Separable Roles for Neur and Ubiquitin in Delta Signalling in the Drosophila CNS Lineages

Area of Science:

• Cellular biology
• Developmental biology
• Biochemistry

Background:

• Notch signaling activation depends on mechanical force from ligand binding.
• Ubiquitin ligases like Neuralized and Mindbomb1 are involved, but ubiquitylation's role is unclear.
• Previous studies suggested Delta-Neur activity doesn't require Neuralized's catalytic domain or Delta's intracellular lysines.

Purpose of the Study:

• To investigate the precise role of ubiquitylation in Delta-Neur ligand-induced Notch signaling.
• To dissect the functional requirements of the Delta-Neur interaction for Notch activation.
• To elucidate the mechanism of Notch signal initiation by the Delta-Neur complex.

Main Methods:

• Utilized a sensitive in vivo assay in newborn *Drosophila* neurons measuring Delta-Notch-dependent Hey gene expression.
• Performed genetic manipulations to assess the necessity of ubiquitylation and specific protein domains for signaling.
• Analyzed the impact of mutations in Delta intracellular lysines and the Neuralized catalytic domain on signaling activity.

Main Results:

• Demonstrated that the physical interaction between Delta and Neuralized (Neur) is essential for Notch signaling, independent of ubiquitylation.
• Showed that while not strictly required, the Neuralized catalytic domain enhances signaling, particularly when Delta lysines are mutated.
• Observed that Hey expression is sensitive to endocytosis perturbations, suggesting a role for this process.

Conclusions:

• The Delta-Neur interaction itself, forming a signaling complex, is the primary driver of Notch activation.
• Ubiquitylation of components other than Delta may enhance Delta-Neur complex function.
• The Delta-Neur complex likely initiates Notch signaling via a force-generating endocytosis event in the receiving cell.