Use of advanced biochemical and bioimaging approaches to decipher the retrograde transport route of immunoglobulin superfamily cell adhesion molecules

Résumé

Endocytosis is a physiological process involving the internalization of cell surface proteins and extracellular cargoes. Following endocytosis, cargoes can either be targeted to the lysosome for degradation or be recycled back directly or indirectly to the plasma membrane. In the latter case, some cargoes are first directed to the Golgi apparatus through the retrograde transport route before being recycled. ALCAM/CD166 and ICAM-1/CD54 are cell adhesion molecules belonging to the immunoglobulin superfamily (IgSF) and both are involved in immune synapse (IS) formation. While the endocytosis of ALCAM is now known to be clathrin-independent and Endophilin-A3 (EndoA3)-dependent, the endocytosis of ICAM-1 is still unclear. Moreover, the intracellular fate of both proteins after internalization remains unknown. In this study, we identified ICAM-1 as a potential new EndoA3-mediated endocytosis cargo and showed that both ALCAM and ICAM-1 follow the retrograde transport route to the Trans-Golgi network (TGN) after internalization. To study the endocytosis of ICAM-1, we first validated our antibody by flow cytometry and microscopy to use it with high resolution confocal microscopy (Airyscan). We also used Total Internal Reflection Fluorescence (TIRF) microscopy with a mScarlet-ICAM-1 construct to study the dynamic colocalization between ICAM-1 and EndoA3 in live cells. Then, to study the retrograde transport of ALCAM and ICAM-1, we used a SNAP-tag based approach developed for this purpose. We first observed that ICAM-1 and EndoA3 dynamically colocalize, suggesting EndoA3-dependent endocytosis. Next, we first confirmed that our SNAP-tag based approach is functional in our cell line. Then, we proved that ALCAM and ICAM-1 follow the retrograde transport route to the TGN and that EndoA3 depletion significantly impairs this process. Finally, for ALCAM, we show that its retrograde transport is dependent on Retromer and the GTPase Rab6, and Syntaxin-16 independent. Altogether, our data unveiled the potential internalization route of ICAM-1 and on the intracellular fate of ALCAM and ICAM-1 after endocytosis, while highlighting some players involved in their transport. These results shine light on the intracellular fate of two members of the IgSF, ALCAM and ICAM-1. As both proteins play a role in IS formation, their intracellular trafficking in cancer cells could affect their recruitment to the IS, thus further affecting immune cell activation. Finally, our data suggest that the members of the IgSF may have a common behaviour in terms of endocytosis and retrograde transport. To validate this hypothesis, ulterior studies on other members, such as L1CAM, will be needed.

la date de réponse	16 janv. 2024
langue originale	Anglais
L'institution diplômante	Universite de Namur
Superviseur	Henri-Francois Renard (Promoteur)