Load Adaptation of Lamellipodial Actin Networks.

Actin filaments polymerizing against membranes power endocytosis, vesicular traffic, and cell motility. In vitro reconstitution studies suggest that the structure and the dynamics of actin networks respond to mechanical forces. We demonstrate that lamellipodial actin of migrating cells responds to mechanical load when membrane tension is modulated. In a steady state, migrating cell filaments assume the canonical dendritic geometry, defined by Arp2/3-generated 70° branch points. Increased tension triggers a dense network with a broadened range of angles, whereas decreased tension causes a shift to a sparse configuration dominated by filaments growing perpendicularly to the plasma membrane. We show that these responses emerge from the geometry of branched actin: when load per filament decreases, elongation speed increases and perpendicular filaments gradually outcompete others because they polymerize the shortest distance to the membrane, where they are protected from capping. This network-intrinsic geometrical adaptation mechanism tunes protrusive force in response to mechanical load.

Unbound Corneocyte Lipid Envelopes in 12R-Lipoxygenase Deficiency Support a Specific Role in Lipid-Protein Cross-Linking.

Loss-of-function mutations in arachidonate lipoxygenase 12B (ALOX12B) are an important cause of autosomal recessive congenital ichthyosis (ARCI). 12R-lipoxygenase (12R-LOX), the protein product of ALOX12B, has been proposed to covalently bind the corneocyte lipid envelope (CLE) to the proteinaceous corneocyte envelope, thereby providing a scaffold for the assembly of barrier-providing, mature lipid lamellae. To test this hypothesis, an in-depth ultrastructural examination of CLEs was performed in ALOX12B-/- human and Alox12b-/- mouse epidermis, extracting samples with pyridine to distinguish covalently attached CLEs from unbound (ie, noncovalently bound) CLEs. ALOX12B--/- stratum corneum contained abundant pyridine-extractable (ie, unbound) CLEs, compared with normal stratum corneum. These unbound CLEs were associated with defective post-secretory lipid processing, and were specific to 12R-LOX deficiency, because they were not observed with deficiency of the related ARCI-associated proteins, patatin-like phospholipase 1 (Pnpla1) or abhydrolase domain containing 5 (Abhd5). These results suggest that 12R-LOX contributes specifically to CLE-corneocyte envelope cross-linking, which appears to be a prerequisite for post-secretory lipid processing, and provide insights into the pathogenesis of 12R-LOX deficiency in this subtype of ARCI, as well as other conditions that display a defective CLE.

Loss of GPNMB Causes Autosomal-Recessive Amyloidosis Cutis Dyschromica in Humans.

Amyloidosis cutis dyschromica (ACD) is a distinct form of primary cutaneous amyloidosis characterized by generalized hyperpigmentation mottled with small hypopigmented macules on the trunks and limbs. Affected families and sporadic case subjects have been reported predominantly in East and Southeast Asian ethnicities; however, the genetic cause has not been elucidated. We report here that the compound heterozygosity or homozygosity of GPNMB truncating alleles is the cause of autosomal-recessive ACD. Six nonsense or frameshift mutations were identified in nine individuals diagnosed with ACD. Immunofluorescence analysis of skin biopsies showed that GPNMB is expressed in all epidermal cells, with the highest staining observed in melanocytes. GPNMB staining is significantly reduced in the lesional skin of affected individuals. Hyperpigmented lesions exhibited significantly increased amounts of DNA/keratin-positive amyloid deposits in the papillary dermis and infiltrating macrophages compared with hypo- or depigmented macules. Depigmentation of the lesions was attributable to loss of melanocytes. Intracytoplasmic fibrillary aggregates were observed in keratinocytes scattered in the lesional epidermis. Thus, our analysis indicates that loss of GPNMB, which has been implicated in melanosome formation, autophagy, phagocytosis, tissue repair, and negative regulation of inflammation, underlies autosomal-recessive ACD and provides insights into the etiology of amyloidosis and pigment dyschromia.

