What is a killer protein doing at the gut barrier?

Project synopsis: The gut is lined by a single layer of cells that are stitched together by tight junctions. This fragile seal is all that separates us from the trillions of microbes in our bowel, and when it fails, inflammation follows. We previously discovered that MLKL, the protein that executes a lytic form of cell death called necroptosis, is also physically and functionally coupled to tight junctions in the gut barrier (Samson et al., 2020 Nature Communications). This project will use advanced imaging and proximity labelling technique to determine when and where human MLKL interacts with the gut barrier, both at steady state and during inflammation. Project in more detail: Necroptosis is a violent form of cell death which relies on the killer protein MLKL moving to cell membranes and disrupting their integrity. Our team found that activated MLKL does not arrive at the cell surface indiscriminately. Instead, it accumulates at the junctions between neighbouring cells and engages the tight junction machinery. This was unexpected and suggests that MLKL may have a second job - one that has nothing to do with killing cells and that may be important when the bowel becomes inflamed. This project will establish the chronology of interactions between MLKL and tight junction proteins in human cells. Using advanced imaging and proximity-based labelling techniques, this project will follow MLKL’s journey from its dormant cytosolic form to its fully active peri-junctional state. Specifically, this project will answer the fundamental question: What is the cue and mechanism by which human MLKL engages the tight junction machinery? And does the peri-junctional arrival of MLKL reinforce the gut barrier or breach it? Key techniques: • advanced fixed- and live-cell imaging • proximity labelling proteomics • human cell culture • barrier function assays and Western blotting Supervisory team: • Dr Andre Samson – Head of the Gut Inflammation Laboratory at the Centre for Inflammatory Diseases (Monash University) • Professor James Murphy – Laboratory Head and Deputy Director of the Walter and Eliza Hall Institute of Medical Research • Dr Sarah Garnish – Postdoctoral Fellow at the Biomedicine Discovery Institute (Monash University) Related work from the team: • Samson et al., (2020) Nature Communications • Samson et al., (2021) Cell Death & Differentiation • Samson & Garnish et al., (2021) Science Signaling • Samson & Murphy (2024) Cell Death & Differentiation • Pang & Al-Ani et al., (2026) Science