Endocytic accessory proteins assemble clathrin while simultaneously destabilizing protein condensates

During endocytosis a dynamic protein network is responsible for assembly of the clathrin coat. Early in the process the Eps15 protein is thought to undergo liquid-liquid phase-separation to form biomolecular condensates. These condensates facilitate clathrin assembly, producing clathrin-coated vesicles that are ultimately excluded from the condensate as the vesicles depart from the plasma membrane. An array of distinct clathrin accessory proteins drive this process, yet their interactions with protein condensates have not been explored. Here we show that clathrin accessory proteins promote the assembly of the clathrin lattice by simultaneously destabilizing protein condensates. By adding diverse accessory proteins to Eps15 condensates, we observed that accessory proteins which cross-linked Eps15 proteins stabilized condensates, while accessory proteins that compete with Eps15-Eps15 interactions destabilized condensates. Interestingly, accessory proteins that destabilized condensates also promoted the assembly of clathrin and its exclusion from condensates. In contrast, accessory proteins that stabilized condensates opposed exclusion of clathrin. Finally, we demonstrated that decreasing condensate stability enhanced clathrin assembly and exclusion, even when no additional clathrin assembly motifs were introduced. Taken together, these results help to explain how condensates can catalyze the assembly of coated vesicles by providing both a substrate for their initiation and a driving force for their exclusion and ultimate departure. Statement of Significance: This work is significant because it reveals how biomolecular condensates could actively drive, rather than merely host, clathrin-mediated endocytosis. By showing that clathrin accessory proteins can tune condensate stability to simultaneously promote clathrin lattice assembly and exclusion, this study uncovers a physical mechanism that could link phase separation to membrane trafficking. The finding that condensate destabilization alone enhances clathrin exclusion reframes condensates as dynamic regulators that could provide both an initiation platform and a force for vesicle maturation and release. More broadly, this work advances our understanding of how cells coordinate assembly and disassembly of complex macromolecular structures through regulated changes in condensate material properties.