Keratin Intermediate Filaments Research Articles

Ectopic Expression of a Truncated Isoform of Hair Keratin 81 in Breast Cancer Alters Biophysical Characteristics to Promote Metastatic Propensity.

Keratins are an integral part of cell structure and function. Here, it is shown that ectopic expression of a truncated isoform of keratin 81 (tKRT81) in breast cancer is upregulated in metastatic lesions compared to primary tumors and patient-derived circulating tumor cells, and is associated with more aggressive subtypes. tKRT81 physically interacts with keratin 18 (KRT18) and leads to changes in the cytosolic keratin intermediate filament network and desmosomal plaque formation. These structural changes are associated with a softer, more elastically deformable cancer cell with enhanced adhesion and clustering ability leading to greater in vivo lung metastatic burden. This work describes a novel biomechanical mechanism by which tKRT81 promotes metastasis, highlighting the importance of the biophysical characteristics of tumor cells.

In vitro effects of wool-derived keratin on human dental pulp-derived stem cells for endodontic applications

In this study we examine the influence of wool-derived keratin intermediate filament proteins (kIFPs) on human dental pulp-derived stem cells (hDPSCs). kIFPs were diluted (10 mg/mL to 0.001 mg/mL) in cell culture media. Effects on hDPSCs proliferation were measured using Alamar blue assay. Keratin concentrations of 1 mg/mL and 0.1 mg/mL were tested for odontogenic differentiation and mineralisation. Alkaline phosphatase (ALP) quantification (7th, 14th, and 21st days), alizarin red S (AR-S) staining and calcium quantification (21st day), reverse transcription polymerase chain reaction (RT-PCR, collagen expression), and immunocytochemical staining for dentin matrix protein (DMP) were performed. hDPSCs showed higher proliferation with kIFPs of 0.1 mg/mL or less (p < 0.0001). The 0.1 mg/mL keratin concentration promoted odontogenic differentiation, confirmed by increased ALP activity, significant calcium deposits (AR-S staining, p < 0.05), up-regulated collagen expression (RT-PCR, p < 0.05), and positive DMP staining. These results suggest that kIFPs could be a potential biomaterial for pulp-dentin regeneration.

Investigation of lncRNA in Bos taurus Mammary Tissue during Dry and Lactation Periods.

This study aims to collect RNA-Seq data from Bos taurus samples representing dry and lactating mammary tissue, identify lncRNA transcripts, and analyze findings for their features and functional annotation. This allows for connections to be drawn between lncRNA and the lactation process. RNA-Seq data from 103 samples of Bos taurus mammary tissue were gathered from publicly available databases (60 dry, 43 lactating). The samples were filtered to reveal 214 dry mammary lncRNA transcripts and 517 lactating mammary lncRNA transcripts. The lncRNAs met common lncRNA characteristics such as shorter length, fewer exons, lower expression levels, and less sequence conservation when compared to the genome. Interestingly, several lncRNAs showed sequence similarity to genes associated with strong hair keratin intermediate filaments. Human breast cancer research has associated strong hair keratin filaments with mammary tissue cellular resilience. The lncRNAs were also associated with several genes/proteins that linked to pregnancy using expression correlation and gene ontology. Such findings indicate that there are crucial relationships between the lncRNAs found in mammary tissue and the development of the tissue, to meet both the animal's needs and our own production needs; these relationships should be further investigated to ensure that we continue to breed the most resilient, efficient dairy cattle.

Alterations promoted by acid straightening and/or bleaching in hair microstructures.

