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Abstract

Clathrin-mediated endocytosis (CME) is one of the major endocytic pathways among eukaryotic organisms. Importantly, this pathway is also hijacked by many pathogens, such as viruses, in order to enter and infect cells. Since the first identification of Clathrin-coated endocytic vesicles, in 1964, CME has been thoroughly characterized and more than 50 proteins have been described to be part of this pathway. Nevertheless, which protein plays a main regulatory function during initiation and which factors are involved in inducing CME activation upon virus binding and internalization is still a matter of debate. Studying the early determinants of virus-cell early interaction and CME recruitment represents an extremely challenging topic due to the fact that such events take place in an extremely narrow time window and are spatially unpredictable. In this work, I describe a novel method to covalently immobilize virus particles onto glass surfaces in order to study early host-pathogens interactions. To specifically address the role of the mechanical vs receptor-mediated properties of viruses in inducing CME activation, latex beads of several sizes were immobilized using the same established approach. By combining surface chemistry, click chemistry and several microscopy techniques (fluorescence live microscopy, super resolution microscopy and electron microscopy) it was possible to unveil new details of early virus–cell interaction. In particular, I could confirm that CME recruitment is dependent on the size of the cargo. Specifically, sizes between 80 to 300 nm in diameter, can favor CME activation independently from receptor binding (mechanical induction). Surprisingly, it was discovered that the maturation process that leads to the formation of Clathrin-coated vesicles (CCVs) is independent from cargo internalization and that the size of the CCVs is imprinted on the Clathrin coat at the early cargo-cell interaction. These results could not be unveiled with canonical cell biology techniques. Interestingly, recruitment of CME can be favored on nanoparticles whose size is below the critical diameter to support mechanical induction (< 80 nm), by artificially inducing receptor engagement/clustering. Taken together these results demonstrate the presence of a fine-tuning between mechanical induction and receptor activation during early virus-cell interaction; this balance plays a major role in virus infection. The established method can be applied in future studies in the field of virology and endocytosis aiming at understanding how different pathogens favor their internalization using certain pathways, which proteins play a major role in endocytosis initiation and which early factors (mechanical VS receptor-mediated) play a role in activating one pathway over the other.