Keratinocytes Communicate with Sensory Neurons via Synaptic-like Contacts.

OBJECTIVE: Pain, temperature, and itch are conventionally thought to be exclusively transduced by the intraepidermal nerve endings. Although recent studies have shown that epidermal keratinocytes also participate in sensory transduction, the mechanism underlying keratinocyte communication with intraepidermal nerve endings remains poorly understood. We sought to demonstrate the synaptic character of the contacts between keratinocytes and sensory neurons and their involvement in sensory communication between keratinocytes and sensory neurons. METHODS: Contacts were explored by morphological, molecular, and functional approaches in cocultures of epidermal keratinocytes and sensory neurons. To interrogate whether structures observed in vitro were also present in the human epidermis, in situ correlative light electron microscopy was performed on human skin biopsies. RESULTS: Epidermal keratinocytes dialogue with sensory neurons through en passant synaptic-like contacts. These contacts have the ultrastructural features and molecular hallmarks of chemical synaptic-like contacts: narrow intercellular cleft, keratinocyte synaptic vesicles expressing synaptophysin and synaptotagmin 1, and sensory information transmitted from keratinocytes to sensory neurons through SNARE-mediated (syntaxin1) vesicle release. INTERPRETATION: By providing selective communication between keratinocytes and sensory neurons, synaptic-like contacts are the hubs of a 2-site receptor. The permanent epidermal turnover, implying a specific en passant structure and high plasticity, may have delayed their identification, thereby contributing to the long-held concept of nerve endings passing freely between keratinocytes. The discovery of keratinocyte-sensory neuron synaptic-like contacts may call for a reassessment of basic assumptions in cutaneous sensory perception and sheds new light on the pathophysiology of pain and itch as well as the physiology of touch. ANN NEUROL 2020;88:1205-1219.

Nabumetone induced photogenotoxicity mechanism mediated by ROS generation under environmental UV radiation in human keratinocytes (HaCaT) cell line.

Nabumetone (NB) is a non-steroidal anti-inflammatory drug (NSAID), prescribed for managing pain associated with acute/chronic rheumatoid arthritis, osteoarthritis and other musculoskeletal disorders. Though some incidences of photosensitivity have been reported, there is limited information available on its phototoxicity potential. In this study, NB photodegraded in a time-dependant manner (0-4 h) under UVA (1.5 mW/cm2), UVB (0.6 mW/cm2) and natural sunlight as observed through UV-vis spectrophotometer and the results were further confirmed with Ultra High-Performance Liquid Chromatography (UHPLC). Photosensitized NB generated reactive oxygen species (ROS) as observed by lipid peroxidation, suggesting oxidative degradation of lipids in cell membrane, thereby resulting in cell damage. MTT and NRU (neutral red uptake) assays revealed that NB induced phototoxicity in concentration-dependent manner (0.5, 1, 5, 10 µg/ml) under UVA, UVB and sunlight exposure (30 min) in human keratinocytes cell line (HaCaT), with significant phototoxicity at the concentration of 5 µg/ml. Photosensitized NB generated intracellular ROS, disrupted mitochondrial and lysosomal membrane integrity, resulting in cell death. UV-induced genotoxicity by NB was confirmed through micronuclei generation, γ-H2AX induction and cyclobutane pyrimidine dimer formation. This is the first study which showed the phototoxicity and photogenotoxicity potential of NB in HaCaT cell line. We also observed that photosensitized NB upregulated inflammatory markers, such as COX-2 and TNFα. This study proposes that sunlight exposure should be avoided by patients using nabumetone and proper guidance should be provided by clinicians regarding photosensitivity of drugs for better safety and efficacy.

Human Epidermal Zinc Concentrations after Topical Application of ZnO Nanoparticles in Sunscreens.