Human hair is a biopolymer constituted mainly of keratin intermediate filaments, lipids, pigments and water. Cosmetic treatments usually interact with the hair at the molecular level, inducing changes in its components and modifying the physicochemical and mechanical properties of the fibers. Here, the effect of acid straightening on the morphology and ultrastructure of Caucasian hair was investigated by a group of complementary experimental methods: wide-, small- and ultra-small-angle X-ray scattering; high-resolution 3D X-ray microscopy; quasi-elastic neutron scattering and inelastic neutron scattering; thermogravimetry-mass spectrometry; and differential scanning calorimetry (DSC). X-ray diffraction patterns showed that acid straightening associated with a flat iron (∼180°C) changed the cortex of the fiber, shown by denaturation of the intermediate filaments (measured by DSC). The increase in the spacing of the lipid layers and the observation of the dehydration behavior of the fiber provided indications that water may be confined between these layers, while neutron spectroscopy showed alterations in the vibration mode of the CH2 groups of the lipids and an increase of the proton (H+) mobility in the hair structure. The latter may be associated with the extremely low pH of the formulation (pH ≃ 1). Additionally, this investigation showed that bleached hair (one-time bleached) is more damaged by the action of acid straightening than virgin hair, which was shown by a threefold increase in the percentage of total porosity of the tresses. The obtained results demonstrate that the investigation approach proposed here can provide very important thermodynamic and structural information on induced changes of hair structure, and certainly can be applied for the evaluation of the action mode and efficiency of cosmetic treatments.

Deacetylation via SIRT2 prevents keratin-mutation-associated injury and keratin aggregation.

Keratin (K) and other intermediate filament (IF) protein mutations at conserved arginines disrupt keratin filaments into aggregates and cause human epidermolysis bullosa simplex (EBS; K14-R125C) or predispose to mouse liver injury (K18-R90C). The challenge for more than 70 IF-associated diseases is the lack of clinically utilized IF-targeted therapies. We used high-throughput drug screening to identify compounds that normalized mutation-triggered keratin filament disruption. Parthenolide, a plant sesquiterpene lactone, dramatically reversed keratin filament disruption and protected cells and mice expressing K18-R90C from apoptosis. K18-R90C became hyperacetylated compared with K18-WT and treatment with parthenolide normalized K18 acetylation. Parthenolide upregulated the NAD-dependent SIRT2, and increased SIRT2-keratin association. SIRT2 knockdown or pharmacologic inhibition blocked the parthenolide effect, while site-specific Lys-to-Arg mutation of keratin acetylation sites normalized K18-R90C filaments. Treatment of K18-R90C-expressing cells and mice with nicotinamide mononucleotide had a parthenolide-like protective effect. In 2 human K18 variants that associate with human fatal drug-induced liver injury, parthenolide protected K18-D89H- but not K8-K393R-induced filament disruption and cell death. Importantly, parthenolide normalized K14-R125C-mediated filament disruption in keratinocytes and inhibited dispase-triggered keratinocyte sheet fragmentation and Fas-mediated apoptosis. Therefore, keratin acetylation may provide a novel therapeutic target for some keratin-associated diseases.

