Examining inflammasome formation using microscopy

The inflammasome consists of a cytosolic NOD-like receptor, an adaptor molecule (ASC) and an effector molecules caspase 1.  Once activated the inflammasome processes inflammatory cytokines such as interleukin 1 beta (IL1B) and IL18 as well as driving an aggressive form of cell death (pyroptosis).  Inflammasomme protein complexes are central to sustaining inflammation in acute diseases (like COVID-19 associated ARDS) or chronic conditions (such as Alzheimer’s Disease, Parkinson’s, diabetes, arthritis).  Patients with rare autoactivating mutations in the NLR proteins have basally active inflammasomes leading to severe autoinflammatory syndromes. How inflammasome complexes form within the cell, particularly in patients with autoactivating mutations in NLRs are poorly understood.  

The aims of this project are as follows:
1.      Identify the molecular mechanisms by which the gain of function mutations causes constitutive activation of the NLRs
2.      Determine why gain of function mutations in different NLRs (NLRP3 and NLRC4) result in differences in inflammasome cytokine production with NLRP3 biased towards IL1B and NLRC4 towards IL18
3.      Visualise how gain of function mutations alter inflammasome formation by visualising the protein complexes at super resolution and atomic resolution

This project will study how the inflammasome forms using state of the art microscopy techniques including live super resolution imaging and cyroelectron microscopy tomography.  The consequences of the gain of function mutations on inflammasome formation will be studied using these techniques in cell lines where the key proteins are tagged and the gain of function mutations introduced by CRISPR/Cas9 (many of which are already available within the laboratory).  This work will be extended to consider cells from patients with these diseases to map back the biology and the imaging onto the cell line models.