Zinc oxide nanoparticle (ZnO NP)-based sunscreens are generally considered safe because the ZnO NPs do not penetrate through the outermost layer of the skin, the stratum corneum (SC). However, cytotoxicity of zinc ions in the viable epidermis (VE) after dissolution from ZnO NP and penetration into the VE is ill-defined. We therefore quantified the relative concentrations of endogenous and exogenous Zn using a rare stable zinc-67 isotope (67Zn) ZnO NP sunscreen applied to excised human skin and the cytotoxicity of human keratinocytes (HaCaT) using multiphoton microscopy, zinc-selective fluorescent sensing, and a laser-ablation inductively coupled plasma-mass spectrometry (LA-ICP-MS) methodology. Multiphoton microscopy with second harmonic generation imaging showed that 67ZnO NPs were retained on the surface or within the superficial layers of the SC. Zn fluorescence sensing revealed higher levels of labile and intracellular zinc in both the SC and VE relative to untreated skin, confirming that dissolved zinc species permeated across the SC into the VE as ionic Zn and significantly not as ZnO NPs. Importantly, the LA-ICP-MS estimated exogenous 67Zn concentrations in the VE of 1.0 ± 0.3 µg/mL are much lower than that estimated for endogenous VE zinc of 4.3 ± 0.7 µg/mL. Furthermore, their combined total zinc concentrations in the VE are much lower than the exogenous zinc concentration of 21 to 31 µg/mL causing VE cytotoxicity, as defined by the half-maximal inhibitory concentration of exogenous 67Zn found in human keratinocytes (HaCaT). This speaks strongly for the safety of ZnO NP sunscreens applied to intact human skin and the associated recent US FDA guidance.

A Role of Epithelial Cells and Virulence Factors in Biofilm Formation by Streptococcus pyogenes In Vitro.

Biofilm formation by Streptococcus pyogenes (group A streptococcus [GAS]) in model systems mimicking the respiratory tract is poorly documented. Most studies have been conducted on abiotic surfaces, which poorly represent human tissues. We have previously shown that GAS forms mature and antibiotic-resistant biofilms on physiologically relevant epithelial cells. However, the roles of the substratum, extracellular matrix (ECM) components, and GAS virulence factors in biofilm formation and structure are unclear. In this study, biofilm formation was measured on respiratory epithelial cells and keratinocytes by determining biomass and antibiotic resistance, and biofilm morphology was visualized using scanning electron microscopy. All GAS isolates tested formed biofilms that had similar, albeit not identical, biomass and antibiotic resistance for both cell types. Interestingly, functionally mature biofilms formed more rapidly on keratinocytes but were structurally denser and coated with more ECM on respiratory epithelial cells. The ECM was crucial for biofilm integrity, as protein- and DNA-degrading enzymes induced bacterial release from biofilms. Abiotic surfaces supported biofilm formation, but these biofilms were structurally less dense and organized. No major role for M protein, capsule, or streptolysin O was observed in biofilm formation on epithelial cells, although some morphological differences were detected. NAD-glycohydrolase was required for optimal biofilm formation, whereas streptolysin S and cysteine protease SpeB impaired this process. Finally, no correlation was found between cell adherence or autoaggregation and GAS biofilm formation. Combined, these results provide a better understanding of the role of biofilm formation in GAS pathogenesis and can potentially provide novel targets for future treatments against GAS infections.

Exosomes serve as novel modes of tick-borne flavivirus transmission from arthropod to human cells and facilitates dissemination of viral RNA and proteins to the vertebrate neuronal cells.

