The stoichiometry and cellular dynamics of Toll-Like Receptor (TLR) signalling has long been a challenge. Previous over-expression models do not reflect physiological conditions resulting in altered stoichiometry and limiting our understanding of Mal/TIRAP in vivo function. Purification models lack the regulatory complexity of the immune cell environment.
Here our research addresses these limitations using CRISPR/Cas9 gene editing and single molecule imaging to visualise endogenous TLR pathway proteins in macrophages. CRISPR/Cas9 homology directed repair was used to endogenously tag Mal/TIRAP, the first intracellular component of the TLR-4 and TLR-2 pathways, in immortalised murine bone marrow derived macrophages (iBMMs). Mal/TIRAP is of particular interest due to its complex role in TLR-4 signalling, mutations in Mal provide both protection and susceptibility in disease.
We observed Mal/TIRAP distributed throughout the cell, in association with the plasma membrane and moving dynamically in the cytoplasm, excluding the nucleus. Strikingly, our data reveals that Mal/TIRAP is dynamically pre-associated to TLR-4 and TLR-2, challenging the current understanding that it is recruited only upon ligand binding (LPS and Pam3CSK4). Approximately 5% of TLR-4 and TLR-2 consistently co-localises with Mal/TIRAP, and, given the low levels of Mal/TIRAP expression, this likely represents a TLR regulatory mechanism. Mal/TIRAP in complex with TLRs is predominately monomeric with occasional dimers and trimers observed. Following LPS stimulation, Mal/TIRAP associates with the Myddosome within five minutes. We propose that the pre-association of Mal/TIRAP with TLRs facilitates the rapid activation of NF-κB signalling in response to pathogens.
Our approach, combining CRISPR/Cas9 with high resolution imaging provides new insights into Mal/TIRAP’s in TLR signalling, supporting a paradigm shift in TLR signalling pathways. We propose a model where signalling is regulated by pre-associated multi-protein complexes, enabling rapid responses through conformational changes, in response to pathogens.