Chemically Triggered Protein-Driven Negative Curvature Enables Endocytosis-like Inward Division

Cell-sized lipid vesicles are powerful platforms for reconstructing biological membrane functions. However, reproducing an endocytosis-like inward division remains a long-standing challenge. Herein, we report a spontaneous protein-driven inward vesicle division that mimics endocytosis. Using cellfree protein synthesis, we selectively inserted an amphiphilic fusion protein, mCherry–oleosin, into the inner leaflet of giant unilamellar vesicles. The insertion of mCherry–oleosin with an optimal hydrophobic length (~62 amino acids) induced the formation of daughter vesicles inside the mother vesicles, driven by the generation of a negative curvature on the inner leaflet. The division efficiency depended on the size of the water-soluble protein domain, demonstrating a molecular size-dependent membrane deformation mechanism. This process enabled the encapsulation of external molecules and other vesicles, thus functioning as a minimal model of endocytosis. Furthermore, endocytosis-like division can be temporally controlled by the external addition of rapamycin to activate split TEV protease and cleave the fusion protein. These findings reveal a previously unexplored pathway for protein-driven negative curvature formation and establish a versatile synthetic platform for studying endocytic processes and designing self-reorganizing artificial cells. Introduction