Molecular determinants and mechanisms of arthropod-borne flavivirus transmission to the vertebrate host are poorly understood. In this study, we show for the first time that a cell line from medically important arthropods, such as ticks, secretes extracellular vesicles (EVs) including exosomes that mediate transmission of flavivirus RNA and proteins to the human cells. Our study shows that tick-borne Langat virus (LGTV), a model pathogen closely related to tick-borne encephalitis virus (TBEV), profusely uses arthropod exosomes for transmission of viral RNA and proteins to the human- skin keratinocytes and blood endothelial cells. Cryo-electron microscopy showed the presence of purified arthropod/neuronal exosomes with the size range of 30 to 200 nm in diameter. Both positive and negative strands of LGTV RNA and viral envelope-protein were detected inside exosomes derived from arthropod, murine and human cells. Detection of Nonstructural 1 (NS1) protein in arthropod and neuronal exosomes further suggested that exosomes contain viral proteins. Viral RNA and proteins in exosomes derived from tick and mammalian cells were secured, highly infectious and replicative in all tested evaluations. Treatment with GW4869, a selective inhibitor that blocks exosome release affected LGTV loads in both arthropod and mammalian cell-derived exosomes. Transwell-migration assays showed that exosomes derived from infected-brain-microvascular endothelial cells (that constitute the blood-brain barrier) facilitated LGTV RNA and protein transmission, crossing of the barriers and infection of neuronal cells. Neuronal infection showed abundant loads of both tick-borne LGTV and mosquito-borne West Nile virus RNA in exosomes. Our data also suggest that exosome-mediated LGTV viral transmission is clathrin-dependent. Collectively, our results suggest that flaviviruses uses arthropod-derived exosomes as a novel means for viral RNA and protein transmission from the vector, and the vertebrate exosomes for dissemination within the host that may subsequently allow neuroinvasion and neuropathogenesis.

TatS: a novel in vitro tattooed human skin model for improved pigment toxicology research.

Reports of tattoo-associated risks boosted the interest in tattoo pigment toxicity over the last decades. Nonetheless, the influence of tattoo pigments on skin homeostasis remains largely unknown. In vitro systems are not available to investigate the interactions between pigments and skin. Here, we established TatS, a reconstructed human full-thickness skin model with tattoo pigments incorporated into the dermis. We mixed the most frequently used tattoo pigments carbon black (0.02 mg/ml) and titanium dioxide (TiO2, 0.4 mg/ml) as well as the organic diazo compound Pigment Orange 13 (0.2 mg/ml) into the dermis. Tissue viability, morphology as well as cytokine release were used to characterize TatS. Effects of tattoo pigments were compared to monolayer cultures of human fibroblasts. The tissue architecture of TatS was comparable to native human skin. The epidermal layer was fully differentiated and the keratinocytes expressed occludin, filaggrin and e-cadherin. Staining of collagen IV confirmed the formation of the basement membrane. Tenascin C was expressed in the dermal layer of fibroblasts. Although transmission electron microscopy revealed the uptake of the tattoo pigments into fibroblasts, neither viability nor cytokine secretion was altered in TatS. In contrast, TiO2 significantly decreased cell viability and increased interleukin-8 release in fibroblast monolayers. In conclusion, TatS emulates healed tattooed human skin and underlines the advantages of 3D systems over traditional 2D cell culture in tattoo pigment research. TatS is the first skin model that enables to test the effects of pigments in the dermis upon tattooing.

Epidermal Regeneration Induced by Comfrey Extract: A Study by Light and Electron Microscopy.

INTRODUCTION: An accelerated healing of superficial wounds was demonstrated in clinical trials with a topical comfrey preparation (Symphytum × uplandicum Nyman). The effect has previously not been examined in skin models. METHODS: An established in vitro model of epidermal cells with the typical strata was used for the observation of effects of applied substances on skin regeneration. Damage corresponding to a typical abrasion was created on day 1 by punching an opening into the epidermal fine structure down to the stratum basale. Samples were either untreated (controls) or exposed to comfrey cream on days 2, 3, 5, and 6. Tissue samples were taken for light and electron microscopy on days 1, 4, and 7. RESULTS AND CONCLUSIONS: Application of comfrey cream led to a quicker regeneration of skin cells and to an earlier differentiation of the cells towards a normal fine structure with a visible distinction of epidermal strata, keratin, and corneocyte formation within 4-7 days. The study covered the early days of skin regeneration and confirms the benefits observed in published clinical trials and non-interventional studies in patients with abrasions.

Crystal Structure of Keratin 1/10(C401A) 2B Heterodimer Demonstrates a Proclivity for the C-Terminus of Helix 2B to Form Higher Order Molecular Contacts.

