Rac1, actin cytoskeleton and microtubules are key players in clathrin-independent endophilin-A3-mediated endocytosis

Endocytic mechanisms actively regulate plasma membrane composition and sustain fundamental cellular functions. Recently, we identified a clathrin-independent endocytic (CIE) modality mediated by the BAR domain protein endophilin-A3 (endoA3), which controls the cell surface homeostasis of the tumor marker CD166/ALCAM. Deciphering the molecular machinery of endoA3-dependent CIE should therefore contribute to a better understanding of its pathophysiological role, which remains so far unknown. Here, we investigate the role in this mechanism of actin, Rho GTPases and microtubules, which are major actors of CIE processes. We show that the actin cytoskeleton is dynamically associated with endoA3- and CD166-positive endocytic carriers and that its perturbation strongly inhibits the uptake process of CD166. We also reveal that the Rho GTPase Rac1, but not Cdc42, is a master regulator of this endocytic route. Finally, we provide evidence that microtubules and kinesin molecular motors are required to potentiate endoA3-dependent endocytosis. Of note, our study also highlights potential compensation phenomena between endoA3-dependent CIE and macropinocytosis. Altogether, our data deepen our understanding of this CIE modality and further differentiate it from other unconventional endocytic mechanisms.