Cytosolic calcium oscillations and gene expression in lymphocytes

Stimulation through phosphoinositide-linked receptors leads to a cytoplasmic calcium ( (Ca{dollar}sp{lcub}2+{rcub}rbracksb{lcub}rm i{rcub}){dollar} rise that is required for many cellular responses including survival, growth and differentiation. Single cell calcium imaging has revealed a kinetic complexity to the (Ca{dollar}sp{lcub}2+{rcub}rbracksb{lcub}rm i{rcub}{dollar} signal that was previously unsuspected. A variety of different (Ca{dollar}sp{lcub}2+{rcub}rbracksb{lcub}rm i{rcub}{dollar} response patterns have been observed at the single cell level including spikes, plateaus and oscillations. In lymphocytes, stimulation through the antigen receptor triggers pronounced (Ca{dollar}sp{lcub}2+{rcub}rbracksb{lcub}rm i{rcub}{dollar} oscillations. The aim of this work was to investigate both the mechanism and the function of (Ca{dollar}sp{lcub}2+{rcub}rbracksb{lcub}rm i{rcub}{dollar} oscillations in these cells. This dissertation is divided into four chapters. The introductory chapter provides an overview of the literature focusing primarily on the mechanisms and the physiological consequences of oscillations in non-excitable cells. The second chapter, describes the results of a study of the mechanism of (Ca{dollar}sp{lcub}2+{rcub}rbracksb{lcub}rm i{rcub}{dollar} oscillations in T lymphocytes using digital calcium imaging, pharmacology and rapid perfusion techniques. Based on this study, I propose that oscillations in these cells are generated by the feedback between calcium release activated Ca{dollar}sp{lcub}2+{rcub}{dollar} (CRAC) channels and the internal Ca{dollar}sp{lcub}2+{rcub}{dollar} stores. The third chapter, is a study of the effects of (Ca{dollar}sp{lcub}2+{rcub}rbracksb{lcub}rm i{rcub}{dollar} spikes and plateaus, on the activation of proinflammatory transcriptional pathways in B lymphocytes. I report that a spike is sufficient to activate NF{dollar}kappa{dollar}B and JNK, but not NFAT and that the pathways have distinct Ca{dollar}sp{lcub}2+{rcub}{dollar} sensitivities. These results reveal a mechanism by which (Ca{dollar}sp{lcub}2+{rcub}rbracksb{lcub}rm i{rcub}{dollar} can achieve specificity in signaling to the nucleus. The fourth and final chapter extends these results to oscillations in T cells by using a novel calcium clamp technique to generate oscillations of a defined frequency and amplitude in a population of cells. I provide the first experimental demonstration that oscillations can selectively activate distinct transcriptional pathways, and that they can increase the efficiency of signaling at low levels of stimulation. Thus in this dissertation I provide evidence for a novel mechanism underlying (Ca{dollar}sp{lcub}2+{rcub}rbracksb{lcub}rm i{rcub}{dollar} oscillations and for the selective decoding of (Ca{dollar}sp{lcub}2+{rcub}rbracksb{lcub}rm i{rcub}{dollar} patterns by transcriptional pathways. The importance of these findings may extend beyond lymphocytes to many other cell types.