Keratin Isotypes Control Desmosome Stability and Dynamics through PKCα

Expression and interaction of desmosomal components and keratins provide stable cell cohesion and protect the epidermis against various types of stress. The differentiation-specific isotype composition of the keratin cytoskeleton and desmosomes is regarded as a major determinant of adhesive strength. In support, wound healing is characterized by a transient decrease in desmosomal adhesion accompanied by increased expression of keratins K6/K16/K17 at the expense of K1/K10. The significance of altered keratin expression for desmosomal composition and adhesion remains incompletely understood at a mechanistic and functional level. Here, we investigated the respective contribution of K5/K14 or K6/K17 to desmosome adhesion, on their stable re-expression in keratinocytes lacking all keratins. This revealed that K5/K14 filaments support stable desmosomes, whereas “wound healing” keratins K6/K17 induce elevated protein kinase C alpha–mediated desmosome disassembly and subsequent destabilization of epithelial sheets. Moreover, our data suggest that K5/K14 sequester protein kinase C alpha in the cytoplasm, whereas K6/K17 or the absence of all keratins enables protein kinase C alpha translocation to the plasma membrane and induction of desmosome disassembly. Gain- and loss-of-function experiments support a major role of K5 in desmosome stability control via protein kinase C alpha. Our data show that keratin isotypes differently and specifically regulate wound healing and invasion by modulating intercellular adhesion

Skin-Specific Expression of ank-393, a Novel Ankyrin-3 Splice Variant

Ankyrins represent a protein family whose members are associated with membrane proteins and the actin cytoskeleton. The principal ankyrin domain structure comprises an amino-terminal membrane-binding, a spectrin-binding, and a regulatory domain, and can be modulated by alternative splicing. In order to investigate the role of ankyrin-3 in skin, we have isolated three complete ankyrin-3 cDNA clones of 5.8 kb, 5.2 kb, and 2.5 kb by reverse transcription–polymerase chain reaction of mouse skin RNA. DNA sequencing confirmed the isolated clones to be splice variants of ankyrin-3. Of these, the smallest cDNA represents a novel ankyrin named ankyrin-393. Surprisingly, this novel ankyrin subtype lacks not only all ankyrin repeats, but also the first 75 amino acids of the spectrin-binding domain. Immuno-fluorescence analysis of mouse skin showed that ankyrin-3 is expressed in all living layers of mouse epidermis. Here, it predominates along the basal and lateral membranes of the basal layer in addition to an even cytoplasmic distribution. In primary mouse keratinocytes grown at elevated Ca2+ levels, ankyrin-393 was localized along the plasma membrane and throughout the cell in a Golgi-like fashion. Depending on fixation conditions, nuclear staining became apparent in many cells. In agreement with previous data, northern blotting revealed a widespread distribution of the two larger ankyrin splice variants. In contrast, the mRNA coding for ankyrin-393 was restricted to mouse skin. Reverse transcription–polymerase chain reaction of mouse skin RNA strongly suggested additional ankyrin isoforms in skin. Our data on ankyrin-393, which lacks a part of the spectrin-binding domain that regulates the affinity to spectrin, suggests a new function for this member of the ankyrin family

Cytoskeleton in motion: the dynamics of keratin intermediate filaments in epithelia

Epithelia are exposed to multiple forms of stress. Keratin intermediate filaments are abundant in epithelia and form cytoskeletal networks that contribute to cell type–specific functions, such as adhesion, migration, and metabolism. A perpetual keratin filament turnover cycle supports these functions. This multistep process keeps the cytoskeleton in motion, facilitating rapid and protein biosynthesis–independent network remodeling while maintaining an intact network. The current challenge is to unravel the molecular mechanisms underlying the regulation of the keratin cycle in relation to actin and microtubule networks and in the context of epithelial tissue function

Keratins Stabilize Hemidesmosomes through Regulation of β4-Integrin Turnover

Epidermal integrity and wound healing depend on remodeling of cell-matrix contacts including hemidesmosomes. Mutations in β4-integrin and plectin lead to severe epidermolysis bullosa (EB). Whether mutations in keratins K5 or K14, which cause EB simplex, also compromise cell-matrix adhesion through altering hemidesmosomal components is not well investigated. In particular, the dependence of β4-integrin endocytosis and turnover on keratins remains incompletely understood. Here, we show that the absence of keratins causes loss of plectin-β4-integrin interaction and elevated β4-integrin phosphorylation at Ser1354 and Ser1362. This triggered a caveolin-dependent endocytosis of β4-integrin but not of other integrins through Rab5 and Rab11 compartments in keratinocytes. Expressing a phospho–deficient β4-integrin mutant reduces β4-integrin endocytosis and rescues plectin localization in keratin–free cells. β4-integrin phosphorylation in the absence of keratins resulted from elevated Erk1/2 activity downstream of increased EGFR and PKCα signaling. Further, increased Erk1/2 phosphorylation and altered plectin localization occur in keratin–deficient mouse epidermis in vivo. Strikingly, expression of the K14-R125P EBS mutant also resulted in plectin mislocalization and elevated β4-integrin turnover, suggesting disease relevance. Our data underscore a major role of keratins in controlling β4-integrin endocytosis involving a plectin-Erk1/2-dependent mechanism relevant for epidermal differentiation and pathogenesis

Induction of Inflammatory Cytokines by a Keratin Mutation and their Repression by a Small Molecule in a Mouse Model for EBS

