Receptor interacting protein kinase 1 (RIPK1) has an essential role in the signalling triggered by death receptors and pattern recognition receptors. RIPK1 is believed to function as a node driving NF-κB-mediated cell survival and inflammation as well as caspase-8 (CASP8)-dependent apoptotic or RIPK3/MLKL-dependent necroptotic cell death. The physiological relevance of this dual function has remained elusive because of the perinatal death of RIPK1 full knockout mice. To circumvent this problem, we generated RIPK1 conditional knockout mice, and show that mice lacking RIPK1 in intestinal epithelial cells (IECs) spontaneously develop severe intestinal inflammation associated with IEC apoptosis leading to early death. This early lethality was rescued by antibiotic treatment, MYD88 deficiency or tumour-necrosis factor (TNF) receptor 1 deficiency, demonstrating the importance of commensal bacteria and TNF in the IEC Ripk1 knockout phenotype. CASP8 deficiency, but not RIPK3 deficiency, rescued the inflammatory phenotype completely, indicating the indispensable role of RIPK1 in suppressing CASP8-dependent apoptosis but not RIPK3-dependent necroptosis in the intestine. RIPK1 kinase-dead knock-in mice did not exhibit any sign of inflammation, suggesting that RIPK1-mediated protection resides in its kinase-independent platform function. Depletion of RIPK1 in intestinal organoid cultures sensitized them to TNF-induced apoptosis, confirming the in vivo observations. Unexpectedly, TNF-mediated NF-κB activation remained intact in these organoids. Our results demonstrate that RIPK1 is essential for survival of IECs, ensuring epithelial homeostasis by protecting the epithelium from CASP8-mediated IEC apoptosis independently of its kinase activity and NF-κB activation.

The development of epithelial appendages, including hairs, glands and teeth starts from ectodermal placodes, and is regulated by interplay of stimulatory and inhibitory signals. Ectodysplasin-A1 (Eda-A1) and Wnts are high in hierarchy of placode activators. To identify direct targets of ectodysplasin pathway, we performed microarray profiling of genes differentially regulated by short exposure to recombinant Eda-A1 in embryonic eda(-/-) skin explants. Surprisingly, there were only two genes with obvious involvement in Wnt pathway: dkk4 (most highly induced gene in the screen), and lrp4. Both genes colocalized with Eda-A1 receptor Edar in placodes of ectodermal organs. They were upregulated upon Edar activation while several other Wnt associated genes previously suggested as Edar targets were unaffected. However, low dkk4 and lrp4 expression was retained in the absence of NF-kappaB signalling in eda(-/-) hair placodes. We provide evidence that this expression was dependent on Wnt activity present prior to Eda-A1/Edar signalling. Dkk4 was recently suggested as a key Wnt antagonist regulating lateral inhibition essential for correct patterning of hair follicles. Several pieces of evidence suggest Lrp4 as a Wnt inhibitor, as well. The finding that Eda-A1 induces placode inhibitors was unexpected, and underlines the importance of delicate fine-tuning of signalling during placode formation.

Epithelial reorganization involves coordinated changes in cell shapes and movements. This restructuring occurs during formation of placodes, ectodermal thickenings that initiate the morphogenesis of epithelial organs including hair, mammary gland, and tooth. Signaling pathways in ectodermal placode formation are well known, but the cellular mechanisms have remained ill defined. We established imaging methodology for live visualization of embryonic skin explants during the first wave of hair placode formation. We found that the vast majority of placodal cells were nonproliferative throughout morphogenesis. We show that cell compaction and centripetal migration are the main cellular mechanisms associated with hair placode morphogenesis and that inhibition of actin remodeling suppresses placode formation. Stimulation of both ectodysplasin/NF-κB and Wnt/β-catenin signaling increased cell motility and the number of cells committed to placodal fate. Thus, cell fate choices and morphogenetic events are controlled by the same molecular pathways, providing the framework for coordination of these two processes.

Whereas the interaction between Tau and the microtubules has been studied in great detail both by macroscopic techniques (cosedimentation, cryo-electron microscopy, and fluorescence spectroscopy) using the full-length protein or by peptide mapping assays, no detailed view at the level of individual amino acids has been presented when using the full-length protein. Here, we present a nuclear magnetic resonance (NMR) study of the interaction between the full-length neuronal protein Tau and paclitaxel-stabilized microtubules (MTs). As signal disappearance in the heteronuclear 1H-15N correlation spectra of isotope-labeled Tau in complex with MTs is due to direct association of the corresponding residue with the solid-like MT wall, we can map directly the fragment in interaction with the MT surface, and obtain a molecular picture of the precise interaction zones. The N-terminal region projects from the microtubule surface, and the lack of chemical shift variations when compared with free Tau proves that this region can regulate microtubular separation without adopting a stable conformation. Amino acids in the four microtubule binding repeats (MTBRs) lose all of their intensity, underscoring their immobilization upon binding to the MTs. The same loss of NMR intensity was observed for the proline-rich region starting at Ser214, underscoring its importance in the Tau:MT interaction. Fluorescence resonance energy transfer (FRET) experiments were used to obtain thermodynamic binding parameters, and led to the conclusion that the NMR defined fragment indeed is the major player in the interaction. When the same Ser214 is phosphorylated by the PKA kinase, the Tau:MT interaction strength decreases by 2 orders of magnitude, but the proline-rich region including the phospho-Ser214 does not gain sufficient mobility in the complex to make it observable by NMR spectroscopy. The presence of an intramolecular disulfide bridge, on the contrary, does lead to a partial detachment of the C-terminus of Tau, and decreases significantly the overloading of Tau on the MT surface.