BG06 Beyond keratin: when simplex is not so simple

Abstract Epidermolysis bullosa simplex (EBS) is a heterogeneous, congenital, mucocutaneous fragility syndrome, characterized ultrastructurally by cleavage within the basal keratinocyte. Seventy-five per cent of cases are attributable to mutations in KRT5 and KRT14, the vast majority of which are dominant negative mutations, resulting in impaired keratin intermediate filament dynamics within the basal layer of the epidermis. The presenting phenotype, including extracutaneous manifestations and overall severity, is often closely linked to the underlying genetic mutation; however, genotype–­phenotype correlation is poorly resolved in a minority of cases, and there is an approximately additional 15% of patients in whom genetic abnormalities are not identified. However, recent advances in next-generation sequencing technologies have expanded the mutational database. We describe two cases of EBS with rare underlying mutations and distinct clinical phenotypes. A 46-year-old man presented with widespread blistering primarily affecting his hands, feet, elbows and knees since childhood. He had unaffected, consanguineous parents and one of his three siblings was affected. Other presenting features included dystrophic nails, focal plantar keratoderma, truncal mottled pigmentation and large areas of hypopigmentation on the back. We considered a diagnosis of ‘EBS with mottled pigmentation’, but the autosomal recessive mode of inheritance and dystrophic nails were atypical. DNA whole-exome sequencing (WES) and bidirectional Sanger sequencing revealed a novel homozygous PLEC mutation [c.94dupG p.(Ala32GlyfsTer3)]. Pathogenic PLEC variants are known to cause autosomal dominant EBS, the Ogna type (MIM131950), autosomal recessive EBS with muscular dystrophy (MIM226670) and autosomal recessive EBS with pyloric atresia (MIM612138). To date, our patient has not manifested any signs of progressive muscle weakness, but the presenting age can vary. The second patient was a 30-year-old woman who presented with blistering on her flexures, feet, hands and friction sites since the age of 4 months. She had unaffected, consanguineous parents and eight unaffected siblings. WES and confirmatory bidirectional Sanger sequencing identified a homozygous nonsense mutation in DST [c.3370C&amp;gt;T p.(Gln1124Ter)]. DST encodes the epithelial isoform of bullous pemphigoid antigen-1, present in the skin, muscle and neuronal tissues. Only 11 patients with DST mutation-induced EBS have been reported to date. The cutaneous phenotype is reported to be blisters, postinflammatory dyspigmentation, scarring, nail dystrophy, keratoderma and hyperhidrosis. If the affected exon is present in neuronal isoforms and neuronal tissue, neurological symptoms are expected. Unlike the patient reported elsewhere who harbours exactly the same mutation, our patient has reported no neurological issues to date. These cases highlight the importance of unravelling genotype–phenotype correlation in rare autosomal recessive forms of EBS, with possible implications for future prognosis and management.

Retromer Chaperones: Potential Therapeutics for Treatment of Skin Disease?

Retromer Chaperones: Potential Therapeutics for Treatment of Skin Disease?

An in vitro analysis of the hemostatic efficacy of fibrinogen precipitation with varied keratin fraction compositions

In preclinical studies, human hair has demonstrated effective hemostatic properties, potentially attributed to keratin proteins facilitating rapid conversion of fibrinogen to fibrin during coagulation. However, the rational use of human hair keratin for hemostasis remains unclear, given its complex mixture of proteins with diverse molecular weights and structures, leading to variable hemostatic capacity. To optimize the rational utilization of human hair keratin for hemostasis, we investigated the effects of different keratin fractions on keratin-mediated fibrinogen precipitation using a fibrin generation assay. Our study focused on high molecular weight keratin intermediate filaments (KIFs) and lower molecular weight keratin-associated proteins (KAPs) combined in various ratios during the fibrin generation. Scanning electron microscope analysis of the precipitates revealed a filamentous pattern with a broad distribution of fiber diameters, likely due to the diversity of keratin mixtures involved. An equal proportion of KIFs and KAPs in the mixture yielded the most extensive precipitation of soluble fibrinogen in an in vitro study, potentially due to structure-induced exposure of active sites. However, all hair protein samples exhibited diverse catalytic behaviors compared to thrombin, highlighting the potential of utilizing specific hair fractions to develop hair protein-based hemostatic materials with optimized capacity.

Architecture and dynamics of a desmosome-endoplasmic reticulum complex.

The endoplasmic reticulum (ER) forms a dynamic network that contacts other cellular membranes to regulate stress responses, calcium signalling and lipid transfer. Here, using high-resolution volume electron microscopy, we find that the ER forms a previously unknown association with keratin intermediate filaments and desmosomal cell-cell junctions. Peripheral ER assembles into mirror image-like arrangements at desmosomes and exhibits nanometre proximity to keratin filaments and the desmosome cytoplasmic plaque. ER tubules exhibit stable associations with desmosomes, and perturbation of desmosomes or keratin filaments alters ER organization, mobility and expression of ER stress transcripts. These findings indicate that desmosomes and the keratin cytoskeleton regulate the distribution, function and dynamics of the ER network. Overall, this study reveals a previously unknown subcellular architecture defined by the structural integration of ER tubules with an epithelial intercellular junction.

