Visualizing Calcium Dynamics in the Regulation of T Lymphocyte Motility and Activation
- Author(s): Dong, Tobias Xiao
- Advisor(s): Cahalan, Michael D
- et al.
Calcium is a ubiquitous cellular messenger that is essential in regulating a wide variety of functions in living organisms. Our understanding of Ca2+ signaling was first made possible by the use of synthetic small-molecule Ca2+ indicators to visualize Ca2+ dynamics within live cells. The subsequent development of genetically encoded Ca2+ indicators opened up new possibilities for in vivo monitoring of Ca2+ signaling, but until recently, the performance of these genetic indicators has always fallen behind those of small-molecule indicators. With the goal of developing new and improved options for Ca2+ imaging studies, we have created a transgenic reporter mouse expressing a novel ratiometric genetically encoded Ca+ indicator, and applied it to the study of T lymphocyte motility and activation.
First, I describe the development of a novel ratiometric genetically encoded Ca2+ indicator, named “Salsa6f”, and demonstrate its use to monitor either cytosolic or near-membrane local Ca2+ signaling. Two-photon imaging is utilized to ensure functionality of Salsa6f in future in vivo studies. Salsa6f and a dominant-negative Orai1 mutant are then used to investigate the mechanism of Orai1-regulated stop-and-go T cell motility. Microfabricated channels are used in an in vitro assay that replicates the confined environmental conditions experienced by T cells in vivo in densely packed lymph nodes. I then describe the development of a transgenic mouse to express Salsa6f in a tissue-specific manner for in vivo studies of Ca2+ signaling in the immune and nervous systems. Lastly, I present two-photon imaging of cellular dynamics within mouse intact lymph node to study unperturbed, endogenous Tregs interacting with conventional T cells and dendritic cells. We demonstrate the crucial involvement of CTLA-4 in determining cellular dynamics among Tregs, conventional T cells, and dendritic cells in vivo. I also lay the groundwork for future in vivo studies utilizing transgenic reporter mice expressing genetically encoded Ca2+ indicators to study the Treg-mediated suppression of Ca2+ signaling in activating conventional T cells.