Topological Assembly of Signalling Proteins in T cells

Background. T cells are adaptive immune cells essential for human immunity, playing a central role in pathogen elimination and tumour surveillance. To recognise harmful antigens, T cells rely on their membrane antigen receptors (TCRs) to scan the surface of antigen-presenting cells (APCs) rapidly and efficiently. After antigen recognition, large changes occur in the contact interface between T cells and APCs, known as the
immunological synapse, triggering a cascade of signalling events that ultimately lead to multiple T cell responses, including proliferation, differentiation, or cell death. One mechanism that seems essential for signal transduction is the spatial organisation of TCRs and other signalling proteins (i.e., cell surface receptors, activating kinases and adaptor proteins) during T cell activation. Fluorescence microscopy techniques have been paramount to observe this mechanism [1]. In activated T cells, signalling proteins form microclusters (multi-molecular aggregates) around phosphorylated TCRs, which are important sites for early signalling. These microclusters then coalesce to form a central supramolecular activation cluster which is surrounded by adhesion proteins and a dense cortical actin mesh to establish an intimate contact between the T cell and the APC [2].
Whilst the main signalling proteins, their interactions and spatial organisation within the immunological synapse are largely known, the mechanisms that mediate protein trafficking to establish microcluster formation in the mature synapse are not completely understood. Actin cytoskeletal remodelling is widely thought to play a key role during the immunological synapse initiation by forming a dense meshwork at the synapse periphery [3,4].
However, there is a lack of understanding of the exact mechanism responsible for modulating membrane protein and clustering.
Aim. Our key hypothesis is that the coalescence of microclusters in larger activation clusters can be explained with the generation of positive topological defects on the membrane due to the dynamic reorganisation of actin in response to antigen-presenting cells. The actin network shows all the characteristic of an inward-growing transportation network, and we believe that the geometrical confinement of this pseudo-2D architecture can influence the plasma membrane topology, which in turns increases dwell time of proteins in local environments aiding microcluster formation. The overall goal of this PhD project is then to use the mathematical formalism of active nematics (active systems made of self-driving elongated units, such as actin filaments [5]) to shed light into the mechanisms that govern the organisation of signalling proteins within the T cell immunological synapse.
In fact, in active nematics, topological defects spontaneously emerge and lead to complex collective self organisation behaviours [5].
Methodology. Experiments will be performed in Simoncelli's lab with the immortal T cell line Jurkat, in which the current T cell signalling paradigm was developed. The Jurkat T cell line (already available) will be cultured and transiently transfected with fluorescently expressing vectors when required (according to standard protocols), using the cell culture facility available in the LCN. To mimic the T cell-APC interface, T cells will
be deposited on planar supported lipid bilayers decorated with suitable proteins and ligands capable of T cell activation. Protein diffusion of different T cell proteins, including the TCR, CD45 and LFA-1 (under resting and activating conditions) will be recorded using a custom-built TIRF microscope (already available). The set up currently counts with two laser lines (647 nm and 488 nm), a scientific complementary metal-oxide-semiconductor (sCMOS) camera, and appropriate optical elements suitable for super-resolution imaging.