Mechanisms of Notch ligands activation by Mib1 using quantitative in vitro and in vivo methods

• Applicant: Professor Dr. Thomas Klein
• Subject Area: Developmental Biology
• Term: since 2025
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 561039599

The Notch signaling pathway mediates short-range cell communication across a variety of developmental, homeostasis, and disease related processes. During signaling, the Notch ligands in the signal-sending cells bind and activate Notch receptors on the signal-receiving cells. In mammals, there are five Notch ligands and four Notch receptors that can all interact with each other. Notch ligand activity has been shown to depend on endocytosis initiated by the E3 ubiquitin ligase Mindbomb1 (MIB1). MIB1 binds to and catalyses the ubiquitylation of the intracellular domains of the Notch ligands. While it has been shown that different ligands have different functional roles in vivo, it is unclear how their distinct activities are encoded at the molecular level and whether MIB1 plays a role in these differences. In this grant application, we will combine quantitative in vivo and in vitro assays to elucidate how MIB1 differentially regulates the activity of mammalian Notch ligands. We will pursue the following three aims: (i) Teasing apart the contributions of MIB1 binding and ubiquitylation efficiency to Notch ligand activity. (ii) Identifying the complex binding modes between MIB1 and different Notch ligands. And, (iii) investigating the possibility for a ‘kiss and run’ mechanism for MIB1 action, where Mib1 dissociates from the ligands upon their ubiquitylation. We will integrate experiments using ‘humanized’ Drosophila, quantitative mammalian cell culture, and bacterial binding and ubiquitylation assays. This project will reveal the molecular mechanisms underlying the interactions of MIB1 with the mammalian Notch ligands and how these interactions promote differential Notch activity in different developmental and disease related contexts.

• DFG Programme: Research Grants
• International Connection: Israel
• Partner Organisation: The Israel Science Foundation
• Cooperation Partner: Professor Dr. David Sprinzak, Ph.D.