General aspects on skin development in vertebrates with emphasis on sauropsids epidermis

General cellular aspects of skin development in vertebrates are presented with emphasis on the epidermis of sauropsids. Anamniote skin develops into a multilayered mucogenic and soft keratinized epidermis made of Intermediate Filament Keratins (IFKs) that is reinforced in most fish and few anurans by dermal bony and fibrous scales. In amniotes, the developing epidermis in contact with the amniotic fluid initially transits through a mucogenic phase recalling that of their anamniotes progenitors. A new gene cluster termed EDC (Epidermal Differentiation Complex) evolved in amniotes contributing to the origin of the stratum corneum. The EDC contains numerous genes coding for over 100 types of corneous proteins (CPs). In sauropsids 2–8 layers of embryonic epidermis accumulate soft keratins (IFKs) but do not form a compact corneous layer. The embryonic epidermis of reptiles and birds produces small amount of other, poorly known proteins in addition to IFKs and mucins. In the following development, a resistant corneous layer is formed underneath the embryonic epidermis that is shed before hatching. The definitive corneous epidermis of sauropsids is mainly composed of CBPs (Corneous beta proteins, formerly indicated as beta-keratins) derived from the EDC. CBPs belong to a gene sub-family of CPs unique for sauropsids, contain an inner amino acid region formed by beta-sheets, are rich in cysteine and glycine, and make most of the protein composition of scales, claws, beaks and feathers. In mammalian epidermis CPs missing the beta-sheet region are instead produced, and include loricrin, involucrin, filaggrin and various cornulins. Small amount of CPs accumulate in the 2–3 layers of mammalian embryonic epidermis and their appendages, that is replaced with the definitive corneous layers before birth. Differently from sauropsids, mammals utilize KAPs (keratin associated proteins) rich in cysteine and glycine for making the hard corneous material of hairs, claws, hooves, horns, and occasionally also scales.

Development and structure of the skin in the Australian lungfish (Neoceratodus forsteri) in relation to epidermal adaptation of tetrapods

AbstractAnalysis of skin development in the Australian lungfish, Neoceratodus forsteri, indicates that keratinocytes form 2 to 3 irregular layers in tadpoles of 2–5 cm in length and are fully stratified (7–12 layers) in juveniles of 16.5–26 cm in length. The epidermis produces mucus and mainly contains intermediate filament keratins of acidic nature but also a minute amount of neutral‐basic keratins (pI 7.0–7.3), which is unique among fish. The latter keratins of 60–62 kDa, are generally present in tetrapods but the specific gene structure and amino sequence of acidic, neutral and basic keratins in N. forsteri are presently unknown. No other epidermal proteins are detected although biochemical data suggest the presence of non‐keratin proteins, including a basic type (pI 8.0–8.2) of around 32 kDa of molecular weight. The spinulated bony layer of elasmoid scales contains glycoproteins involved in mineralization, such as osteonectin and osteopontin. Also, alkaline phosphatase, involved in calcium precipitation onto the collagen matrix, has been detected in scales of juveniles up to 26 cm in length. The present review concludes that the knowledge of the data derived from epidermal and dermal protein annotation from the recently sequenced genome of this fish species will contribute to address the study on the evolution of tetrapod skin.

Mechanical anisotropy of hair affected by genetic diseases highlights structural information related to differential crosslinking in keratins.

Previous work with Atomic Force Microscope (AFM) nanoindentation, on longitudinal and cross-sections of the human hair fibre, allowed for the derivation of a model for the mechanical behaviour of human hair, called the Anisotropic Index. Expanding that research further, and by applying this model, the nanomechanical behaviour of hairs from patients with the disease Trichothiodystrophy (TTD) has been examined and structural insights, gained from combining the AFM results with Differential Scanning Calorimetry (DSC) experiments and tensile measurements, suggests that TTD-affected hairs have a relatively increased amount of Keratin Intermediate Filaments, contained in compartments of differing crosslinking extent. The associated calculations of axial and transverse Young's Moduli deliver values in good agreement with the measured fibre mechanics. Furthermore, comparing these findings with the results previously obtained from the study of hairs from patients with the disease Monilethrix, it is shown that the Anisotropic Index correlates well with the known deficiencies in both hair types obtained from such patients and allows for discerning between the Control hair and from those affected by the two diseases. AFM nanoindentation along and across the fibre axis and the Anisotropic Index thus appear to reveal structural details of hair not otherwise acquirable, whilst DSC may offer a quick and simple method for distinguishing between different severities of TTD.

