A study of SNARE-mediated autophagosome clearance using fluorescence lifetime microscopy
Saleeb, Rebecca S.
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Cell survival requires the turnover of toxic cellular material and recycling of biomolecules in low nutrient conditions. An efficient degradation system is therefore essential for disease prevention and its dysfunction has been linked to both neurodegeneration and oncogenesis. Bulk degradation is accomplished through the collection of cytoplasmic material in a unique sequestration vesicle, which forms de novo and subsequently deposits cargo in the lysosome for degradation. This process, known as autophagy, therefore requires membrane fusion between the autophagosomal vesicle and the lysosome. SNARE proteins mediate membrane fusion events and therefore their careful regulation ensures the proper organisation of the membrane trafficking network. The SNARE proteins governing autophagosome clearance have been identified as syntaxin 17, SNAP29 and VAMP8 and SNARE assembly appears to be positively regulated by VPS33A. This well established model of SNARE-mediated autophagosome clearance has not, however, been demonstrated within the spatiotemporal framework of the cell and little is known about how VPS33A modulates SNARE function. The research presented in this thesis therefore aims to determine the applicability of the proposed SNARE model within the cellular environment and to investigate the regulatory mechanisms controlling syntaxin 17 function. To accomplish this, carefully validated fluorescence colocalisation and time-resolved fluorescence lifetime imaging techniques were primarily employed. The limitations of these techniques were also considered for data interpretation and a novel prototype SPAD array technology, designed for high-speed time-correlated single photon counting, was trialled for widefield FLIM-FRET. FLIM-FRET revealed that VAMP8 has been incorrectly assigned as the dominant autophagosomal R-SNARE and VPS33A studies evidence a multi-modal regulation of Stx17 that diverges from other studied syntaxin family modulation mechanisms. A new model of SNAREmediated autophagosome clearance is therefore proposed, where syntaxin 17 engages with SNAP29 and VAMP7 to drive membrane fusion with the endolysosome in a manner governed by VPS33A and dependent on the phosphorylation status of syntaxin 17.