Elucidating the role of ARF GTPases in complement receptor-mediated mast cell function

• Applicant: Dr. Pia Hagenbach
• Subject Area: Cell Biology ; Immunology
• Term: since 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 545019013

Mast cells (MCs) are specialized immune cells mainly known for their role in allergy. They can react to external stressors by rapidly triggering exocytosis of secretory granules (SGs) that are filled with prestored inflammatory mediators for defense. This is a heavily regulated process that involves ligand binding mediated by receptors on the plasma membrane (PM) of MC, followed by receptor endocytosis and vesicular trafficking of SGs for fusion to the PM. The role of ARF GTPases, which are major regulators of intracellular trafficking, in orchestrating trafficking in MCs is poorly understood. In order to shed light on ARF-dependent trafficking in MC, I plan to investigate the role of ARF1 and ARF6 in the trafficking of the complement-mediated G protein-coupled receptors (GPCRs), C3aR and C5aR. Understanding trafficking mechanisms of GPCRs is crucial as their signaling activity is regulated via their subcellular location. Interestingly, both ARF1 and ARF6 have been implicated in post-Golgi trafficking. This together with further scientific evidence are hinting at a role in endocytic recycling of receptors and SG biogenesis and exocytosis. Employing CRISPR/Cas9 gene editing, dynamic confocal and stimulated emission depletion (STED) super-resolution microscopy, perturbation via acute degradation and trafficking assays we aim to pinpoint the trafficking mechanisms of C3aR and C5aR and the function of ARF GTPases in this process. In particular, we aim to 1) generate endogenously tagged knock in MC cell lines to be used for trafficking analysis, 2) delineate the endocytic trafficking mechanisms of the receptors upon ligand binding, 3) gain insights into anterograde trafficking of the receptors from ER-to-PM and lastly 4) elucidate the role of ARF1 and ARF6 in trigger-induced internalization and exocytosis of the receptors. With this approach, we will additionally gain insights into the mechanisms of SG biogenesis, a long unanswered question of MC research. Overall, this project will allow us to elucidate the spatiotemporal dynamics of C3aR and C5aR at a nanoscale resolution and to acquire an understanding of ARF GTPases in complement receptor-mediated MC activation and MC function in general.

• DFG Programme: WBP Position