Visualisation of innate immune signalling complexes in cells by cryo-electron tomography

Abstract

Innate immune signalling is a vital immune response to various infections and cell damage. A common feature of innate signalling complexes is forming a signalling platform through oligomerisation in cells. RIG-I-like receptors (RLRs), RIG-I, MDA5 and LGP2 sense cytosolic viral dsRNA and then promote oligomerisation of the mitochondrial antiviral- signalling protein (MAVS) on the outer mitochondrial membrane. MAVS signalling induces activation of cytosolic kinases, which in turn activates transcription factors IRF3 and NF-kB to promote interferon production. Similarly, activation of NLRP3, a sensor protein to various danger-associated molecular patterns (DAMPs) and pathogen-associated molecular patterns (PAMPs), induces oligomerisation of adaptor protein ASC into large puncta. Caspase-1 is recruited and then cleaved to initiate caspase-1 dependent pyroptosis and the release of pro-inflammatory cytokines. Previous structural studies have shown mechanistic models of the signalling components involved. However, the structure of the innate signalling complex has not been studied in cells so far. In this thesis, I tested fluorescence labelling strategies and employed live-cell confocal imaging techniques to visualise MAVS proteins in cells. In addition, I used cellular signalling assays to test the functionality of tagged-MAVS. Although the results has not yielded the desired functional and fluorescently labelled MAVS yet, it provides solid foundation for future labelling experiments of MAVS. I optimised a cryo-correlative light electron microscopy (cryo-CLEM) workflow to attain high-resolution structural insights into the ASC signalling complexes in cells. My cryo-ET reconstructions, obtained in unstained and fully hydrated conditions, show that the speck is formed of a filamentous network consisting of hollow-tube branched filaments with the dimensions predicted for ASC filaments, based on structural studies of purified ASC PYD filaments and full-length monomeric ASC. The structural organisation of the ASC filament network allows ribosomes and small TGN-like vesicles to be retained in the puncta, or to permeate through them. The filament branching and packing density within ASC puncta provide structural integrity while allowing downstream signalling molecules to diffuse freely and bind at high density within the network.