Endophilin-A3 (endoA3) is a BAR domain protein that mediates endoA3-dependent endocytosis, a type of clathrin-independent endocytosis (CIE). EndoA3 has previously been shown to be required for internalisation of the glycoprotein and cell adhesion molecule CD166; however, the molecular players involved in this process are not known. Using CD166 as a model cargo, Henri-François Renard and colleagues (Tyckaert et al., 2022) investigate the role of the actin cytoskeleton, microtubules and Rho GTPases in endoA3-dependent endocytosis. Here, they find that actin dynamically associates with CD166- and endoA3-positive endocytic structures. Chemical disruption of the actin cytoskeleton perturbs the membrane distribution of CD166 and reduces its uptake. Similarly, inhibition of microtubule formation significantly reduces CD166 internalisation. Concordantly, silencing of kinesin motor proteins, such as kinesin-14 and KIF5B, also diminishes CD166 uptake, suggesting a potential role for microtubule-based transport of nascent endoA3-positive endocytic vesicles. Finally, using mutant constructs, RNA interference and small-molecule inhibitors, the authors demonstrate that the Rho GTPase Rac1, but not Cdc42, specifically promotes CD166 uptake via endoA3-dependent endocytosis and not other types of Rac1-mediated CIE. Together, these findings identify new molecular players in endoA3-dependent endocytosis and also clarify the mechanisms underlying CD166 membrane dynamics and turnover.
