Calcium-dependence of cytotoxic granule endocytosis depends on the Flower protein

Abstract

Abstract Cytotoxic T lymphocytes (CTLs) kill their targets by secreting perforin and granzymes from cytotoxic granules (CGs) at the interface between CTL and target cells, the immunological synapse (IS). Retrieval of membrane proteins at the IS plays a crucial role in CTL effector function by impacting the serial killing ability of CTLs. However, the cellular and molecular mechanism of CG endocytosis is largely unknown. In neuronal synapses, it has been shown that calcium influx is instrumental to trigger endocytosis, but the identity of calcium channel remains unclear. Flower protein has been reported as a potential candidate to regulate synaptic endocytosis in drosophila. We investigated a potential calcium-dependence of CG endocytosis and whether Flower plays a role in this process. We first confirmed that Flower protein is expressed in WT mouse CD8+ CTLs by western blot. We then generated Flower-deficient mice to further study Flower function. While we observed no change of degranulation and CG secretion in Flower-deficient CTLs, CG endocytosis at the IS was entirely blocked upon target cell contact. Re-introduction of Flower protein or elevation of extracellular calcium rescued the endocytosis defect in Flower-deficient CTLs. Interestingly, live imaging data showed that Flower localizes on intracellular vesicles which polarize to the IS upon target cell contact. We conclude from our data that endocytosis of CGs is calcium-dependent and that the Flower protein is providing the required calcium, probably through release from intracellular organelles.