Matthias Rübsam

Tropism-modified AAV Vectors Overcome Barriers to Successful Cutaneous Therapy

Autologous human keratinocytes (HK) forming sheet grafts are approved as skin substitutes. Genetic engineering of HK represents a promising technique to improve engraftment and survival of transplants. Although efficacious in keratinocyte-directed gene transfer, retro-/lentiviral vectors may raise safety concerns when applied in regenerative medicine. We therefore optimized adeno-associated viral (AAV) vectors of the serotype 2, characterized by an excellent safety profile, but lacking natural tropism for HK, through capsid engineering. Peptides, selected by AAV peptide display, engaged novel receptors that increased cell entry efficiency by up to 2,500-fold. The novel targeting vectors transduced HK with high efficiency and a remarkable specificity even in mixed cultures of HK and feeder cells. Moreover, differentiated keratinocytes in organotypic airlifted three-dimensional cultures were transduced following topical vector application. By exploiting comparative gene analysis we further succeeded in identifying αvβ8 integrin as a target receptor thus solving a major challenge of directed evolution approaches and describing a promising candidate receptor for cutaneous gene therapy.

E-cadherin integrates mechanotransduction and EGFR signaling to control junctional tissue polarization and tight junction positioning

Generation of a barrier in multi-layered epithelia like the epidermis requires restricted positioning of functional tight junctions (TJ) to the most suprabasal viable layer. This positioning necessitates tissue-level polarization of junctions and the cytoskeleton through unknown mechanisms. Using quantitative whole-mount imaging, genetic ablation, and traction force microscopy and atomic force microscopy, we find that ubiquitously localized E-cadherin coordinates tissue polarization of tension-bearing adherens junction (AJ) and F-actin organization to allow formation of an apical TJ network only in the uppermost viable layer. Molecularly, E-cadherin localizes and tunes EGFR activity and junctional tension to inhibit premature TJ complex formation in lower layers while promoting increased tension and TJ stability in the granular layer 2. In conclusion, our data identify an E-cadherin-dependent mechanical circuit that integrates adhesion, contractile forces and biochemical signaling to drive the polarized organization of junctional tension necessary to build an in vivo epithelial barrier.In multi-layered epithelia tight junctions (TJ) are confined to the most suprabasal viable layer. Here the authors show that this is regulated by ubiquitously localized E-cadherin tuning junctional tension and EGFR activity to inhibit TJ formation in lower layers while promoting TJ stability in the granular layer 2.

Adhesion forces and cortical tension couple cell proliferation and differentiation to drive epidermal stratification

To establish and maintain organ structure and function, tissues need to balance stem cell proliferation and differentiation rates and coordinate cell fate with position. By quantifying and modelling tissue stress and deformation in the mammalian epidermis, we find that this balance is coordinated through local mechanical forces generated by cell division and delamination. Proliferation within the basal stem/progenitor layer, which displays features of a jammed, solid-like state, leads to crowding, thereby locally distorting cell shape and stress distribution. The resulting decrease in cortical tension and increased cell–cell adhesion trigger differentiation and subsequent delamination, reinstating basal cell layer density. After delamination, cells establish a high-tension state as they increase myosin II activity and convert to E-cadherin-dominated adhesion, thereby reinforcing the boundary between basal and suprabasal layers. Our results uncover how biomechanical signalling integrates single-cell behaviours to couple proliferation, cell fate and positioning to generate a multilayered tissue.Mechanics of epidermal differentiation Miroshnikova et al. find that during embryonic development, epidermal basal layer crowding generates local changes in cell shape, cortical tension, and adhesion that initiate differentiation and delamination

Adherens Junctions and Desmosomes Coordinate Mechanics and Signaling to Orchestrate Tissue Morphogenesis and Function: An Evolutionary Perspective

Cadherin-based adherens junctions (AJs) and desmosomes are crucial to couple intercellular adhesion to the actin or intermediate filament cytoskeletons, respectively. As such, these intercellular junctions are essential to provide not only integrity to epithelia and other tissues but also the mechanical machinery necessary to execute complex morphogenetic and homeostatic intercellular rearrangements. Moreover, these spatially defined junctions serve as signaling hubs that integrate mechanical and chemical pathways to coordinate tissue architecture with behavior. This review takes an evolutionary perspective on how the emergence of these two essential intercellular junctions at key points during the evolution of multicellular animals afforded metazoans with new opportunities to integrate adhesion, cytoskeletal dynamics, and signaling. We discuss known literature on cross-talk between the two junctions and, using the skin epidermis as an example, provide a model for how these two junctions function in concert to orchestrate tissue organization and function.

