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Abstract 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 (EDP) as a VASP binding partner and establish novel non-canonical interactions between EDP and the EVH1 and EVH2 domains of VASP. The major FEME regulators EDP and lamellipodin (LPD) interact multivalently with VASP undergoing liquid-liquid phase separation both in solution and on lipid membranes. We show that priming complex mimicking condensates localise actin polymerization, with LPD-VASP promoting and EDP antagonising actin assembly, suggesting a novel role for EDP during the priming step of FEME. Finally, we show that LPD and EDP recruits and clusters VASP on lipid membranes mimicking the plasma membrane’s inner leaflet to locally assemble actin filaments. Our results support a model where actin polymerization in FEME is spatiotemporally initiated by the depletion of EDP, mediated by receptor activation. Teaser Liquid-like condensates formed by actin polymerases and endocytic proteins, localise and regulate actin polymerization.
Narayan et al. (Fri,) studied this question.