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
