431 Tropomyosin-receptor kinase fused gene (TFG) induces collagen synthesis in human dermal fibroblasts

S | Growth Factors, Cell Adhesion & Matrix Biology 427 Development of senescence corresponds to the loss of sphingomyelin-enriched submicrometric domains and their associated EGFR-signaling in epidermal keratinocytes A Mound, V Lozanova, D Tyteca, C Guere, K Vie, C Lambert de Rouvroit, F ChainiauxDebacq and Y Poumay 1 Narilis, University of Namur, Namur, Belgium, 2 De Duve Institute UCL, Brussels, Belgium and 3 Laboratoires Clarins, Paris, France Normal somatic cells in vitro exhibit replicative senescence which slows down, then blocks the cell cycle. In cultured human keratinocytes, proportion of cells exhibiting senescenceassociated b-galactosidase activity, over-expressing p16, and exhibiting reduced response to mitotic stimuli (e.g. EGF) is increased. Cholesterol-depletion from keratinocyte plasma membrane has been shown to disrupt lipid microdomains and to activate several signaling pathways. Whereas cholesterol and sphingomyelin in plasma membrane are believed to play roles in cell perception of its environment, demonstration of specialized lipid domains is still debated. Therefore, demonstration of lipid domains was hereby performed in keratinocytes and further analyzed during replicative senescence, as well as in signaling pathways. Despite unaltered expression of receptors and downstream signaling intermediates, senescent keratinocytes become unable to signal through EGFR and p38. Vital imaging of pre-senescent and senescent keratinocytes were performed to characterize location, distribution and composition of lipid microdomains. This report illustrates usefulness of inserting exogenous fluorescent sphingomyelin (BODIPY-SM) to analyze lipid submicrometric domains in keratinocytes. Similarly, fluorescent lysenin and theta toxin fragments fused to mCherry were demonstrated as specific, sensitive and quantitative SMor cholesterol-specific probes. Keratinocytes treated with Y27632 ROCK inhibitor to delay senescence, as well as immortalized keratinocytes were identically analyzed. Data indicate that lipid domains are detected in pre-senescent keratinocytes, nevertheless SM-enriched domains gradually disappear with replicative senescence but are maintained in Y27632-treated and immortalized keratinocytes. Thus, alteration of lipid microdomains is likely involved during epidermal senescence. S76 Journal of Investigative Dermatology (2016), Volume 136 428 A novel function for JNK in regulating desmosome assembly and adhesion N Li, M Park, Z Liu and LA Diaz Dermatology, University of North Carolina, Chapel Hill, NC Desmosomes are intercellular adhesive junctions that play a critical role in keratinocyte cohesion. Our understanding of how desmosomes assembly and maturation are regulated is limited. The c-Jun N-terminal kinase (JNK) is a stress-activated protein kinase that is involved in various functions such as cytoskeleton remodeling, cell motility, and apoptosis. Here we investigated the potential role of JNK on desmosome assembly and adhesion in cultured human keratinocytes. We found that pharmacological inhibition of unstimulated, basal activity of JNK accelerates cell border translocation of desmosomal cadherins and desmoplakins following high Ca switch. Remarkably, inhibition of JNK initiates cell-cell contact and induces recruitment of desmosomal proteins to intercellular junctions under a low Ca (0.1 mM) culture condition. Knockdown of JNK by siRNA silencing also induces localization of desmosomal proteins on intercellular borders in low Ca. The steady state levels of desmosomal cadherins are elevated in response to JNK inhibition or siRNA silencing. Cycloheximide chase assay reveals an increased half-life of desmosomal cadherins in JNKinhibited keratinocytes, suggesting that elevated level of desmosomal cadherins is at least in part attributed to increased stability of the proteins. The effects of JNK inhibition on desmosomal adhesion was next examined by calcium depletion assay. The results show that JNK-inhibited keratinocytes are resistant to EGTA-induced loss of desmosomal components from the cell border, whereas little or no desmosomal proteins remain on cell borders in control cells following calcium depletion. Furthermore, dispase assays demonstrate that JNK inhibition significantly enhances intercellular adhesive strength of keratinocytes. Collectively, this study discovers a novel role of JNK in regulating desmosome assembly and adhesion. 