The need to spatially organize molecules to regulate reaction conditions is thematic in biology, particularly in cell-cell communication. One example of such a biological system is the immune synapse (IS), the specialized junction formed between a T cell and an antigen-presenting cell (APC) in the lymphatic system. The physical chemistry occurring at the IS allows for recognition of and sensitive discrimination between “self” and “foreign” antigens, imparting immunity to an entire organism when it works properly, and resulting in autoimmunity (i.e., lupus, arthritis, diabetes, MS) or immunodeficiency (i.e., AIDS) when it does not. Proteins of various functions must assemble into highly-organized supramolecular activation clusters (SMAC) to initiate the cellular program that leads to an immune response. Recent work has illustrated the crucial role of the cytoskeleton in achieving this organization, and it is my goal to investigate how cytoskeletal control over molecular organization on the cell surface regulates signal transduction pathways. In particular, I wish to examine the forces generated by specific scaffold and motor proteins on distinct classes of molecules at the IS.

The IS is an organized assembly of proteins involved in adhesion, antigen presentation and recognition, as well as costimulation and signaling. Upon contact with an APC, leukocyte function-associated antigen 1 (LFA-1, on the T cell), and intercellular adhesion molecule 1 (ICAM-1, APC) chiefly mediate initial adhesion. Small clusters of T cell receptor (TCR) and its antigen-presenting ligand, the major histocompatibility complex (pMHC), form in the periphery of the nascent junction (Figure 1A). In thymocytes, this arrangement persists, leading to selection against self-reactive T cells, whereas in helper and cytotoxic T cells, the TCR-pMHC cluster coalesce within 10 minutes, forming a “mature IS” (Figure 1B), and inducing signaling that leads T cell activation and cytolytic killing, respectively.

This reorganization is partially influenced by the disparity in size between the two receptor-ligand pairs (LFA-1-ICAM-1 ~ 42 nm; TCR-pMHC ~ 15 nm); the mature IS, in which membrane bending is minimized, is the energetically favored configuration. However, cytoskeletal disruptions of many types have been shown to prevent IS maturation, as well as any subsequent immune function. Furthermore, by replacing passive APCs with nanopatterned, glass-supported lipid bilayers (SLBs) containing lipid-linked versions of the cognate ligands, the Groves Lab has observed a dominant, centrally-biased force acting on the receptors (Figure 1C). SLBs have the dual benefit of improving imaging of the IS by firmly defining the junction plane, and of allowing discriminate control over the composition and structure of the surface that is presented to the T cell. In our experiments, chromium patterns, deposited onto the glass support by electron beam lithography, act as barriers to lateral diffusion of lipids, lipid-linked proteins, and in turn, associated receptor proteins on the T cell surface, allowing us to dissect the synapse as it matures, and directly visualize the reorganization-driving forces in a way never before attempted.


Imminent Future Work

I plan to take our observations to the cell interior, observing the effects of this physical perturbation, both by direct fluorescence imaging of the cytoskeletal structure beneath the junction in this “frustrated” state, and by observation of changes in surface receptor patterns in the presence of cytoskeleton-inhibiting drugs. At present, I have had success with a model system (HEK293 cells), staining them with fluorescently tagged phalloidin (actin-specific probe), as well as observing morphological changes upon drugging them with cytochalasin D (an actin polymerization inhibitor). This confirms that upon integration with the T cell system, the desired observations should be obtainable. Further work with inhibitors of myosin and kinesin (motor proteins) is planned. Results of this study will contribute to our understanding of the forces at work in biological systems, and the manner in which spatial control leads to the important chemistry at cell-cell junctions.





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