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Final Report Summary - BIOPHIS (Biophysical mechanisms regulating early T cell signalling events)

The goal of this project was to investigate how biophysical mechanisms regulate the activation of immune cells, specifically Helper T cells. The project sought to draw on the latest advanced in super-resolution fluorescence microscopy methods – those that were awarded the 2014 Nobel Prize in Chemistry.

Specifically, the project utilised Structured Illumination Microscopy (SIM) in a novel combination with Spatio-Temporal Image Correlation Spectroscopy (STICS) to quantify the structure and dynamics of the cortical actin meshwork in T cell synapses. We found that the cortical actin mesh flowed in a retrograde fashion towards the synapse centre and that this flow was coupled to the flow of the plasma membrane itself. Figure 1a) shows an example of the actin structure in green with membranes in red, together with a region selected and STICS overlay of actin flow. The flow was dependent on actin polymerisation, and, using CRISPR-Cas9 technology, we observed that the coupling between cortical actin and the plasma membrane was dependent on the actin cross-linking protein alpha-actinin. Thus, the flow of actin, and the linker proteins may represent a novel mechanism to regulate the previously observed retrograde flow of signalling microclusters at the T cell synapse. These flows have been shown to be critical for the correct activation of immune cells in response to pathogens (during an immune response) and for inhibiting T cell activation elsewhere (to limit autoimmunity). The novel method combination was published in 2014 (Ashdown et al, Biophysical Journal) with the main biological results published in 2017 (Ashdown et al, Biophysical Journal).

In parallel to the above description of actin and membrane flow, we have also developed methods to quantify the clustering of plasma membrane proteins and applied those methods to the T cell synapse. Using these techniques, published in Nature Methods (Rubin-Delanchy et al 2015), we show that the clustering of the plasma membrane proteins ZAP70 and TCRzeta are altered upon T cell activation. Impact is generated through dissemination of the tools themselves, which are now in use in several labs around the world and also the biological results, which give new insight into the mechanisms controlling the immune response.

Finally, we have imaged membrane lipid order in the plasma membrane of T cells using the environmentally sensitive membrane dye di-4-ANEPPDHQ. Membrane order is thought to be an important parameter in controlling the lateral segregation and diffusion of membrane proteins. We were able to show that the T cell synapse contains numerous sub-synaptic membranous vesicles which have diverse lipid order. The vesicle populations, distinguished by their lipid order were found to exhibit diverse behaviour and selective cargo transport.

Taken together, the project has a) developed new tools for use in the life sciences. These include methods for the accurate quantification of molecular flow and the quantification of nanoscale protein clustering and b) has shed new insight into the biological factors regulating T cell activation. These include potential roles for the coupling of cortical acting and the plasma membrane, differential nanoscale protein clustering and membrane lipid order in sub-synaptic T cell vesicles. Ultimately, the human immune response to pathogens is dependent on the correct activation of T cells and many autoimmune diseases are caused by the inappropriate activation of these cells. This project has therefore contributed to our understanding of the role of T cell activation in health and disease.

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United Kingdom


Life Sciences
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