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 non-canonical interactions between Endophilin and VASP. The major FEME regulators Endophilin and Lamellipodin interact multivalently with VASP to form liquid-like condensates both in solution and on lipid membranes that localize actin polymerization with the extent of actin polymerized regulated by multivalent Endophilin-Lamellipodin interactions. We identify a novel function for Endophilin condensates in bundling actin filaments and show that Endophilin directly binds filamentous actin. Our findings support a model that explains the connection between local actin polymerization and dynamic formation and dissolution of endocytic priming patches in FEME.