The epidermal polarity protein Par3 is a non–cell autonomous suppressor of malignant melanoma

Melanoma, an aggressive skin malignancy with increasing lifetime risk, originates from melanocytes (MCs) that are in close contact with surrounding epidermal keratinocytes (KCs). How the epidermal microenvironment controls melanomagenesis remains poorly understood. In this study, we identify an unexpected non–cell autonomous role of epidermal polarity proteins, molecular determinants of cytoarchitecture, in malignant melanoma. Epidermal Par3 inactivation in mice promotes MC dedifferentiation, motility, and hyperplasia and, in an autochthonous melanoma model, results in increased tumor formation and lung metastasis. KC-specific Par3 loss up-regulates surface P-cadherin that is essential to promote MC proliferation and phenotypic switch toward dedifferentiation. In agreement, low epidermal PAR3 and high P-cadherin expression correlate with human melanoma progression, whereas elevated P-cadherin levels are associated with reduced survival of melanoma patients, implying that this mechanism also drives human disease. Collectively, our data show that reduced KC Par3 function fosters a permissive P-cadherin–dependent niche for MC transformation, invasion, and metastasis. This reveals a previously unrecognized extrinsic tumor-suppressive mechanism, whereby epithelial polarity proteins dictate the cytoarchitecture and fate of other tissue-resident cells to suppress their malignant outgrowth.

Assessing the In Vivo Epidermal Barrier in Mice: Dye Penetration Assays

INTRODUCTION The formation of a properly functioning epidermal barrier is a prerequisite for terrestrial life. The epidermis not only protects from external influences such as pathogens, chemicals, and UV light, but also prevents dehydration. To maintain proper barrier function, the epidermis undergoes a dynamic turnover driven by proliferating keratinocytes in the basal layer that, upon induction of differentiation, will move upward through the stratum spinosum and stratum granulosum while undergoing a terminal differentiation process to ultimately form the stratum corneum (SC). This layer is in direct contact with the outside environment and provides important structural and innate immune barrier properties. Barrier function is initiated when stratum granulosum keratinocytes start secreting and crosslinking specific proteins and lipids into the intercellular space and at the same time transform their membrane into a so-called cornified envelope (e.g., by linking different structural proteins such as loricrin to the inner side of the cell membrane). Consequently, keratinocytes shed their nucleus to become corneocytes forming the SC. In recent years it has become clear that the SC is not the sole structure providing barrier properties to the epidermis. It is now clear that specialized intercellular junctions, called tight junctions (TJs), are present between cells of the granular layer, where they ensure proper barrier function (Furuse et al., 2002; Tunggal et al., 2005). TJs have been well characterized in simple epithelia where they form a paracellular sizeand ion-specific barrier to separate tissue compartments. The size and ion selectivity of TJs is mainly based on which members of the claudin family of transmembrane proteins are located in the TJs of different tissues. The epidermis expresses several claudins, and loss of claudin-1 in mice results in perinatal lethality attributable to rapid dehydration (Furuse et al., 2002). In addition, TJs are crucial for immune surveillance by Langerhans cells (Kubo et al., 2009). The importance of the two barriers is further confirmed by observations that mutations in SC proteins such as filaggrin are linked to diseases characterized by an impaired skin barrier, such as atopic dermatitis and ichthyosis vulgaris (Palmer et al., 2006; Smith et al., 2006). In addition, human claudin-1 mutations are associated with neonatal sclerosing cholangitis associated with ichthyosis (Hadj-Rabia et al., 2004). However, the exact molecular mechanisms of how TJs and the SC contribute to the formation, maintenance, and restoration of the skin barrier are not well understood. In addition, it is not known whether and how TJs and the SC cooperate to form a fully functional skin barrier. Transgenic mouse models have been helpful tools to investigate how proteins implicated in epidermal barrier function contribute to the formation and maintenance of skin barrier function. Next to assessing whether such mice have a BENEFITS OF DYE PENETRATION ASSAYS

E-cadherin binds to desmoglein to facilitate desmosome assembly

Desmosomes are adhesive junctions composed of two desmosomal cadherins: desmocollin (Dsc) and desmoglein (Dsg). Previous studies demonstrate that E-cadherin (Ecad), an adhesive protein that interacts in both trans (between opposing cells) and cis (on the same cell surface) conformations, facilitates desmosome assembly via an unknown mechanism. Here we use structure-function analysis to resolve the mechanistic roles of Ecad in desmosome formation. Using AFM force measurements, we demonstrate that Ecad interacts with isoform 2 of Dsg via a conserved Leu-175 on the Ecad cis binding interface. Super-resolution imaging reveals that Ecad is enriched in nascent desmosomes, supporting a role for Ecad in early desmosome assembly. Finally, confocal imaging demonstrates that desmosome assembly is initiated at sites of Ecad mediated adhesion, and that Ecad-L175 is required for efficient Dsg2 and desmoplakin recruitment to intercellular contacts. We propose that Ecad trans interactions at nascent cell-cell contacts initiate the recruitment of Dsg through direct cis interactions with Ecad which facilitates desmosome assembly.