Required minimal protein domain of flower for synaptobrevin2 endocytosis in cytotoxic T cells

Abstract

Flower, a highly conserved protein, crucial for endocytosis and cellular ftness, has been implicated in cytotoxic T lymphocyte (CTL) killing efciency through its role in cytotoxic granule (CG) endocytosis at the immune synapse (IS). This study explores the molecular cues that govern Flower-mediated CG endocytosis by analyzing uptake of Synaptobrevin2, a protein specifc to CG in mouse CTL. Using immunogold electron microscopy and total internal fuorescence microscopy, we found that Flower translocates in a stimulus-dependent manner from small vesicles to the IS, thereby ensuring specifcity in CG membrane protein recycling. Using confocal live-cell imaging, we assessed the ability of a range of naturally occurring mouse, human and Drosophila isoforms to rescue defective endocytosis in Flower KO CTLs. This analysis demonstrated that the N-terminal portion of the protein, encompassing amino acids 1–106 in mice, is the minimal domain necessary for Synaptobrevin2 endocytosis. Additionally, we identifed two pivotal sites through site-specifc mutation: a putative AP2- binding site, and a tyrosine at position 104 in mouse Flower. These fndings provide insights into Flower's specifc functional domain essential for CG endocytosis, which is a key process in mediating T cell serial killing required for the efective fght against cancer.