Endophilin-lamellipodin-VASP, key components in fast endophilin-mediated endocytosis, control actin polymerization within liquid-like condensates.

Actin polymerization is essential in several clathrin-independent endocytic pathways including fast endophilin-mediated endocytosis (FEME); however, the actin machinery involved in FEME has been elusive. Here, we show that the actin polymerase VASP colocalizes and interacts directly with the FEME priming complex. We identify endophilin as a VASP binding partner and establish novel noncanonical interactions between the SH3 domain of endophilin and the Ena/VASP homology 1 or 2 (EVH1 and EVH2) domains of VASP. We show that liquid-like condensates formed by endophilin, lamellipodin, and VASP localize actin polymerization both in solution and on lipid membranes and result in the formation of torus-shaped condensates in the bulk. We establish a new role for multivalent endophilin-lamellipodin interactions in the regulation of actin polymerization, and we identify a novel function for endophilin as a promoter of actin bundling. Our findings support a model that explains the connection between local actin polymerization and dynamic formation and dissolution of endocytic priming patches in FEME.