Pathological mutations reveal the key role of the cytosolic iRhom2 N-terminus for phosphorylation-independent 14-3-3 interaction and ADAM17 binding, stability, and activity.

The protease ADAM17 plays an important role in inflammation and cancer and is regulated by iRhom2. Mutations in the cytosolic N-terminus of human iRhom2 cause tylosis with oesophageal cancer (TOC). In mice, partial deletion of the N-terminus results in a curly hair phenotype (cub). These pathological consequences are consistent with our findings that iRhom2 is highly expressed in keratinocytes and in oesophageal cancer. Cub and TOC are associated with hyperactivation of ADAM17-dependent EGFR signalling. However, the underlying molecular mechanisms are not understood. We have identified a non-canonical, phosphorylation-independent 14-3-3 interaction site that encompasses all known TOC mutations. Disruption of this site dysregulates ADAM17 activity. The larger cub deletion also includes the TOC site and thus also dysregulated ADAM17 activity. The cub deletion, but not the TOC mutation, also causes severe reductions in stimulated shedding, binding, and stability of ADAM17, demonstrating the presence of additional regulatory sites in the N-terminus of iRhom2. Overall, this study contrasts the TOC and cub mutations, illustrates their different molecular consequences, and reveals important key functions of the iRhom2 N-terminus in regulating ADAM17.

Mechanical activation of lung epithelial cells through the ion channel Piezo1 activates the metalloproteinases ADAM10 and ADAM17 and promotes growth factor and adhesion molecule release.

In the lung, pulmonary epithelial cells undergo mechanical stretching during ventilation. The associated cellular mechanoresponse is still poorly understood at the molecular level. Here, we demonstrate that activation of the mechanosensitive cation channel Piezo1 in a human epithelial cell line (H441) and in primary human lung epithelial cells induces the proteolytic activity of the metalloproteinases ADAM10 and ADAM17 at the plasma membrane. These ADAMs are known to convert cell surface expressed proteins into soluble and thereby play major roles in proliferation, barrier regulation and inflammation. We observed that chemical activation of Piezo1 promotes cleavage of substrates that are specific for either ADAM10 or ADAM17. Activation of Piezo1 also induced the synthesis and ADAM10/17-dependent release of the growth factor amphiregulin (AREG). In addition, junctional adhesion molecule A (JAM-A) was shed in an ADAM10/17-dependent manner resulting in a reduction of cell contacts. Stretching experiments combined with Piezo1 knockdown further demonstrated that mechanical activation promotes shedding via Piezo1. Most importantly, high pressure ventilation of murine lungs increased AREG and JAM-A release into the alveolar space, which was reduced by a Piezo1 inhibitor. Our study provides a novel link between stretch-induced Piezo1 activation and the activation of ADAM10 and ADAM17 in lung epithelium. This may help to understand acute respiratory distress syndrome (ARDS) which is induced by ventilation stress and goes along with perturbed epithelial permeability and release of growth factors.

Bioprinting-associated pulsatile hydrostatic pressure elicits a mild proinflammatory response in epi- and endothelial cells.

During nozzle-based bioprinting, like inkjet and microextrusion, cells are subjected to hydrostatic pressure for up to several minutes. The modality of the bioprinting-related hydrostatic pressure is either constant or pulsatile depending on the technique. We hypothesized that the difference in the modality of hydrostatic pressure affects the biological response of the processed cells differently. To test this, we used a custom-made setup to apply either controlled constant or pulsatile hydrostatic pressure on endothelial and epithelial cells. Neither bioprinting procedure visibly altered the distribution of selected cytoskeletal filaments, cell-substrate adhesions, and cell-cell contacts in either cell type. In addition, pulsatile hydrostatic pressure led to an immediate increase of intracellular ATP in both cell types. However, the bioprinting-associated hydrostatic pressure triggered a pro-inflammatory response in only the endothelial cells, with an increase of interleukin 8 (IL-8) and a decrease of thrombomodulin (THBD) transcripts. These findings demonstrate that the settings adopted during nozzle-based bioprinting cause hydrostatic pressure that can trigger a pro-inflammatory response in different barrier-forming cell types. This response is cell-type and pressure-modality dependent. The immediate interaction of the printed cells with native tissue and the immune system in vivo might potentially trigger a cascade of events. Our findings, therefore, are of major relevance in particular for novel intra-operative, multicellular bioprinting approaches.

Small behavioral adaptations enable more effective prey capture by producing 3D-structured spider threads.

Spiders are known for producing specialized fibers. The radial orb-web, for example, contains tough silk used for the web frame and the capture spiral consists of elastic silk, able to stretch when prey impacts the web. In concert, silk proteins and web geometry affects the spider's ability to capture prey. Both factors have received considerable research attention, but next to no attention has been paid to the influence of fiber processing on web performance. Cribellate spiders produce a complex fiber alignment as their capture threads. With a temporally controlled spinneret movement, they connect different fibers at specific points to each other. One of the most complex capture threads is produced by the southern house spider, Kukulcania hibernalis (Filistatidae). In contrast to the so far characterized linear threads of other cribellate spiders, K. hibernalis spins capture threads in a zigzag pattern due to a slightly altered spinneret movement. The resulting more complex fiber alignment increased the thread's overall ability to restrain prey, probably by increasing the adhesion area as well as its extensibility. Kukulcania hibernalis' cribellate silk perfectly illustrates the impact of small behavioral differences on the thread assembly and, thus, of silk functionality.

Improvements of Microcontact Printing for Micropatterned Cell Growth by Contrast Enhancement.

Patterned neuronal cell cultures are important tools for investigating neuronal signal integration, network function, and cell-substrate interactions. Because of the variable nature of neuronal cells, the widely used coating method of microcontact printing is in constant need of improvements and adaptations depending on the pattern, cell type, and coating solutions available for a certain experimental system. In this work, we report on three approaches to modify microcontact printing on borosilicate glass surfaces, which we evaluate with contact angle measurements and by determining the quality of patterned neuronal growth. Although background toxification with manganese salt does not result in the desired pattern enhancement, a simple heat treatment of the glass substrates leads to improved background hydrophobicity and therefore neuronal patterning. Thirdly, we extended a microcontact printing process based on covalently linking the glass surface and the coating molecule via an epoxysilane. This extension is an additional hydrophobization step with dodecylamine. We demonstrate that shelf life of the silanized glass is at least 22 weeks, leading to consistently reliable neuronal patterning by microcontact printing. Thus, we compared three practical additions to microcontact printing, two of which can easily be implemented into a workflow for the investigation of patterned neuronal networks.