429 Nanoscale molecular organization of the desmosome as revealed by super-resolution microscopy SN Stahley, EI Bartle, CE Atkinson, AP Kowalczyk and AL Mattheyses 1 Cell Biology, Emory University School of Medicine, Atlanta, GA, 2 Biochemistry and Molecular Biology, University of Chicago, Chicago, IL and 3 Dermatology, Emory University School of Medicine, Atlanta, GA The desmosome is a macromolecular complex responsible for providing strong adhesion between epidermal keratinocytes. Due in part to the size and molecular complexity of the desmosome, the precise organization of proteins within the intact complex and how desmosome ultrastructure relates to adhesion has been challenging to study. We addressed this challenge with super-resolution direct stochastic optical reconstruction microscopy (dSTORM). In contrast to electron microscopy, which shows desmosomes as pairs of electron dense plaques, dSTORM reveals the distribution of fluorescently labeled proteins. The 20 nm spatial resolution of dSTORM together with the inherent mirror symmetry of the desmosome and specific protein labeling allowed us to analyze desmosomal protein organization. Using domain specific antibodies, we mapped the molecular arrangement of proteins in the desmosome and measured desmosome length. This analysis was performed for desmoglein 3, plakoglobin and desmoplakin in cultured primary human keratinocytes. Changes in the molecular arrangement of desmoplakin but not plakoglobin correlated with enhanced adhesive function. Additionally, desmosomes were identified with dSTORM in human skin sections. Our results reveal desmosomal protein organization in vitro and in vivo while additionally demonstrating the power of super-resolution imaging for investigating cell adhesion structures. This work provides a foundation for individual desmosome analysis that can be leveraged to study the molecular details of desmosome assembly, maturation and disruption in human disease. 430 Tissue polarization of mechano-adhesive signals, cytoskeleton and EGFR signaling controls skin barrier formation M Ruebsam, A Mertz, G Goranci, V Horsley, E Dufresne, W Ziegler, A Kubo, M Amagai and CM Niessen 1 Dermatology, University of Cologne, Cologne, Germany, 2 Yale University, New Haven, CT, 3 Hannover Medical School, Hannover, Germany, 4 Dermatology, Keio University School of Medicine, Tokyo, Japan and 5 CECAD Cologne, University of Cologne, Cologne, Germany Skin barrier function critically depends on the presence of functional tight junctions (TJ) in the stratum granulosum. How polarization of epidermal TJ barrier function across the tissue is controlled is not known. Using high resolution microscopy on whole mount epidermis we examined if and how polarization of junctions and cytoskeleton is important for barrier formation. Our data show that even though adherens junctions (AJ) are found in all layers, vinculin positive mechano-sensitive AJs are only present in the granular layer. There these AJs form a basolateral network that on the apical side interacts with a continuous TJ network that is strongly positive for F-actin, with lower layers showing much less F-actin organization. To ask whether cadherin dependent mechanotransduction was important we inactivated E-cadherin, which greatly impaired initial junction formation and altered tension as judged by traction force microscopy. In vivo loss of E-cadherin disrupted the polarized organization of vinculin and F-actin and resulted in discontinuous and leaky tight junctions. Although inactivation of vinculin disturbed initial junction formation more severely, the TJs and skin barrier dysfunction was less extensive than loss of E-cadherin. E-cadherin has also been implicated in the regulation of EGFR signaling. Surprisingly, we found that the EGFR not only localized to basal cells but was also enriched at TJs in the SG, which was lost upon E-cadherin inactivation, accompanied by increased receptor activation. Indeed, EGFR inhibition was able to rescue TJ barrier function in E-cadherin keratinocytes. In conclusion, our data identify a key role for E-cadherin dependent mechanotransduction in coordinating the polarized organization of junctions, cytoskeleton and EGFR receptor activity crucial for skin barrier function. 431 Tropomyosin-receptor kinase fused gene (TFG) induces collagen synthesis in human dermal fibroblasts C Kim, S Kong, J Ha, Y Chang, Y Lee and J Lee 1 Chungnam National University, Daejeon, Korea (the Republic of) and 2 LG Household and Healthcare, Daejeon, Korea (the