We previously determined the crystal structure of the wild-type keratin 1/10 helix 2B heterodimer at 3.3 Å resolution. We proposed that the resolution of the diffraction data was limited due to the crystal packing effect from keratin 10 (K10) residue Cys401. Cys401K10 formed a disulfide-linkage with Cys401 from another K1/10 heterodimer, creating an "X-shaped" structure and a loose crystal packing arrangement. We hypothesized that mutation of Cys401K10 to alanine would eliminate the disulfide-linkage and improve crystal packing thereby increasing resolution of diffraction and enabling a more accurate side chain electron density map. Indeed, when a K10 Cys401Ala 2B mutant was paired with its native keratin 1 (K1) 2B heterodimer partner its x-ray crystal structure was determined at 2.07 Å resolution; the structure does not contain a disulfide linkage. Superposition of the K1/K10(Cys401Ala) 2B structure onto the wild-type K1/10 2B heterodimer structure had a root-mean-square-deviation of 1.88 Å; the variability in the atomic positions reflects the dynamic motion expected in this filamentous coiled-coil complex. The electrostatic, hydrophobic, and contour features of the molecular surface are similar to the lower resolution wild-type structure. We postulated that elimination of the disulfide linkage in the K1/K10(Cys401Ala) 2B structure could allow for the 2B heterodimers to bind/pack in the A22 tetramer configuration associated with mature keratin intermediate filament assembly. Analysis of the crystal packing revealed a half-staggered anti-parallel tetrameric complex of 2B heterodimers; however, their register is not consistent with models of the A22 mode of tetrameric alignment or prior biochemical cross-linking studies.

Herpetiform pemphigus with characteristic transmission electron microscopic findings of various-sized ballooning vacuoles in keratinocytes without acantholysis.

We report a unique case of a Japanese woman with herpetiform pemphigus (HP) who had IgG autoantibodies reactive with nondesmosomal sites of keratinocytes and presented characteristic transmission electron microscopic (TEM) findings of various-sized vacuoles in keratinocytes without acantholysis. The patient presented with pruritic annular oedematous erythemas with small blisters lining the margins on the trunk and extremities. Histopathological examinations showed intraepidermal blisters with prominent infiltrations of eosinophils. Direct and indirect immunofluorescence tests revealed the presence of in vivo bound and circulating IgG autoantibodies to the keratinocyte cell surfaces. However, enzyme-linked immunosorbent assays for desmoglein (Dsg) 1, Dsg3 and desmocollins 1-3 showed negative results. Immunoblotting using the full-length human Dsg1 recombinant protein showed a positive band. TEM examination showed various-sized vacuoles squashing the nuclei in many keratinocytes, resulting in rupture of the cells. Immunoelectron microscopic examination revealed IgG deposition over the entire keratinocyte cell surfaces, which spared the desmosomes. IgG antibodies were also present on the inside walls of the vacuoles around the nuclei of keratinocytes and on the cell surfaces of infiltrating eosinophils. This patient also had marked eosinophilia and high levels of thymus and activation-regulated chemokine and interleukin-5 in the serum. These results indicated a novel autoantigen on the nondesmosomal keratinocyte cell surfaces and the pathogenesis of bullous spongiotic change with inflammation in HP.

Protection of glucotoxicity by a tripeptide derivative of α-melanocyte-stimulating hormone in human epidermal keratinocytes.

