Complex multivalent protein interactions involving endophilin, lamellipodin and VASP regulate membrane shaping phenomena and actin assembly.

Abstract

Lamellipodin (LPD) is a peripheral membrane protein that plays an initiatory role in endocytic pathways such as Fast endophilin mediated endocytosis by the recruitment of Endophilin, and regulatory roles in lamellipodia and filopodia by recruiting ena/VASP proteins. The processes of endocytosis and cellular motility depend on actin polymerization and involve inward and outward deformation of the plasma membrane respectively. The question thus can be asked: how does LPD recruit its binding partners, the curvature generating BAR protein endophilin and the actin polymerase VASP, to specifically trigger the aforementioned phenomena and concomitant directional membrane remodelling? In this thrust, we probe the complex multi-protein interplay by reconstituting these interactions in vitro. First, we show that VASP and LPD associate multivalently to undergo liquid like phase separation, a fundamental physical process ubiquitously observed in biology. We find that actin polymerization is exclusively initiated in the LPD-VASP rich clusters, and that elongating filaments deform the clusters resulting in local protein reorganization and the formation of large scale VASP-LPD facilitated actin networks. Our observation along with the fact that growing actin filaments deform the adjacent lipid bilayer, introduces a new intermediate step for membrane remodelling, a protein-protein phase separation mediated actin network formation. Next, we investigated the role of Endophilin in the above-mentioned system. We have previously shown that endophilin and LPD interact multivalently in the priming of FEME patches. Here we find that endophilin participates in VASP-LPD phase separation and influences actin filament morphology and connectivity. We thus anticipate that the initiation and the direction of curvature generation are governed by complex multivalent interactions that regulate actin polymerization.