Regulation of isolation membrane remodelling during autophagosome biogenesis by sorting nexins

Autophagy describes the intriguing and essential process of cellular self-eating. It is found in organisms ranging from yeast to man, further demonstrating its importance for cells across the animal, plant and fungal Kingdoms. Cells use autophagy to generate energy and materials in order to survive when conditions become unfavourable. They also use this process to dispose of damaged and potentially toxic cellular components. This is particularly important in long-lived cells such as the neurons in our brains which have to cope over many years with the cumulative damage that occurs as a normal consequence of cellular function. We now know much about how autophagy is triggered in cells and the molecules responsible for the formation of autophagosomes - the membrane-bound vehicles that are the functional units of the autophagy pathway - but we still do not understand how these structures are assembled within cells. There exists a large family of proteins called sorting nexins (SNXs) that play important roles in remodelling existing membranes within our cells. We have preliminary evidence for a role for some of these in autophagy, and now wish to understand in detail how these proteins act. We will use state-of-the-art microscopy and biochemical assays to describe how SNXs regulate autophagy in cells that are undergoing starvation or are attempting to remove toxic inclusions. These studies will significantly advance our understanding of membrane dynamics during autophagy.

Macroutophagy (autophagy) is an essential catabolic process that isolates cytoplasmic material within double membrane autophagosomes that deliver their contents to lysosomes for degradation. It is upregulated during starvation and stress, and is responsible for the elimination of misfolded protein aggregates (aggrephagy) and damaged mitochondria (mitophagy). At present, the membrane remodelling events required to generate autophagosomes, and in particular the factors that control the curvature of the autophagic template membrane (the isolation membrane), remain elusive. Evidence in yeast suggests that a PI(3)P-binding sorting nexin (snx4p) is required for different forms of autophagy, but its precise roles are not known. The sorting nexins (SNXs) are a family of proteins with membrane remodelling and membrane sorting roles in the endocytic network. They are characterised by the presence of a phox homology (PX) domain, through which they can bind the phosphoinositide PI(3)P, and include a SNX-BAR sub-family (Bin/Amphiphysin/Rvs) with roles in the control of membrane curvature. We have found that one SNX-BAR protein, SNX4, associates with a fraction of LC3-decorated autophagic structures in mammalian cells, and that its siRNA suppression inhibits autophagy. In this proposal, we will use dynamic live-cell imaging and fixed cell fluorescence imaging to describe the timing of SNX4 localisation to the isolation membrane. We will then use our published expertise in immuno EM to define the precise localisation of SNX4 at the autophagosome initiation site, and importantly whether SNX4 PX and BAR domains control isolation membrane remodelling. We will then functionally characterise the molecular basis for SNX4 regulation of autophagy - including the identification its cognate SNX-BAR partner. Finally, we will use our human SNX siRNA library to test whether SNX4 or any other SNXs are required for aggrephagy and mitophagy using characterised, cell-based model systems.

Communications and Engagement: This research has important implications for our understanding of autophagy, a process that can go wrong in many human and animal diseases. As a basic Cell Biology research problem, it is essential that our findings be disseminated appropriately to the general public. The Department and University websites publicise grants and important research papers, with key achievements given press releases. We work closely with press officers both within the University and within grant awarding bodies to coordinate press releases. I maintain my own website, detailing our research in both scientific and layman's terms. I am a member of the University 'Directory of Experts', and have appeared in the Times HES in my capacity as Research Fellow. I am involved in science policy awareness initiatives such as SET for Britain and SET for Europe and I have personally attended the House of Commons to present the work of my group to groups of MPs. As an RCUK Academic Fellow, I am actively involved with public engagement initiatives. These include the biennial 'Science Alive' fair held in central Bristol as part of Science awareness week. Here, research staff and graduate students from the department present hands-on stalls with examples of active research in the Department and how that relates to our understanding of important human diseases. Our annual Sixth Form Schools Week in Biochemistry is attended by approximately 500 sixth-form students from the Bristol area, where they take part in practical sessions and lectures introducing basic concepts of Biochemistry. Departmental staff and graduate students also take part in the annual Sutton Schools' week, which aims to improve widening participation. This drives interest in science engineering and technology as a career, and helps disseminate our work to large audiences. Members of staff, myself included, regularly host local students on work experience programmes, and have contributed to NAGTY Summer Schools held at Bristol University. I also participate in widening participation through my membership on the Cell Biology Committee of the Royal Microscopical Society, who champion outreach activities. Capability: The PI and any researchers engage in communication work to the public as described above. Communication through press releases is coordinated by dedicated staff, trained to tailor outputs to the relevant audiences, and press release content is an iterative process involving press office staff and the PI. The PI has received local training in science communication, and attends public engagement for a (including the University 'Engaged' programme) to improve awareness of public outreach resources and initiatives. For other staff involved in the grant, the University runs regular courses in - science communication - including a Graduate skills training course 'Speaking at Scientific Meetings' co-presented by the PI. Staff members are also encouraged to engage with similar programmes run by the Royal Society and the Wellcome Trust, as well as benefiting from Research Council Programmes such as VITAE. Resource for the activity: No additional resource required