BACKGROUND: Chronic skin ulcers are a major complication and a therapeutic challenge in patients with diabetes mellitus. Glucose-induced accumulation of reactive oxygen species (ROS) is considered to be an important pathogenetic factor in diabetes. OBJECTIVES: To characterize the impact of high glucose (HG) on normal human keratinocytes (NHKs) and examine if Lys-d-Pro-Thr (KdPT), a tripeptide derived from α-melanocyte-stimulating-hormone, has protective effects. METHODS: We investigated the key functions of NHKs under HG conditions with or without KdPT in vitro as well as ex vivo employing a skin organ culture model. RESULTS: HG impaired metabolic activity, cell proliferation, viability and migration of NHKs. As shown by atomic force microscopy HG altered the biophysical properties of NHKs, i.e. cell size and elasticity. Glucotoxicity in NHKs was paralleled by the induction of intracellular ROS and endoplasmic reticulum stress. KdPT attenuated HG-induced oxidative stress and antagonized the effects of glucose on cell viability, metabolic activity and migration. Importantly, KdPT also antagonized the suppressive effect of HG on epidermal migration in wounded human skin organ cultures. CONCLUSIONS: Our findings highlight a novel effect of KdPT that could be exploited for the future therapy of diabetic skin ulcers.

Keratinocyte Monolayers on Hyaluronic Acid Membranes as "Upside-Down" Grafts Reconstitute Full-Thickness Wounds.

BACKGROUND Skin replacement by means of cultured epithelial keratinocytes is a well-accepted method. However, several clinical drawbacks of sheet autografts (CEA - cultured epithelial autografts) have stimulated various efforts to optimize cell culture and cell delivery. Recent developments include use of cell monolayers instead of a fully differentiated epithelium, as well as use of various biomaterials to grow and transport the cultured cells. To optimize the transfer of human keratinocytes directly to the recipient wound bed, we used an "upside-down" technique, delivering cultured cells directly to the wound with the carrier material on top. MATERIAL AND METHODS Subconfluent second-passage human keratinocyte monolayers on esterified hyaluronic acid membranes (KHAMC - Keratinocyte-Hyaluronic-Acid-Membrane-Composites) were transplanted either as upside-down grafts or as upside-up grafts onto standardized full-thickness wounds in athymic nude mice versus controls with the cell-free membrane alone. RESULTS In the upside-down group, 14 days after grafting, a multi-layered, differentiating epidermis was found, whereas the wounds in the upside-up group and in the control group were not completely closed up to day 21. Persistence of human keratinocytes was shown in the upside-down group only, from day 7 until day 35 after grafting. CONCLUSIONS This study confirms that upside-down grafting of subconfluent monolayers of serum-free cultured human keratinocytes on esterified hyaluronic acid membranes is a suitable means to transfer actively proliferative keratinocytes, and reduces wound contraction. Compared to standard grafting protocols of cultured epithelium, such as CEA sheet grafts, it is easier to apply, does not need enzymatic detachment of cells from the culture dish, and limits the number of production steps required.

Lysosome Alterations in the Human Epithelial Cell Line HaCaT and Skin Specimens: Relevance to Psoriasis.

Despite the constantly updated knowledge regarding the alterations occurring in the cells of patients with psoriasis, the status and the role of the lysosome, a control center of cell metabolism, remain to be elucidated. The architecture of the epidermis is largely regulated by the action of lysosomes, possibly activating signaling pathways in the cellular crosstalk of keratinocytes-epidermal cells-with infiltrating immune cells. Thus, in the present study, lysosome alterations were examined in vitro and in situ using a two-dimensional (2D) keratinocyte model of HaCaT cells with "psoriasis-like" inflammation and skin specimens, respectively. Specific fluorescence and immunohistochemical staining showed an augmented level of acidic organelles in response to keratinocyte activation (mimicking a psoriatic condition while maintaining the membrane integrity of these structures) as compared with the control, similar to that seen in skin samples taken from patients. Interestingly, patients with the most pronounced PASI (Psoriasis Area and Severity Index), BSA (Body Surface Area), and DLQI (Dermatology Life Quality Index) scores suffered a high incidence of positive lysosomal-associated membrane protein 1 (LAMP1) expression. Moreover, it was found that the gene deregulation pattern was comparable in lesioned (PP) and non-lesioned (PN) patient-derived skin tissue, which may indicate that these alterations occur prior to the onset of the characteristic phenotype of the disease. Changes in the activity of genes encoding the microphthalmia family (MiT family) of transcription factors and mammalian target of rapamycin complex 1 (MTORC1) were also observed in the in vitro psoriasis model, indicating that the biogenesis pathway of this arm is inhibited. Interestingly, in contrast to the keratinocytes of HaCaT with "psoriasis-like" inflammation, LAMP1 was up-regulated in both PP and PN skin, which can be a potential sign of an alternative mechanism of lysosome formation. Defining the molecular profile of psoriasis in the context of "the awesome lysosome" is not only interesting, but also desired; therefore, it is believed that this paper will serve to encourage other researchers to conduct further studies on this subject.

