Immobilized DLL4-induced Notch signaling is mediated by dynamics of the actin cytoskeleton.

Notch signaling, which is essential for tissue development and homeostasis, has received attention as an attractive target for cancer therapy, tissue engineering and regenerative medicine. For signal activation, the Notch receptor undergoes proteolysis after binding to its ligand. This process is mediated by a mechanical pulling force, and receptor trans-endocytosis is known to play a central role in supplying the force. On the other hand, Notch ligands immobilized on carrier materials also induce artificial Notch activation. However, the mechanism of signal activation by immobilized ligand proteins is not fully understood. Here, we found that the actin cytoskeleton in Notch1-expressing cells contributes to signal activation induced by immobilized DLL4 (Delta-like ligand 4), and the results showed that pharmacological inhibition of actin dynamics impaired Notch signaling induced by DLL4-coated beads. Moreover, inhibition of actin dynamics remarkably impaired cell migration and was correlated with Notch signaling activity. We also investigated the contribution of Notch cis-endocytosis (the endocytosis of Notch receptor into signal-receiving cells) as an actin-mediated cell biological process to further explore the mechanism of Notch activation by DLL4-coated beads. Compromising the receptor cis-endocytosis pathway with the dynamin inhibitor did not alter DLL4-coated bead-induced Notch signaling, indicating that signal activation is not mediated by dynamin-dependent receptor cis-endocytosis. These findings suggest that Notch activation by immobilized ligands is primarily driven by actin-based cell movement, which might supply a sufficient mechanical force for receptor cleavage, but not by receptor cis-endocytosis.

Exhaustive identification of conserved upstream open reading frames with potential translational regulatory functions from animal genomes.

Upstream open reading frames (uORFs) are present in the 5'-untranslated regions of many eukaryotic mRNAs, and some peptides encoded by these regions play important regulatory roles in controlling main ORF (mORF) translation. We previously developed a novel pipeline, ESUCA, to comprehensively identify plant uORFs encoding functional peptides, based on genome-wide identification of uORFs with conserved peptide sequences (CPuORFs). Here, we applied ESUCA to diverse animal genomes, because animal CPuORFs have been identified only by comparing uORF sequences between a limited number of species, and how many previously identified CPuORFs encode regulatory peptides is unclear. By using ESUCA, 1517 (1373 novel and 144 known) CPuORFs were extracted from four evolutionarily divergent animal genomes. We examined the effects of 17 human CPuORFs on mORF translation using transient expression assays. Through these analyses, we identified seven novel regulatory CPuORFs that repressed mORF translation in a sequence-dependent manner, including one conserved only among Eutheria. We discovered a much higher number of animal CPuORFs than previously identified. Since most human CPuORFs identified in this study are conserved across a wide range of Eutheria or a wider taxonomic range, many CPuORFs encoding regulatory peptides are expected to be found in the identified CPuORFs.