The cell biology of the calcium release-activated calcium channel in T cells

Division Of Basic Sciences - Nci

Investigators

Abstract

It has been known for years that TCR engagement leads to elevation of intracellular calcium. The biphasic nature of this process has also been long-studied. The initial peak of intracellular calcium follows TCR induced tyrosine kinase activation, which leads to recruitment of phospholipase-Cgamma to the phosphorylated LAT adapter molecule along with other adapters and enzymes. Activation of this enzyme in the LAT complex results in breakdown of phosphoinositides in the plasma membrane leading to second messenger production (IP3) and release of calcium stores from the endoplasmic reticulum. It has also been known that depletion of these stores is sensed in the cells and that this depletion leads to influx of external calcium via calcium release-activated calcium (CRAC) channels. The molecular components of this channel had been long sought, but recently the calcium detector STIM1 and the channel protein Orai1 were identified and characterized in a number of cell types. Upon depletion of intracellular calcium, the two proteins interact and calcium influx ensues. We were interested in the localization and activity of these proteins in T cells. We obtained fluorescently tagged versions of the proteins and expressed them in T cells. We detected the clustering and colocalization of these two molecules in punctae upon TCR engagement. These structures are analogous to those seen by others in non-lymphoid cells. Surprisingly we also observed the colocalization of these molecules at the opposite pole of the activated T cells in cap-like structures. The formation of the caps depended on TCR engagement, did not involve restructuring of the entire endoplasmic reticulum and was affected by disruption of the cytoskeleton. The caps were also very dynamic and their content was observed to migrate to the sites of interaction of the T cell with antigen presenting cells. The function of these caps is under investigation.