| Summary: | Natural killer (NK) cells kill abnormal cells through the exocytosis of secretory lysosomes containing the cytotoxic proteins perforin and granzymes. Secretory lysosome exocytosis requires the movement of secretory lysosomes along microtubules to cluster at the microtubule organising centre (MTOC), before polarisation to the immunological synapse and fusion with the plasma membrane. The aims of this thesis were to investigate the roles of syntaxin 11, Rab7 and Arl8b in secretory lysosome exocytosis. Familial haemophagocytic lymphohistiocytosis subset 4 (FHIA) is caused by mutation of syntaxin 11, a soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein. NK cells from FHL4 patients have a defect in secretory lysosome exocytosis. Analysis of syntaxin 11 in the NK cell line, YTS and primary NK cells demonstrated that this SNARE partially localises to early endosomes, and that it was membrane associated and palmitoylated. Membrane localisation in YTS cells was abolished ill the syntaxin 11 FHIA mutants Gln268X, Leu194ProfsX2 and Va1l24fsX60, which may be due to loss of the potentially palmitoylated C-tenninal region of this SNARE. Consistent with this, syntaxin 11 in which C-terminal cysteine residues were mutated was mislocalised in YTS cells, whilst additional disruption of SNARE domain interactions abrogated membrane localisation. Rab7 promotes lysosome movement along microtubules such that they cluster around MTOC, whilst Arl8b promotes lysosome movement towards the cell periphery. Rab7 and Arl8b may therefore play antagonistic roles in secretory lysosome movement along microtubules in NK cells. Overexpression of Rab7 promoted secretory lysosome clustering in YTS cells, suggesting an increase in MTOC-directed movement along microtubules, although a dominant negative Rab7 mutant did not affect secretory lysosome exocytosis in primary NK cells. Overexpression of dominant negative Arl8b mutant promoted secretory lysosome clustering in YTS cells, and increased secretory lysosome exocytosis in primary NK cells. This suggests Arl8b may inhibit secretory lysosome exocytosis in NK cells.
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