The Story of the Finest Armor: Developmental Aspects of Reptile Skin.

The reptile skin is a barrier against water loss and pathogens and an armor for mechanical damages. The integument of reptiles consists of two main layers: the epidermis and the dermis. The epidermis, the hard cover of the body which has an armor-like role, varies among extant reptiles in terms of structural aspects such as thickness, hardness or the kinds of appendages it constitutes. The reptile epithelial cells of the epidermis (keratinocytes) are composed of two main proteins: intermediate filament keratins (IFKs) and corneous beta proteins (CBPs). The outer horny layer of the epidermis, stratum corneum, is constituted of keratinocytes by means of terminal differentiation or cornification which is a result of the protein interactions where CBPs associate with and coat the initial scaffold of IFKs. Reptiles were able to colonize the terrestrial environment due to the changes in these epidermal structures, which led to various cornified epidermal appendages such as scales and scutes, a beak, claws or setae. Developmental and structural aspects of the epidermal CBPs as well as their shared chromosomal locus (EDC) indicate an ancestral origin that gave rise to the finest armor of reptilians.

Abstract A016: Aging-dependent emergent mechanical properties of single epithelial cells exploited for detection of breast cancer susceptibility

Abstract Age is the major risk factor in most carcinomas, yet, little is known about the specific reasons aging increases cancer susceptibility. In the mammary gland, luminal epithelial cells rank high as the putative breast cancer cell of origin. Dysregulation of keratin intermediate filament proteins exemplifies a hallmark age-dependent change in luminal cells, which implicates mechanical states unique to cancer susceptible cells. We implemented mechano-node-pore sensing (mechano-NPS), a multi-parametric single-cell analysis that simultaneously measures cell diameter, resistance to compressive deformation, transverse deformation under constant strain, and recovery time after deformation. We demonstrated that the epithelial lineages, chronological ages, and stages of cancer progression of primary human mammary epithelial cells (HMEC) exhibited discrete mechanical phenotypes. We trained a machine learning model that accurately predicted the chronological age of average risk HMEC cells based exclusively on mechanical properties. Application of the model to cells from women who are germline carriers of high-risk cancer-causing mutations showed that they are mechanically old irrespective of their chronological age, suggesting that mechanical states could be a window into detection and prevention of cancer susceptible states. Indeed, this mechano-age model detected high-risk women with &amp;gt;90% accuracy. Mass spectrometry and cell-based functional assays in mammary epithelia revealed that cytoskeleton related proteins keratin 14 (KRT14) and pseudopodium enriched atypical kinase 1 (PEAK1) were key drivers of age-dependent mechanical signatures. Pharmacological and gene silencing approaches that targeted KRT14 and PEAK1 modulated the mechanical age of HMEC and, in the case of PEAK1 modulation, ablated luminal epithelial cells in an age- and lineage- dependent manner. We define an intersection between mechanical phenotypes and novel age-dependent changes in cytoskeleton-related proteins that we hypothesize can be exploited to assess an individual’s breast cancer susceptibility and provide new targets for cancer-prevention strategies. Citation Format: Stefan Hinz, Masaru Miyano, Antigoni Manousopoulou, Rosalyn W. Sayaman, Kristina Y. Aguilera, Michael E. Todhunter, Jennifer C. Lopez, Leo D. Wang, Lydia L. Sohn, Mark A. LaBarge. Aging-dependent emergent mechanical properties of single epithelial cells exploited for detection of breast cancer susceptibility [abstract]. In: Proceedings of the AACR Special Conference: Aging and Cancer; 2022 Nov 17-20; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_1):Abstract nr A016.

