CSL/Su(H) as a regulatory node for immune stress resilience by fine tuning Notch activity in Drosophila melanogaster

• Applicant: Privatdozentin Dr. Anja Christina Nagel
• Subject Area: Developmental Biology
• Term: since 2025
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 563470962

Notch signalling activity controls key processes such as cell differentiation, proliferation or stress response in vertebrates and insects alike. Here, CSL/Su(H) serves as transcriptional regulator of Notch target genes. We recently discovered a novel regulatory mechanism of Notch signalling activity: phosphorylation of Su(H) at Serine 269 interferes with its DNA binding and thus its activity. This mechanism is important for blood cell homeostasis, and is used in the immune response to parasitoid wasp infestation. Here, Notch activity must be inhibited to allow the formation of encapsulation-active blood cell types necessary to fend off the wasp attack. In extensive screens, we have identified Pkc53E (i.e. human PKCα) as the kinase that phosphorylates Su(H) during wasp defence. However, neither the exact molecular trigger of Pkc53E activation is known, nor whether other stress-induced scenarios modify Notch via this mechanism. In addition, we found three other kinases by mass spectrometry that target the neighbouring Threonine 271 in the DNA-binding domain of Su(H), suggesting a network of kinases may be involved in a context specific downregulation of Notch activity. In our project, we use Drosophila as a model system that, thanks to its genetic accessibility, allows specifically activating or knocking down genes in a given tissue, and following gene activity or cell types with appropriate reporters in vivo. Three objectives are pursued: Firstly, we want to investigate how Pkc53E is activated during a wasp attack. We suspect that reactive oxygen species (ROS) are involved in this process, which we want to test with suitable experiments. To this end, we plan to use new tools for in vivo studies, e.g. the Affimer technology to detect pS269-Su(H). Secondly, we want to know whether Pkc53E turns down Notch activity in other stress situations, for example in response to injury using live imaging, or to bacterial infections by analysing intestinal regeneration with appropriate cell markers. Also, the impact of systemic bacterial infection or other stressors on pkc53E and Su(H) phospho-mutants is of interest. Thirdly, we want to investigate the possible link between immune-metabolism and Notch regulation in Drosophila. We plan to study metabolic changes in immune tissues in response to wasp infestation, and also the effects of Notch-activity changes in metabolically active tissues on specific parameters (immune cells dynamics, cytokine secretion etc.). Eventually, transcriptome analyses shall uncover expression changes in Su(H) phospho-mutants in response to parasitism. In sum, our studies aim to unravel the molecular network underlying the inhibition of Notch signal transduction via CSL/Su(H) phosphorylation in the immune stress resilience. The high conservation of CSL homologues raises the possibility that phosphorylation of CSL is also utilized in mammals for a context-specific modulation of Notch activity.

• DFG Programme: Research Grants