T-cell calcium dynamics visualized in a ratiometric tdTomato-GCaMP6f transgenic reporter mouse

Abstract

Calcium is an essential cellular messenger that regulates numerous functions in living organisms. Here, we describe development and characterization of ‘Salsa6f’, a fusion of GCaMP6f and tdTomato optimized for cell tracking while monitoring cytosolic Ca ²⁺, and a transgenic Ca ²⁺ reporter mouse with Salsa6f targeted to the Rosa26 locus for Cre-dependent expression in specific cell types. The development and function of T cells was unaffected in Cd4-Salsa6f mice. We describe Ca ²⁺ signals reported by Salsa6f during T cell receptor activation in naive T cells, helper Th17 T cells and regulatory T cells, and Ca ²⁺ signals mediated in T cells by an activator of mechanosensitive Piezo1 channels. Transgenic expression of Salsa6f enables ratiometric imaging of Ca ²⁺ signals in complex tissue environments found in vivo. Two-photon imaging of migrating T cells in the steady-state lymph node revealed both cell-wide and localized sub-cellular Ca ²⁺ transients (‘sparkles’) as cells migrate.

eLife digest

To help protect the body from disease, small immune cells called T lymphocytes move rapidly, searching for signs of infection. These signs are antigens – processed pieces of proteins from invading bacteria and viruses – which are displayed on the surface of so-called antigen-presenting cells. To visit as many different antigen-presenting cells as possible, T cells move quickly from one to the next in an apparently random manner. How T cells are programmed to move in this way is largely unknown.

The entry of calcium ions into cells, through channel proteins, triggers characteristic actions in many cells throughout the body. As such it is possible that the T cells’ movements are related to calcium signals too. However, it was technically challenging to directly measure the amount of calcium in moving cells within the body.

To overcome this issue, Dong, Othy et al. genetically engineered mice to produce a new calcium-sensitive reporter protein in their T cells. The reporter, which was named Salsa6f, consisted of a red fluorescent protein fused to another protein that glows green when it binds to calcium ions. Measuring the ratio of red and green fluorescence gives a measure of the concentration of calcium ions inside the cell. In the absence of calcium signaling, the cells can still be tracked via the red fluorescence of Salsa6f. Importantly, the reporter did not affect the development or activity of the T cells in the mice. In a related study, Dong, Othy et al. then used their transgenic mice to ask whether calcium signals guide moving T cells as they search for antigens.

Future studies could use these transgenic mice to track the calcium ion concentration in numerous cell types. This would enable new approaches to relate the inner workings of cells to their behaviors in many different organ systems throughout the body.