Water-Soluble Fullerenol C60(OH)36 toward Effective Anti-Air Pollution Induced by Urban Particulate Matter in HaCaT Cell.

Particulate matter (PM), a widespread air pollutant, consists of a complex mixture of solid and liquid particles suspended in air. Many diseases have been linked to PM exposure, which induces an imbalance in reactive oxygen species (ROS) generated in cells, and might result in skin diseases (such as aging and atopic dermatitis). New techniques involving nanomedicine and nano-delivery systems are being rapidly developed in the medicinal field. Fullerene, a kind of nanomaterial, acts as a super radical scavenger. Lower water solubility levels limit the bio-applications of fullerene. Hence, to improve the water solubility of fullerene, while retaining its radical scavenger functions, a fullerene derivative, fullerenol C60(OH)36, was synthesized, to examine its biofunctions in PM-exposed human keratinocyte (HaCaT) cells. The PM-induced increase in ROS levels and expression of phosphorylated mitogen-activated protein kinase and Akt could be inhibited via fullerenol pre-treatment. Furthermore, the expression of inflammation-related proteins, cyclooxygenase-2, heme oxygenase-1, and prostaglandin E2 was also suppressed. Fullerenol could preserve the impaired state of skin barrier proteins (filaggrin, involucrin, repetin, and loricrin), which was attributable to PM exposure. These results suggest that fullerenol could act against PM-induced cytotoxicity via ROS scavenging and anti-inflammatory mechanisms, and the maintenance of expression of barrier proteins, and is a potential candidate compound for the treatment of skin diseases.

Interplay between FGFR2b-induced autophagy and phagocytosis: role of PLCγ-mediated signalling.

Signalling of the epithelial splicing variant of the fibroblast growth factor receptor 2 (FGFR2b) induces both autophagy and phagocytosis in human keratinocytes. Here, we investigated, in the cell model of HaCaT keratinocytes, whether the two processes might be related and the possible involvement of PLCγ signalling. Using fluorescence and electron microscopy, we demonstrated that the FGFR2b-induced phagocytosis and autophagy involve converging autophagosomal and phagosomal compartments. Moreover, the forced expression of FGFR2b signalling mutants and the use of specific inhibitors of FGFR2b substrates showed that the receptor-triggered autophagy requires PLCγ signalling, which in turn activates JNK1 via PKCδ. Finally, we found that in primary human keratinocytes derived from light or dark pigmented skin and expressing different levels of FGFR2b, the rate of phagocytosis and autophagy and the convergence of the two intracellular pathways are dependent on the level of receptor expression, suggesting that FGFR2b signalling would control in vivo the number of melanosomes in keratinocytes, determining skin pigmentation.

Lamellipodial wrinkles in fish keratocytes as markers of imperfect coordination between extension and retraction during cell migration.

Cell migration involves the precise coordination between extension at the front of the cell and retraction at the rear. This coordination is particularly evident in fast moving cells such as fish keratocytes, where it leads to highly stable gliding motion, propelled at the front by broad, 0.1-0-2 µm thick lamellipodia. Transient uncoupling between extension and retraction can occur if the rear is temporarily stuck, which might eventually lead to cell shape instabilities. We have frequently observed in fish keratocytes the presence of lamellipodial radial wrinkles, detected by confocal, scanning electron and side-view microscopy as folds in the lamellipodium up to 2 µm in height. Using a linear finite elements analysis, we simulated the displacement of cells either with perfect coordination between extension and retraction or with the rear transiently stuck while the front continues extending, and we observed that in this last condition compression stresses arise in the lamellipodium which predict the formation of the observed pattern of lamellipodial wrinkles. In support of the numerical modeling findings, we observed that the transient halting of retraction at the rear using micromanipulation induced the formation of lamellipodial wrinkles in previously flat lamellipodia. The obtained results suggest that the conspicuous lamellipodial wrinkles observed in migrating fish keratocytes are the product of transient imbalances between front and rear displacements, and are therefore useful markers of the short scale dynamics of extension and retraction coordination during cell migration.

