Lysosome

Natural killer (NK) cells are an important component of the innate arm of the immune system, killing infected and tumorigenic cells. NK cells kill through the exocytosis of secretory lysosomes containing perforin, which facilitates the entry of granzymes, enzymes which trigger apoptosis, into target cells. Patients affected by Familial Haemophagocytic Lymphohistiocytosis (FHL), a rare genetic disease, display low NK cell cytotoxicity and as a result display an uncontrolled inflammatory response to infection. Four genes have been shown to be mutated in patients affected by FHL (PRF1 (FHL2), UNC13D (FHL3), STX11 (FHL4) and STXBP2 (FHL5)), all of which encode proteins involved in the exocytosis of secretory lysosomes. The precise role of the SNARE Syntaxin 11 (STX11), mutated in FHL-4, is still unknown. We have used live cell confocal microscopy to demonstrate the importance of membrane association for syntaxin 11 in facilitating NK cell cytotoxicity. Using a disease associated C-terminal truncation mutant (268x) and by mutating the 5 C-terminal cysteines to alanines (5CysA), we highlight the importance of this region in the membrane association and palmitoylation of syntaxin 11. We have also used the biotin switch assay to confirm that syntaxin 11 is membrane associated via a palmitoylation site. Finally we have used flow cytometry and RNA interference to investigate the importance of interactions between syntaxin 11 and other proteins involved in secretory lysosome exocytosis, all of which will help to elucidate the molecular mechanisms involved in the killing of infected and tumorigenic cells, and inform treatments for secretory lysosome-related diseases.

Lysosome exocytosis plays a major role in resealing plasma membrane (PM) disruptions. This process involves two sequential steps. First, lysosomes are recruited to the periphery of the cell and then fuse with the damaged PM. However, the trafficking molecular machinery involved in lysosome exocytosis and PM repair (PMR) is poorly understood. We performed a systematic screen of the human Rab family to identify Rabs required for lysosome exocytosis and PMR. Rab3a, which partially localizes to peripheral lysosomes, was one of the most robust hits. Silencing of Rab3a or its effector, synaptotagmin-like protein 4a (Slp4-a), leads to the collapse of lysosomes to the perinuclear region and inhibition of PMR. Importantly, we have also identified a new Rab3 effector, nonmuscle myosin heavy chain IIA, as part of the complex formed by Rab3a and Slp4-a that is responsible for lysosome positioning at the cell periphery and lysosome exocytosis.

Amyloid diseases are characterised by the aggregation of peptides or proteins into insoluble fibrils. In a number of amyloid diseases including; Alzheimer’s, Parkinson’s and type 2 diabetes mellitus, cell death and tissue damage is associated with amyloid formation, but the precise nature of the cytotoxic species in vivo is poorly understood. In vitro studies suggest that pre-fibrillar oligomers cause membrane disruption and are cytotoxic, although other studies have shown that fibrils may also be cytotoxic. The aim of this project is to study the mechanisms of amyloid cytotoxicity. We have shown that amyloid fibril preparations including those generated from disease specific amyloid; β2-m (dialysis-related amyloidosis), α-synuclein (Parkinson’s), Aβ1-40 (Alzheimer’s) and amylin (type 2 diabetes), functional (E. coli curli fibrils) and sequences not found in nature (ccβ-peptide) cause dysfunction in cultured cells and disrupt liposomal membranes, moreover fragmented fibril samples cause more dysfunction than non-fragmented samples. We have also shown, using β2-m amyloid fibrils as a model, that fibrils are internalised into lysosomes whereupon they promote lysosomal dysfunction. In future work we will examine whether lysosomal dysfunction is a common mechanism of amyloid cytotoxicity.