et al., 2013). Report of KRT5 Mutation Underlying Acantholytic Dowling-Degos Disease with Together these findings point to a misregulation of 14-3-3 and YAP as likely contributors to the epidermal phenotype exhibited in the ear and tail skin of Krt14 C373A mice. However, this attractive proposition will not take off if the functional benefits of the material cannot be proven. This comparison makes use of the sequences from the head and central rod domain for each keratin. Approx. Type I and II subtypes of keratin form polymers in an epithelial cell-type-specific manner, which is also influenced by developmental stage, cellular differentiation, and pathophysiology. Epidermolysis Bullosa Simplex. Coulombe, C.-H. Lee, in Encyclopedia of Biological Chemistry (Second Edition), 2013. In the fiber cortex is a bundle of protein strands mainly composed of -keratins (50%60%), -keratins (20%30%), and melanin granules trapped inside the cortex strands, which accounts for 70%95% of the total mass of the whole hair. Ng, in Comprehensive Biomaterials II, 2017. (A) Toluidine blue-stained sections (1 mm thick from epoxy-embedded skin of young adult WT and Krt14 C373A mice. In hair, the form is most commonly associated with a relaxed state, while the form is induced when keratin is extended or stretched in the presence of higher humidity and temperature. Scale bar, 20 m. After nucleofection, cells were plated on collagen-coated coverglass and processed for analysis. Br J Dermatol. N=3 biological replicates. Keratins are a separate family of fibrous structural proteins that make up the main constituents of hair, nails, and hooves of other mammals. Functional analysis identified tissue-specific differences in leakage patterning and response to the leakage agonist histamine. 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). 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. There is a strong precedent for interactions between 14-3-3 proteins and keratins, including K18 (Liao and Omary, 1996; Ku et al., 1998), K17 (Kim et al., 2006), which occur in a phosphorylation-dependent fashion. 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). We next assessed markers relevant to keratinocyte proliferation to identify possible causes for the defective barrier of Krt14 C373A skin relative to WT. Keratinocytes from 1 or 2 day old C57Bl/6 newborn mouse skin were isolated as described (Wang et al., 2016), and cultured in FAD medium (low calcium, 0.07 mM) for 1 day. 2010 Jul;163(1):197-200. doi: If the phenotypes are also present in backskin, this would complicate the conclusions. The distribution of cysteine residues in mouse K14 protein is schematized in Figure 1A. 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. 1.0 g RNA was reverse-transcribed with the iScript cDNA Synthesis kit (1708891BUN, Bio-Rad Laboratories) using the manufacturers protocol. 2005 Aug;125(2):239-43. and de novo mutations in the KRT5 and KRT14 genes, phenotype/genotype The YAP1 and TEAD genes (human) are themselves poised to be positively regulated by YAP in progenitor keratinocytes (Figure 7figure supplement 1; Supplementary file 2). We also observed a greater frequency (~3 fold) of TUNEL-positive nuclei in Krt14 C373A epidermis compared to WT, with apoptotic cell death confined to the suprabasal compartment (Figure 3C,D). Besides, the editors and reviewers should agree that much remains to be learned about the distribution and evolution of forces, on various scales, as progenitor keratinocytes initiate terminal differentiation and delaminate to enter the suprabasal compartment. In addition, a disulfide bonding-rich perinuclear network of keratin filaments could also afford protection to the nucleus and the genome during delamination (Lee et al., 2012), thus extending the known role of K5-K14 IFs in providing mechanical support in basal keratinocytes (Coulombe et al., 1991a; Ramms et al., 2013). 1) The reviewers felt that the analysis of mechanics (mostly data in Figure 6) were underdeveloped and gave a few suggestions for how this might be strengthened experimentally. The latter yielded compelling results that have been added to Figure 6. We have performed stainings of skin tissue sections using antibodies directed at vinculin and the -catenin a-18 (mechanically-sensitive) epitope. Students t test: n.s., no statistical difference; ***p<0.005. Our data clearly establish that all three elements work in concert in this setting, with keratin dependent-disulfide bonding and the resulting impact on YAP subcellular partitioning and activity revealed as both a novel and unexpected determinant of these processes. Epub 2011 Feb 17. Monitoring the status of such elements provides a test for altered mechanosensing or mechanotransduction. MedlinePlus also links to health information from non-government Web sites. Nuclei are stained with DAPI in frames A, C, E and F. Scale bars, 20 m. Mutations that cause severe forms of the disorder badly disrupt the assembly of keratin intermediate filaments, while mutations that result in milder forms impair keratin filament assembly to a lesser degree. 