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Deciphering the mechanism of enclysis: new tools for cell biology in live human tissues

Primary Supervisor: Dr Zania Stamataki, Institute of Immunology and Immuotherapy

Secondary supervisor: Professor Jon Preece

PhD project title: Deciphering the mechanism of enclysis: new tools for cell biology in live human tissues.

University of Registration: University of Birmingham

Project outline:

Background. We recently discovered that hepatocytes, epithelia that make up 80% of the liver, can preferentially engulf and delete T cell subsets that dampen inflammation (Tregs). We termed this process enclysis (enclosure + lysis) and it is more similar to macropinocytosis (cell internalisation of large amounts of fluid) than the phagocytosis of dead cells, when we investigate cell membrane alterations. Our team has also established methodology to preserve human livers ex vivo with continuous perfusion, in a manner that enables time lapse multiphoton imaging without harming hepatocytes.

Project Aim. To adapt cell biology techniques designed to study membrane alterations, intracellular vesicle communication and molecular mechanisms of endocytosis, to study live human hepatocytes in situ in human liver wedges.

Our team. To define the mechanism of the newly described biological phenomenon of enclysis, we have composed a team that brings together expertise from Biosciences, Chemistry, Cell biology and Immunology, from Universities of Birmingham and Warwick. Dr. Zania Stamataki is a liver immunologist and senior lecturer at the University of Birmingham (MDS, UoB) that discovered enclysis (Davies et al., in press, Cell Reports), and collaborates with Prof. Jon Preece (Chemistry, UoB) who generated novel fluorochromes ideal for use in perfused human liver tissue in multi-photon microscopy. We established a high content assay to demonstrate mitochondrial activity in hepatocytes, and validated it for use in small molecule screening for enclysis inhibitors. The timing for this project is perfect as Prof. Jason Mercer, an expert in macropinocytosis and high content imaging, is moving to UoB from MRC-LMCB at UCL and has a long track record of bioinformatic analyses of large datasets. Prof. Steve Royle from University of Warwick (UoW), brings cell biology expertise in endocytosis. We also collaborate with Prof. David Hodson (MDS, UoB), who is a biophotonics expert with expertise in cell metabolism. This studentship is a great opportunity to bring together a multi-disciplinary team to understand the mechanism of a new cellular process.

Project milestones:

  1. Multiphoton training -imaging live human liver
  • Select optimal fluorochrome combinations for T cell and hepatocyte viability
  • Evaluate readouts for cell migration, metabolic activity (speed, trajectory, mitochondrial activity, endocytic processes)
  • Outcomes: validate new fluorochromes in human tissue, bioinformatics training
  1. Enclysis mechanism -cytoskeletal and membrane processes
  • Perform high content assays to identify the effects of known endocytosis modulators in enclysis
  • Validate targets in authentic human disease livers perfused ex vivo
  • Outcomes: Understand biology -biochemistry/high content imaging
  1. Enclysis mechanism -surface receptors and signalling
  • Establish trafficking of fluorescently tagged enclysis receptors in super-resolution imaging (ICAM-1, beta-catenin, others)
  • Inhibit enclysis receptors and signalling molecules in vitro and in ex vivo perfused livers (blocking antibodies, small molecules)
  • Outcomes: Understand biology -genetic modification/super-resolution imaging

Training. The student will have access to the world-class imaging facilities and associated analyses tools through Birmingham Advanced Light Microscopy Centre, the Centre of Membrane Proteins and Receptors (COMPARE) (UoB) and Centre for Mechanochemical Cell Biology (UoW). Based in the largest solid organ transplant centre in Europe, the student will have access to ~2 explant livers per week, which is unparalleled access to human tissue. The primary supervisor has supervised three award-winning PhD students to completion as a first supervisor, is a trained “Mental Health for PGR” champion and a co-founder or the MDS mentoring scheme.

References:

  1. Davies et al. … Stamataki , in press 2019 Cell Reports
  2. Mercer and Helenius: Nat Cell Biol. 2009 May;11(5):510-20.

BBSRC Strategic Research Priority: Understanding the Rules of Life: Immunology

Techniques that will be undertaken during the project:

  • Multiphoton imaging
  • Bioinformatics
  • Biochemistry
  • High content imaging
  • Molecular biology
  • Cell biology
  • Immune cell isolation
  • Cell culture

Contact: Dr Zania Stamataki, University of Birmingham