Epidermolysis bullosa simplex (EBS) is a skin disorder caused by mutations in keratin (K) 5 or K14 genes. It is widely regarded as a mechanobullous disease, resulting from a weakened cytoskeleton, causing extensive cytolysis. It was postulated by others that certain K14 mutations induce tumor necrosis factor-α (TNF-α) and increase apoptosis. Here, we report that in K5−/− mice and in a cell culture model of EBS, the mRNA and protein levels of TNF-α remain unaltered. Transcriptome analysis of K5−/− mice revealed, however, that the proinflammatory cytokines IL-6 and IL-1β were significantly upregulated at the mRNA level in K5−/− mouse skin. These results were confirmed by TaqMan real-time PCR and ELISA assays. We hypothesize that keratin mutations contribute to EBS in a mouse model by inducing local inflammation that mediates a stress response. Following clinical reports, we applied the small molecule doxycycline to K5−/− mice. We demonstrate that doxycycline extended the survival of neonatal K5−/− mice from less than 1 to up to 8hours. Microarray and TaqMan real-time PCR showed a downregulation of matrix metalloproteinase 13 and IL-1β, indicating an effect of doxycycline on transcription. Our data offer a novel small molecule-based therapy approach for EBS

IMECE2008-68137 FRACTIONAL ORDER MODELS FOR VISCOELASTICITY OF SOFT BIOLOGICAL TISSUES

ABSTRACT Dynamic mechanical properties of soft tissues provide information that may be used in medical diagnosis. Developing a better fundamental understanding of the governing constitutive relations could improve diagnostic techniques. The mechanical behavior of soft tissues and tissue mimicking phantoms, such as gels, can be represented by viscoelastic material models. Static loading of viscoelastic materials yields information related to elasticity, creep and stress relaxation. However, a broader measure of ratedependent properties that affect mechanical wave propagation and wave attenuation in such materials can only be extracted from measured response to dynamic excitation. The well known linear viscoelastic material models of Voigt, Maxwell and Kelvin cannot represent the more complicated frequency dependency of these materials over a broad spectral range. Therefore, fractional calculus methods have been considered to model the viscoelastic behavior of soft tissue-like materials. Fractional order models capture the viscoelastic material behavior using fractional orders of differential equation

Keratins regulate protein biosynthesis through localization of GLUT1 and -3 upstream of AMP kinase and Raptor

Removal of the entire keratin family of intermediate filament proteins from embryonic epithelia has surprising implications for mTOR signaling

Development of a Nitridation Gas-Surface Boundary Condition for High-Fidelity Hypersonic Simulations

Gas-surface interaction phenomena have a strong impact on the heat flux experienced by atmospheric entry bodies in the hypersonic regime. Numerically, they can be expressed as a boundary condition to be imposed to the Navier-Stokes equations to achieve predictive engineering simulations. The mass and energy conservation can be abstracted in a thin layer containing both the solid and the gas phases. Such a balance was implemented in the open source MUTATION++ library. It is convenient to easily plug verified models in any type of CFD solver to model the response of material surfaces. We have extended the library to accommodate a state-of-the-art nitridation and nitrogen recombination mechanisms derived from beam experiments. MUTATION++ was coupled to US3D, a high-fidelity finite-volume flow solver, to simulate an experimental campaign conducted in the VKI Plasmatron facility. The experiment consists in applying a subsonic high-enthalpy nitrogen flow over an axi-symmetric ablative material sample. The simulation results on the stagnation line were compared to those obtained using a one-dimensional solver. Both results showed good agreement, verifying the implementation of the boundary condition. The computational model predicts a lower mass blowing rate than the experimental value. The catalytic behaviour of the mechanism, in agreement with the beam experiment predictions, induces higher heat flux values than those expected for the testing conditions of the Plasmatron facility

Assessing neuraxial microstructural changes in a transgenic mouse model of early stage Amyotrophic Lateral Sclerosis by ultra‐high field MRI and diffusion tensor metrics

bjective: Cell structural changes are one of the main features observed during the development of amyotrophic lateral sclerosis (ALS). In this work, we propose the useof diffusion tensor imaging (DTI) metrics to assess specific ultrastructural changes in the central nervous system during the early neurodegenerative stages of ALS.Methods: Ultra-high field MRI and DTI data at 17.6T were obtained from fixed, excised mouse brains, and spinal cords from ALS (G93A-SOD1) mice.Results: Changes in fractional anisotropy (FA) and linear, planar, and spherical anisotropy ratios (CL, CP, and CS, respectively) of the diffusion eigenvalues were measured in white matter (WM) and gray matter (GM) areas associated with early axonal degenerative processes (in both the brain and the spinal cord). Specifically, in WM structures (corpus callosum, corticospinal tract, and spinal cord funiculi) as the disease progressed, FA, CL, and CP values decreased, whereas CS values increased.In GM structures (prefrontal cortex, hippocampus, and central spinal cord) FA and CP decreased, whereas the CL a nd C values were unchanged or slightly smaller.Histological studies of a fluorescent mice model (YFP, G93A-SOD1 mouse) corroborated the early alterations in neuronal morphology and axonal connectivity measured by DTI.Conclusions: Changes in diffusion tensor shape were observed in this animal model at the early, nonsymptomatic stages of ALS. Further studies of CL, CP, and CSas imaging biomarkers should be undertaken to refine this neuroimaging tool for future clinical use in the detection of the early stages of ALSFil: Gatto, Rodolfo G.. University Of Illinois. Deparment Of Biological Science; Estados UnidosFil: Weissmann, Carina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; ArgentinaFil: Amin, Manish. University of Florida; Estados UnidosFil: Finkielsztein, Ariel. Northwestern University; Estados UnidosFil: Sumagin, Ronen. Northwestern University; Estados UnidosFil: Mareci, Thomas H.. University of Florida; Estados UnidosFil: Uchitel, Osvaldo Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; ArgentinaFil: Magin, Richard L.. University Of Illinois. Deparment Of Biological Science; Estados Unido