Evidence indicates that endosomal entry promotes signaling by the Notch receptor, but the mechanisms involved are not clear. In a search for factors that regulate Notch activation in endosomes, we isolated mutants in Drosophila genes that encode subunits of the vacuolar ATPase (V-ATPase) proton pump. Cells lacking V-ATPase function display impaired acidification of the endosomal compartment and a correlated failure to degrade endocytic cargoes. V-ATPase mutant cells internalize Notch and accumulate it in the lysosome, but surprisingly also show a substantial loss of both physiological and ectopic Notch activation in endosomes. V-ATPase activity is required in signal-receiving cells for Notch signaling downstream of ligand activation but upstream of γ-secretase-dependent S3 cleavage. These data indicate that V-ATPase, probably via acidification of early endosomes, promotes not only the degradation of Notch in the lysosome but also the activation of Notch signaling in endosomes. The results also suggest that the ionic properties of the endosomal lumen might regulate Notch cleavage, providing a rationale for physiological as well as pathological endocytic control of Notch activity.

Three-dimensional biofabrication using photo-crosslinkable hydrogel bioscaffolds has the potential to revolutionize the need for transplants and implants in joints, with articular cartilage being an early target tissue. However, to successfully translate these approaches to clinical practice, several barriers must be overcome. In particular, the photo-crosslinking process may impact on cell viability and DNA integrity, and consequently on chondrogenic differentiation. In this review, we primarily explore the specific sources of cellular cytotoxicity and genotoxicity inherent to the photo-crosslinking reaction, the methods to analyze cell death, cell metabolism, and DNA damage within the bioscaffolds, and the possible strategies to overcome these detrimental effects. Significance statement Several hurdles need to be addressed before the clinical translation of articular cartilage regeneration procedures using photo-crosslinkable hydrogels. Cellular cytotoxicity and genotoxicity need to be identified and carefully detected to provide an indication of the safety of the repair treatment approach in patients.

Notch signaling in prominently involved in growth regulation in metazoan tissues. Because of this, Notch is often upregulated in cancer and current efforts point to developing drugs that block its activation. Notch receptor endocytosis towards acidic compartments is a recently appreciated determinant of signaling activation. Vacuolar H+ ATPase (V‐ATPase) is responsible for acidification of endocytic organelles and mutants in V‐ATPase subunit encoding genes in model organisms have been recently shown to display loss of Notch signaling. Here, we show that administration of BafilomycinA1 (BafA1), a highly specific V‐ATPase inhibitor decreases Notch signaling during Drosophila and Zebrafish development, and in human cells in culture. In normal breast cells, we find that BafA1 treatment leads to accumulation of Notch in the endo‐lysosomal system, and reduces its processing and signaling activity. In Notch‐addicted breast cancer cells, BafA1 treatment reduces growth in cells expressing membrane tethered forms of Notch, while sparing cells expressing cytoplasmic forms. In contrast, we find that V‐ATPase inhibition reduces growth of leukemia cells, without affecting Notch activatory cleavage. However, consistent with the emerging roles of V‐ATPase in controlling multiple signaling pathways, in these cells Akt activation is reduced, as it is also the case in BafA1‐treated breast cancer cells. Our data support V‐ATPase inhibition as a novel therapeutic approach to counteract tumor growth via signaling pathways regulated at the endo‐lysosomal level.

Barrier-to-Autointegration Factor (BAF) involvement in prelamin A-related chromatin organization changes