2003 Apr;21(4):447. Review. We also find that the immunofluorescence signal for -catenin, lamin A/C, F-actin stress fibers and phosphorylated myosin light chain II (pMLC Ser19) are each increased in Krt14 C373A relative to WT (Figure 6E,F). In summary, keratins have presented themselves as exciting new biomaterials which are distinctly different from the suite of natural biomaterials that are currently being used. For decades, scientists have been developing ways to solubilize these hard structures, paving the way for the possibility to reassemble keratins into a wide variety of templates from a bottoms-up approach. Dysfunctional and leaky blood vessels resulting from disruption of the endothelial cell (EC) barrier accompanies numerous diseases. Cryosections were allowed to thaw in PBS buffer at room temperature and incubated with primary antibodies followed by Alexa Fluorconjugated secondary antibodies (Thermo Fisher Scientific), counterstained in DAPI (1;5, 000 in PBS; D1306, Thermo Fisher Scientific), and mounted in FluorSave Reagent mounting medium (345789, Calbiochem) for indirect immuno-fluorescence (Hobbs et al., 2015; Kerns et al., 2016). Such species could non-enzymatically mediate K14-dependent disulfide bonding in the perinuclear cytoplasm of keratinocytes (Suzuki et al., 2019). Unraveling how these keratins interact with one another could have huge benefits in terms of selecting the right combinations as biomaterials to create a desired structure and environment. In basal keratinocytes of WT epidermis, keratin IFs typically occur as bundles near the nucleus. Importantly, immunostaining for keratin may assist the differential diagnosis of lung neoplasms. (F) Quantification of the number of p63-positive nuclei per mm of epidermis (left) and their distance from the basal lamina (right). Bogaert K, Wenzel J, Braun-Falco M, Rutten A, Rogers MA, Ruzicka T, Nthen MM, This new data is consistent with the reviewers prediction and with the observation that there is considerably less evidence for K14-dependent disulfide bonding in adult back skin tissue as reported in Figure 1C. Keratins play an important role in biological functioning of cells. The model proposes an identical role for the conserved cysteine in coil 2 of keratin 10, which is expressed early during terminal differentiation, thereby maintaining YAP1s sequestration to the cytoplasm and active Hippo signaling. Five random fields were sampled for each of 3 mice per genotype. (E) Relative mRNA levels (qRT-PCR) for YAP target genes Ccn1, Zeb1, Ccn2, and Snail2 in adult WT and Krt14 C373A tail skin. Invest Dermatol. Indirect immuno-fluorescence microscopy for -catenin, desmoglein one and lamin A/C. 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. The sloughing is removing both old cells, oils, and keratin protein in the process. Amemiya A, editors. The intermediate filament protein keratin 14 (K14) provides vital structural support in basal keratinocytes of epidermis. Normalized expression values for each target gene in all qRT-PCR experiments were derived from at least three biological replicates. We next assessed the distribution of 14-3-3 and YAP using indirect immunofluorescence of tissue sections prepared from WT and Krt14 C373A tail skin. RNA was harvested using TRIzol reagent (15596018, Thermo Fisher Scientific) and purified using the Nucleospin RNA kit (740955.250, Machery Nagel) according to the manufacturers instructions. **p<0.01, *p<0.05, n.s., non-significant. Third, the nucleus is known to function as a mechanosensor (Cao et al., 2017), and local forces impacted by the perinuclear network of keratin filaments could alter the mechanical gating of YAP across nuclear pores (Elosegui-Artola et al., 2017). The EC barrier is established through endothelial cell tight and adherens junctions. They are encoded by 54 evolutionarily conserved genes (28 type I, 26 type II) and regulated in a pairwise and tissue type-, differentiation-, and context-dependent manner. First, KRT10 (human) and Krt10 (mouse) expression is turned on at the earliest stage of terminal differentiation (Woodcock-Mitchell et al., 1982; Schweizer et al., 1984), and the K10 protein features a structurally and biochemically equivalent Cys residue at position 401 and is capable of interacting with 14-3-3 (Wilker et al., 2007; Huang et al., 2010). The left ears of age-matched WT and Krt14 C373A mice (23 months old) were topically treated with 40 l acetone twice daily for 7 days (Denda et al., 1996). The multifaceted roles of SEKs are increasingly appreciated due to findings obtained from transgenic mouse models and human studies that identified SEK variants in several digestive diseases. Short term studies suggest that keratin is well tolerated by tissues in vivo, and that degradation is slow. The diverse keratin intermediate filament family evolved from a common ancestral gene and shares a common structural plan. pBK-CMV His-GFP-K14WT or cysteine variants (Feng and Coulombe, 2015a; Feng and Coulombe, 2015b) were transfected into Krt14-/- skin keratinocytes using P1 Primary Cell 4D-Nucleofector X Kit (V4XP-1024, Lonza). sgRNA, single guide RNA; PAM, protospacer adjacent motif. What happens to the thickness, proliferation and YAP/14-3-3 localization? The KRT5 gene provides instructions for making a protein called keratin 5. (D) Indirect immunofluorescence for YAP in WT and Krt14 C373A tail skin sections. Samples were post-fixed in osmium tetroxide, counter-stained with uranyl acetate, and embedded in epoxy resin as previously described(Lessard et al., 2013). F. Costa, A.R. Researchers believe that keratin 5 interacts with pigment-producing cells called melanocytes to transport melanosomes, which are cellular structures within melanocytes that carry pigment called melanin.

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