Changes in nano-mechanical properties of human epidermal cornified cells depending on their proximity to the skin surface.

During formation of the stratum corneum (SC) barrier, terminally differentiated keratinocytes continue their maturation process within the dead superficial epidermal layer. Morphological studies of isolated human corneocytes have revealed differences between cornified envelopes purified from the deep and superficial SC. We used atomic force microscopy to measure the mechanical properties of native human corneocytes harvested by tape-stripping from different SC depths. Various conditions of data acquisition have been tested and optimized, in order to obtain exploitable and reproducible results. Probing at 200 nN allowed us to investigate the total stiffness of the cells (at 50 nm indentation) and that of the cornified envelopes (at 10 to15 nm), and lipid envelopes (at 5 to 10 nm). The obtained data indicated statistically significant differences between the superficial (more rigid) and deep (softer) corneocytes, thus confirming the existence of depth and maturation-related morphological changes within the SC. The proposed approach can be potentially used for minimally invasive evaluation of various skin conditions such as aging, skin hydration, and pathologies linked to SC.

In Vitro Replication of Chelonid Herpesvirus 5 in Organotypic Skin Cultures from Hawaiian Green Turtles (Chelonia mydas).

Fibropapillomatosis (FP) is a tumor disease of marine turtles associated with chelonid herpesvirus 5 (ChHV5), which has historically been refractory to growth in tissue culture. Here we show, for the first time, de novo formation of ChHV5-positive intranuclear inclusions in cultured green turtle cells, which is indicative of active lytic replication of the virus. The minimal requirements to achieve lytic replication in cultured cells included (i) either in vitro cultures of ChHV5-positive tumor biopsy specimens (plugs) or organotypic cultures (rafts) consisting of ChHV5-positive turtle fibroblasts in collagen rafts seeded with turtle keratinocytes and (ii) keratinocyte maturation induced by raising raft or biopsy cultures to the air-liquid interface. Virus growth was confirmed by detailed electron microscopic studies that revealed intranuclear sun-shaped capsid factories, tubules, various stages of capsid formation, nuclear export by budding into the perinuclear space, tegument formation, and envelopment to complete de novo virus production. Membrane synthesis was also observed as a sign of active viral replication. Interestingly, cytoplasmic particles became associated with keratin filaments, a feature not seen in conventional monolayer cell cultures, in which most studies of herpesvirus replication have been performed. Our findings draw a rich and realistic picture of ChHV5 replication in cells derived from its natural host and may be crucial not only to better understand ChHV5 circulation but also to eventually complete Koch's postulates for FP. Moreover, the principles described here may serve as a model for culture of other viruses that are resistant to replication in conventional cell culture.IMPORTANCE A major challenge in virology is the study of viruses that cannot be grown in the laboratory. One example is chelonid herpesvirus 5 (ChHV5), which is associated with fibropapillomatosis, a globally distributed, debilitating, and fatal tumor disease of endangered marine turtles. Pathological examination shows that ChHV5 is shed in skin. Here we show that ChHV5 will grow in vitro if we replicate the complex three-dimensional structure of turtle skin. Moreover, lytic virus growth requires a close interplay between fibroblasts and keratinocytes. Finally, the morphogenesis of herpesviral growth in three-dimensional cultures reveals a far richer, and likely more realistic, array of capsid morphologies than that encountered in traditional monolayer cell cultures. Our findings have applications to other viruses, including those of humans.