Required Minimal Protein Domain of Flower for Synaptobrevin2 Endocytosis in Cytotoxic T Cells

Overview

Journal Cell Mol Life Sci

Publisher Springer

Specialty Biology

Date 2024 Dec 18

PMID 39694922

Authors

Keerthana Ravichandran

Claudia Schirra

Katja Urbansky

Szu-Min Tu

Nadia Alawar

Stefanie Mannebach

Elmar Krause

David Stevens

C Roy D Lancaster

Veit Flockerzi

Jens Rettig

Hsin-Fang Chang

Ute Becherer

Affiliations

Soon will be listed here.

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.

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