Thesis presented October 06, 2023

Abstract:
Extracellular vesicles (EVs) are recognized to play an important role in physiological and pathological intercellular communication processes. EVs carry lipids, proteins, and microRNAs. which can be shuttled between cells, thereby allowing intercellular communications. The transfer of biologically active EVs cargoes into receiving cells begins with endocytosis of the EVs which are thought to fuse with the endosomal membrane. This is analogous to the content delivery of some enveloped viruses which requires their fusion with the endosomal membrane in a way dependent on acidic pH and the protein Alix.
The aim of our work is to characterize the molecular mechanisms driving the fusion of EVs with target membranes of cells or liposomes. For this, we used luciferase complementation assay to follow the fusion of EVs to membranes of receiving cells and fluorescence membrane-mixing assay to quantify EV membrane fusion to liposomes. We also intend to test if alike viruses, Alix is required for fusion of EVs with endosomal membranes. For this, we used recipient cells expressing LgBit, an inactive subunit of nanoluciferase that activates upon binding to a small peptide, HiBit was fused to the EV cargo proteins and luminescence should only be emitted once HiBit is delivered to the cytoplasm of LgBit recipient cells. We could demonstrate the interaction of HiBit-containing EVs with LgBit-receiving cells but no increase in luminescence, suggesting that no fusion occurs. While in the presence of VSV-G protein, luminescence was enhanced, showing that our method is capable of detecting fusion of EVs to membranes of receiving cells. Importantly, the presence of Alix in recipient cells did not seem to be crucial for this fusion, as it also occurred in Alix ko cells.
However, using fluorescence membrane-mixing assay, our results demonstrated the fusion of stained EVs with unlabeled liposomes in the presence of recombinant Alix at low pH, simulating the acidic conditions found in endosomes.
Finally, we examined how Alix is associated with EVs, as the protein had been reported to be both cytosolic and extracellular suggesting that it can cross membranes. In summary, my thesis work tends to show that EV have the capacity to fuse with membrane in an Alix-dependent process invitro. However, we were unable to establish the role of Alix in EV fusion with endosomal membrane in vivo.

Keywords:
ESCRT, Alix, extracellular vesicles, endosomes, Virus