Role Of Clustering In Determining Spatial Organization At The Immunological Synapse

Role of Clustering in Determining Spatial Organization at the Immunological Synapse

Micron-scale assemblies of molecules are thematic in biology, although their mechanism of formation and exact functional role are oftentimes unknown. The immunological synapse (IS)--the gateway event to the body's initiation of an immune response against infection--is a hallmark example. T cell detection of pathogenic invasion on an antigen-presenting cell leads to the arrangement of receptor-ligand pairs into well-defined concentric zones referred to as supramolecular activation clusters (SMACs). The main signaling molecule, the T cell receptor (TCR), binds its specific foreign peptide-presenting ligand, major histocompatibility complex (pMHC). These complexes form a central cluster in the central SMAC (cSMAC) at the center of the intermembrane junction. Immediately surrounding the cSMAC is the peripheral SMAC (pSMAC), populated by a ring of leukocyte function associated antigen-1 (LFA-1) bound to intercellular adhesion molecule-1 (ICAM-1). In this dissertation, we determine how the final IS pattern emerges from a uniform distribution of receptor-ligand pairs. It is known that the actin cytoskeleton drives the centripetal transport of these proteins, but it is unclear how actin sorts them into their final destinations. We postulate that the large-scale sorting of proteins into the different SMACs is a natural consequence of smaller scale protein sorting into microclusters, which may contain hundreds of molecules. To test this, we increase the LFA-1 cluster size two additional degrees beyond its native state with antibody crosslinkers. We either crosslink LFA-1 directly or indirectly with antibodies against its ICAM-1 ligand, which is presented on a supported membrane with the activating pMHC. Progressively more central localization of LFA-1 proportional to the degree of crosslinking results until LFA-1 occupies the cSMAC with TCR. Based on these results, we propose a sorting mechanism based on frictional protein coupling to actin. In the frictional force coupling model, the extent of radial protein transport by actin is determined by the specific coupling chemistry and the protein cluster size. This model predicts cluster size-based protein sorting across the IS. Using fluorescence fluctuation measurements and a small illumination area, we detect a gradient of LFA-1:ICAM-1 cluster sizes across the pSMAC in the native IS, as predicted by our model. Thus, we demonstrate that the well-regulated event of protein clustering is a critical parameter in regulating spatial patterning in the IS.

Molecular Dynamics at the Immunological Synapse

The immunological synapse (IS) is a specialised cell-cell adhesion that mediates antigen acquisition and regulates the activation of lymphocytes. Initial studies of the IS showed a structure composed of stable supra-molecular activation clusters (SMAC) organised during the interaction of helper T lymphocytes with B lymphocytes, working as antigen presenting cells. A central SMAC of coalesced T cell receptors (TCRs) and a peripheral SMAC for cell-cell adhesion were observed. IS with similar structure was later described during antigen acquisition by B cells and during the interaction of NK cells with target and healthy cells. More recent research developed with microscopy systems that improve the spatial and temporal resolution has showed the complex molecular dynamics at the IS that governs lymphocyte activation. Currently, the IS is seen as a three-dimensional structure where signalling networks for lymphocyte activation and endosomal and cytoskeleton machinery are polarised. A view has emerged in which dynamic microclusters of signalling complexes are composed of molecular components attached to the plasma membrane and other components conveyed on sub-synaptic vesicles transported to the membrane by cytoskeletal fibers and motor proteins. Much information is nonetheless missing about how the dynamics of the endosomal compartment, the cytoskeleton, and signalling complexes are reciprocally regulated to achieve the function of lymphocytes. Experimental evidence also suggests that the environment surrounding lymphocytes exposed to different antigenic challenge regulates IS assembly and functional output, making an even more complex scenario still far from being completely understood. Also, although some signalling molecular components for lymphocyte activation have been identified and thoroughly studied, the function of other molecules has not been yet uncovered or deeply characterised. This research topic aims to provide the reader with the latest information about the molecular dynamics governing lymphocyte activation. These molecular dynamics dictate cell decisions. Thus, we expect that understanding them will provide new avenues for cell manipulation in therapies to treat different immune-related pathologies.

The Role of Tissue Resident NK Cells During Homeostasis, Primary and Secondary Infection