1. Required minimal protein domain of flower for synaptobrevin2 endocytosis in cytotoxic T cells.
- Author
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Ravichandran, Keerthana, Schirra, Claudia, Urbansky, Katja, Tu, Szu-Min, Alawar, Nadia, Mannebach, Stefanie, Krause, Elmar, Stevens, David, Lancaster, C. Roy D., Flockerzi, Veit, Rettig, Jens, Chang, Hsin-Fang, and Becherer, Ute
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CYTOTOXIC T cells ,SERIAL murders ,LIFE sciences ,CYTOLOGY ,PROTEIN domains ,ENDOCYTOSIS - Abstract
Flower, a highly conserved protein, crucial for endocytosis and cellular fitness, has been implicated in cytotoxic T lymphocyte (CTL) killing efficiency 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 specific to CG in mouse CTL. Using immunogold electron microscopy and total internal fluorescence microscopy, we found that Flower translocates in a stimulus-dependent manner from small vesicles to the IS, thereby ensuring specificity 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 identified two pivotal sites through site-specific mutation: a putative AP2-binding site, and a tyrosine at position 104 in mouse Flower. These findings provide insights into Flower's specific functional domain essential for CG endocytosis, which is a key process in mediating T cell serial killing required for the effective fight against cancer. [ABSTRACT FROM AUTHOR]
- Published
- 2024
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