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Role of Clustering in Determining Spatial Organization at the Immunological Synapse

  • Author(s): Hartman, Nina Caculitan
  • Advisor(s): Groves, Jay T
  • et al.
Abstract

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.

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