T cell receptors (TCRs) on developing thymocytes are screened for appropriate recognition of MHC molecules and self-reactivity before differentiating into CD4, CD8, or other non-conventional T cell subsets prior to exiting the thymus. However, how thymocyte interactions and T cell receptor signaling relate to developmental outcomes during these selection processes is unclear. Dynamic re-localization of proteins within cells can provide information about cell polarity and cell signaling. Here we use fluorescent fusion proteins to identify the subcellular localization of cell polarity proteins (PKCζ) and markers of TCR signaling (LAT) in conjunction with time-lapse two-photon microscopy of thymic lobes. In our current study, we have generated chimeras with bone marrow engineered to express fluorescent fusion proteins, and observe the re-localization of these proteins during thymocyte migration and intercellular interactions. We present a semi-automated approach to quantify fluorescent fusion protein distribution in cells of thymus tissue and show asymmetric distribution of the PKCζ-GFP fusion protein during migration as well as differential accumulation of the LAT-GFP fusion protein during developmental checkpoints. These observations may lead us to an understanding of how the quality of TCR interactions in the thymus affects developmental outcome.
- Copyright © 2011 by The American Association of Immunologists, Inc.