Quantifications of FCHo2 interaction with in vitro and in cellulo systems

<p>Clathrin-mediated endocytosis (CME) is a central trafficking pathway in eukaryotic cells regulated by phosphoinositides. The plasma membrane phosphatidylinositol-4,5-bisphosphate (PI(4,5)P₂) plays an instrumental role in driving CME initiation. The F-BAR domain only protein 1 and 2 complex (FCHo1/2) is among the early proteins that reach the plasma membrane, but the exact mechanisms triggering its recruitment remain elusive. Here, we show the molecular dynamics of FCHo2 self-assembly on membranes by combining minimal reconstituted <i>in vitro</i> and cellular systems. Our results indicate that PI(4,5)P₂ domains assist FCHo2 docking at specific membrane regions, where it self-assembles into ring-like shape protein patches. We show that the binding of FCHo2 on cellular membranes promotes PI(4,5)P₂ clustering at the boundary of cargo receptors and that this accumulation enhances clathrin assembly. Thus, our results provide a mechanistic framework that could explain the recruitment of early PI(4,5)P₂-interacting proteins at endocytic sites.</p>