Multiple O-glucosylation sites on Notch function as a buffer against temperature-dependent loss of signaling.

Mutations in Drosophila rumi result in a temperature-sensitive loss of Notch signaling. Rumi is a protein O-glucosyltransferase that adds glucose to EGF repeats with a C-X-S-X-P-C consensus sequence. Eighteen of the 36 EGF repeats in the Drosophila Notch receptor contain the consensus O-glucosylation motif. However, the contribution of individual O-glucose residues on Notch to the regulation of Notch signaling is not known. To address this issue, we carried out a mutational analysis of these glucosylation sites and determined their effects on Notch activity in vivo. Our results indicate that even though no single O-glucose mutation causes a significant decrease in Notch activity, all of the glucose residues on Notch contribute in additive and/or redundant fashions to maintain robust signaling, especially at higher temperatures. O-glucose motifs in and around the ligand-binding EGF repeats play a more important role than those in other EGF repeats of Notch. However, a single O-glucose mutation in EGF12 can be compensated by other O-glucose residues in neighboring EGF repeats. Moreover, timecourse cell aggregation experiments using a rumi null cell line indicate that a complete lack of Rumi does not affect Notch-Delta binding at high temperature. In addition, rumi fully suppresses the gain-of-function phenotype of a ligand-independent mutant form of Notch. Our data suggest that, at physiological levels of Notch, the combined effects of multiple O-glucose residues on this receptor allow productive S2 cleavage at high temperatures and thereby serve as a buffer against temperature-dependent loss of Notch signaling.

Drosophila embryonic 'fibroblasts': extending mutant analysis in vitro.

The in vivo analysis of Drosophila using genetics, with almost a hundred year history, has produced an immense body of knowledge about biology. In vitro analysis, while arguably the poor cousin to its in vivo relative, has a utility--in biochemical analyses and in cell-based screening, for example, with RNAi. A major block to the development of in vitro analysis has been the lack of an efficient genetic method to derive cell lines from mutant Drosophila strains. We recently discovered that expression of activated Ras (Ras(V12)) provides cells in vitro with both a survival and a proliferative advantage and hence promotes the generation of cell lines. In this addendum, we provide new data describing the genesis of seven cell lines corresponding to a rumi mutant, which demonstrate that the method can be used to derive lines and study genetic mutants in vitro.