AP-1 functions in Notch signaling and cell polarity – APiNotch

Asymmetric cell division is a conserved mechanism by which cell fate diversity is generated during Metazoan development. How one cell can generate two daughter cells with different identities and how defects in this asymmetry can contribute to pathologies are the fundamental questions we are addressing in Drosophila. We are investigating this process in the context of asymmetric cell division of neural precursor cells called Sensory Organ Precursor (SOP). These latter undergo four rounds of asymmetric division, in which mother cells generate distinct daughters via the unequal segregation of the cell-fate determinants Numb and Neuralized (Neur) at mitosis. At each division binary cell fate decision are regulated by Delta-Notch dependent cell-cell signalling. Notch receptor is activated by the ligand Delta present at the surface of adjacent signal-sending cell (trans-activation). Numb is an endocytic protein that can bind to Notch and Sanpodo (Spdo), a four pass transmembrane protein required for Notch activation in SOP lineage, thereby preventing Notch activation in the cells inheriting Numb. Neur acts in SOPs and pIIb cells, the signal-sending cells, to regulate the endocytosis and signalling activity of Delta, thereby promoting Notch activation. Despite intensive studies, the mechanism by which Neur regulates Delta activity is not known. The nature of the ligand activation and the subcellular localisation where recycled Delta could interact with Notch to produce signalling remained unknown. Similarly, endocytosis of Notch is proposed to play both positive and negative roles on the signaling. Our research aims to understand how intracellular trafficking regulates the spatio-temporal regulation of Notch-dependent fate decision.
We have performed a genetic screen that led to the identification of novel potential regulators of Notch signaling. Among them, we identified the clathrin adaptor AP-1 complex. While mammalian AP-1 is involved in lysosome-related organelle biogenesis and in polarized sorting of membrane proteins to the basolateral plasma membrane, the function of Drosophila AP-1 was largely unknown. We reported that AP-1 localized at the trans-Golgi Network and in recycling endosomes, acts as a negative regulator of Notch signalling. Inactivation of AP-1 causes ligand-dependent activation of Notch leading to a fate transformation within sensory organs. Loss of AP-1 causes apical accumulation of the Notch activator Sanpodo and stabilization of both Sanpodo and Notch at the interface between SOP daughter cells, where DE-Cadherin is localized. Our data point towards a specific DE-Cadherin-rich junctional contact containing Notch and Sanpodo that could serve as a launching platform from where ligands are trafficked for signalling. While our results have begun to shed light on the function of AP-1 in the control of Notch signaling, several important issues remained unsolved. We here propose to investigate the molecular mechanisms by which AP1 complex regulate Notch signaling. To this aim, our research proposal is aimed at:
1- identifying the site and mechanism of action of AP-1 at both light and electron microscopy level. We propose to develop and adapt correlative light and electron microscopy to clonal analyses in Drosophila
2- characterizing novel interactors of AP-1 identified by genetic (our screen) and biochemical (collaboration with Pr. B. Hoflack, Dresden) means
3- investigating the link between AP-1-dependent E-Cadherin junctional domain and Notch signaling (launching platform).
To reach this goal, we propose to combine Drosophila genetics to cutting edged cell biology approaches to investigate in molecular terms the respective role of regulators identified in the screens as well as the role of DE-Cadherin junctional domain in the regulation of Notch-dependent binary fate decisions following asymmetric cell division.