Polymorphism of keratin intermediate filament type- i gene and its association with wool quality traits in Magra sheep

A study was conducted to identify the polymorphism of keratin intermediate filament type-I (KIF-I) gene and its association with wool quality traits in Magra sheep. Blood samples and greasy fleece yield data of 162 adult males and females of Magra sheep (2 to 4 yr old) were collected. Following isolation of genomic DNA, genotyping of KIF-I gene was done by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). The PCR-RFLP of KIF-I gene at locus KRT1.2 generated three genotype viz., AA, AB and BB with genotype frequencies of 0.49, 0.43 and 0.08, respectively. The frequencies for A and B allele were 0.71 and 0.29, respectively. Hardy-Weinberg equilibrium indicated that the population was in genetic equilibrium with respect to KIF-I gene. In conclusion, greater frequencies of allele A and genotype AA and AB are suggestive of its importance in quality of wool traits in Magra sheep.

Regionally specific levels and patterns of keratin 8 expression in the mouse embryo visceral endoderm emerge upon anterior-posterior axis determination.

The mechanical properties of the different germ layers of the early mammalian embryo are likely to be critical for morphogenesis. Cytoskeleton components (actin and myosin, microtubules, intermediate filaments) are major determinants of epithelial plasticity and resilience to stress. Here, we take advantage of a mouse reporter for Keratin 8 to record the pattern of the keratin intermediate filaments network in the first epithelia of the developing mouse embryo. At the blastocyst stage, Keratin 8 is strongly expressed in the trophectoderm, and undetectable in the inner cell mass and its derivatives, the epiblast and primitive endoderm. Visceral endoderm cells that differentiate from the primitive endoderm at the egg cylinder stage display apical Keratin 8 filaments. Upon migration of the Anterior Visceral Endoderm and determination of the anterior-posterior axis, Keratin 8 becomes regionally distributed, with a stronger expression in embryonic, compared to extra-embryonic, visceral endoderm. This pattern emerges concomitantly to a modification of the distribution of Filamentous (F)-actin, from a cortical ring to a dense apical shroud, in extra-embryonic visceral endoderm only. Those regional characteristics are maintained across gastrulation. Interestingly, for each stage and region of the embryo, adjacent germ layers display contrasted levels of keratin filaments, which may play a role in their adaptation to growth and morphological changes.

185 aPKC is a rheostat that controls basal junctional dynamics and suprabasal cytoskeletal organization essential for epidermal dynamics and integrity

The stratified skin epidermis needs dynamic cell rearrangement to self-renew while maintaining tissue integrity to guarantee barrier function in the face of continuous mechanical stress. The contractile actomyosin cytoskeleton connects with intercellular adheres junctions to enable cellular rearrangements and migration, whereas the physical properties of the keratin intermediate filament cytoskeleton through its interaction with intercellular desmosomes mechanically strengthen tissues. How cells coordinate resilience and cellular reorganization to maintain tissue integrity during renewal while adapting to mechanical stress is not known.

Cortical tension regulates desmosomal morphogenesis.

