In non-stimulated control samples (-Tg), much weaker, but clearly detectable signals were obtained suggesting preformed STIM1/ORAI1 clusters in non-stimulated Jurkat T cells (Fig. studies. RCAN1 Furthermore, within the first second of T cell receptor (TCR) activation, Ca2+ microdomain figures increase in the subplasmalemmal space, an effect not observed upon genetic deletion of or or upon antagonism of the Ca2+ mobilizing second messenger nicotinic acid adenine dinucleotide phosphate (NAADP). Taken together, while preformed clusters of STIM and ORAI1 allow for local Ca2+ access events in non-stimulated cells, upon TCR activation, NAADP-evoked Ca2+ release via RYR1, in tight interplay with Ca2+ access via ORAI1 and STIM, rapidly increases the quantity of Ca2+ microdomains, thereby initiating spread of Ca2+ signals deeper into the cytoplasm to promote full T cell activation. One Sentence Summary In T cells, initial Ca2+ microdomains are evoked by preformed clusters of ORAI and STIM in a tight interplay with RyR1. Introduction In non-excitable cells such as T cells sustained Ca2+ signaling is usually mediated by store-operated Ca2+ access (SOCE) that consists of two phases: upon T cell receptor (TCR) activation, second messengers nicotinic acid adenine dinucleotide phosphate (NAADP), D-and T cells, less cells displayed spontaneous Ca2+ microdomains and the number of such Ca2+ microdomains was markedly and significantly decreased (Fig 1A, ?,B).B). Interestingly, in T cells number and amplitude of Ca2+ microdomains was not different from wt cells (Fig 1A, ?,D).D). Moreover, blockade of NAADP evoked Ca2+ release by the specific antagonist BZ194 (10) did not alter spontaneous Ca2+ microdomains, indicating that the NAADP/RYR1 signaling axis is not involved in spontaneous Ca2+ microdomains. Open in a separate windows Fig. 1. Spontaneous Ca2+ microdomains in non-stimulated T cells are dependent on expression of ORAI1 and STIM1/2(A) Ca2+ microdomains in a non-stimulated wt T cell (top), T cell (middle) and T cell (bottom). Arrow heads show (-)-DHMEQ Ca2+ microdomains directly at the PM. (B) Characteristics of Ca2+ microdomains in main murine wt (n=69), (n=28), (n=24), (n=39) and (n=46) in a 5s time period. Comparison of the number of signals per cell and frame (data represent mean SEM). Statistically significant differences are marked by asterisks (* p<0.05, ** p<0.01, *** p<0.001, Kruskal-Wallis Test). (C, D) Kinetic analysis of the 5s time period. Statistical analysis was performed with wt against all other genotypes (as (-)-DHMEQ indicated) with Mann-Whitney-U-Test across all time points (**p<0.005, ***p<0.001). Further, subsecond kinetics of spontaneous Ca2+ microdomain formation was analyzed in the subplasmalemmal space. The analyzed layer is usually ~ 423 32 nm (mean SD, n= 206) in depth and comprises an area spanning a 90 angle of the confocal plane analyzed (Fig 1 C, ?,D,D, insets; for analysis of T cells using dartboard segments observe also suppl Fig S1). In this layer, almost no Ca2+ microdomains were present in and deletion on spontaneous subplasmalemmal Ca2+ microdomains was observed (Fig 1A lower panel, ?panel,D).D). Consistently, a marked and statistically significant difference in the mean free cytosolic Ca2+ concentration ([Ca2+]i) was observed between wt and or quiescent T (-)-DHMEQ cells (203 nM, n=68, vs 142nM or 52 nM, n= 45, p<0.001, Kruskal-Wallis test), but not between wt and T cells (283nM, n=67, vs 225nM, n=31, p>0.05, Kruskal-Wallis test). These results again suggest basal Ca2+ access driven microdomains by pre-activated ORAI1 and STIM1/STIM2 clusters of non-stimulated T cells, while RYR1 or NAADP do not appear to be of major importance in this process. As independent approach, spontaneous Ca2+ microdomains were analyzed in Jurkat T cells stably transfected with ORAI1 fused to a genetically (-)-DHMEQ encoded Ca2+ indication for optical imaging (GECO) (Fig 2), that detects Ca2+ access via ORAI1 channels (11). Spontaneous local and oscillatory Ca2+ access events via ORAI1 were observed in the presence of 1 mM extracellular Ca2+ (Fig 2 A, ?,BB upper panels). Chelating free extracellular Ca2+ with EGTA markedly decreased these spontaneous Ca2+ access events (Fig 2 A, ?,B,B, 2nd upper panels). Involvement of ORAI1 was further confirmed by inhibiting Ca2+ access (-)-DHMEQ using the specific CRAC channel inhibitor Synta 66 (Fig 2 A, ?,B,B, middle panels). Rapid chelation of free cytosolic Ca2+ by pre-loading the cells with 5 M BATPA-AM also largely diminished ORAI1-GECO signals, indicating that the freely diffusible BAPTA free acid competes effectively with the GECO construct for free Ca2+ ions entering the cell through ORAI1 (Fig 2 A, ?,B,B, 2nd lower panels). Inhibition of NAADP signaling by BZ194 (10) experienced no effect on spontaneous Ca2+ microdomains (Fig 2 A, ?,B,B, bottom panels). Quantification of spontaneous local Ca2+ entry.