Mechanical stability is a fundamental and essential property of epithelial cell sheets. It is in large part determined by cell-cell adhesion sites that are tightly integrated by the cortical cytoskeleton. An intimate crosstalk between the adherens junction-associated contractile actomyosin system and the desmosome-anchored keratin intermediate filament system is decisive for dynamic regulation of epithelial mechanics. A major question in the field is whether and in which way mechanical stress affects junctional plasticity. This is especially true for the desmosome-keratin scaffold whose role in force-sensing is virtually unknown. To examine this question, we inactivated the actomyosin system in human keratinocytes (HaCaT) and canine kidney cells (MDCK) and monitored changes in desmosomal protein turnover.Partial inhibition of myosin II by para-nitro-blebbistatin led to a decrease of the cells' elastic modulus and to reduced desmosomal protein turnover in regions where nascent desmosomes are formed and, to a lower degree, in regions where larger, more mature desmosomes are present. Interestingly, desmosomal proteins are affected differently: a significant decrease in turnover was observed for the desmosomal plaque protein desmoplakin I (DspI), which links keratin filaments to the desmosomal core, and the transmembrane cadherin desmoglein 2 (Dsg2). On the other hand, the turnover of another type of desmosomal cadherin, desmocollin 2 (Dsc2), was not significantly altered under the tested conditions. Similarly, the turnover of the adherens junction-associated E-cadherin was not affected by the low doses of para-nitro-blebbistatin. Inhibition of actin polymerization by low dose latrunculin B treatment and of ROCK-driven actomyosin contractility by Y-27632 treatment also induced a significant decrease in desmosomal DspI turnover. Taken together, we conclude that changes in the cortical force balance affect desmosome formation and growth. Furthermore, they differentially modulate desmosomal protein turnover resulting in changes of desmosome composition. We take the observations as evidence for a hitherto unknown desmosomal mechanosensing and mechanoresponse pathway responding to an altered force balance.

Expression of vimentin alters cell mechanics, cell-cell adhesion, and gene expression profiles suggesting the induction of a hybrid EMT in human mammary epithelial cells.

Vimentin is a Type III intermediate filament (VIF) cytoskeletal protein that regulates the mechanical and migratory behavior of cells. Its expression is considered to be a marker for the epithelial to mesenchymal transition (EMT) that takes place in tumor metastasis. However, the molecular mechanisms regulated by the expression of vimentin in the EMT remain largely unexplored. We created MCF7 epithelial cell lines expressing vimentin from a cumate-inducible promoter to address this question. When vimentin expression was induced in these cells, extensive cytoplasmic VIF networks were assembled accompanied by changes in the organization of the endogenous keratin intermediate filament networks and disruption of desmosomes. Significant reductions in intercellular forces by the cells expressing VIFs were measured by quantitative monolayer traction force and stress microscopy. In contrast, laser trapping micro-rheology revealed that the cytoplasm of MCF7 cells expressing VIFs was stiffer than the uninduced cells. Vimentin expression activated transcription of genes involved in pathways responsible for cell migration and locomotion. Importantly, the EMT related transcription factor TWIST1 was upregulated only in wild type vimentin expressing cells and not in cells expressing a mutant non-polymerized form of vimentin, which only formed unit length filaments (ULF). Taken together, our results suggest that vimentin expression induces a hybrid EMT correlated with the upregulation of genes involved in cell migration.

Allele-Specific Inactivation of an Autosomal Dominant Epidermolysis Bullosa Simplex Mutation Using CRISPR-Cas9.

Epidermolysis bullosa simplex (EBS) is a rare mechanobullous disease caused by dominant-negative mutations in either keratin 5 (KRT5) or keratin 14 (KRT14) genes. Until now, there is no cure for EBS and the care is primarily palliative. The discovery of the clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 system raised hope for the treatment of EBS and many other autosomal dominant diseases by mutant allele-specific gene disruption. In this study, we aim to disrupt the mutant allele for the heterozygous EBS pathogenic variation c.449T>C (p.Leu150Pro) within KRT5. This mutation generates, naturally, a novel protospacer-adjacent motif for the endonuclease Streptococcus pyogenes Cas9. Thus, we designed a single-guide RNA that guides the Cas9 to introduce a DNA cleavage of the mutant allele in patient's keratinocytes. Then, transfected cells were single-cell cloned and analyzed by deep sequencing. The expression of KRT5 and KRT14 was quantified, and the keratin intermediate filament stability was assessed. Results showed successful stringent mutant allele-specific knockout. An absence of synthesis of mutant transcript was further confirmed indicating permanent mutant allele-specific inactivation. Edited EBS patient keratinocytes produced a lower amount of K5 and K14 proteins compared with nonedited EBS cells, and no disturbance of cellular properties was observed.