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. Author manuscript; available in PMC: 2019 Sep 5.
Published in final edited form as: Sci Signal. 2018 Dec 18;11(561):eaat0358. doi: 10.1126/scisignal.aat0358

Fig. 5. RYR1, but not RYR3 or TPC1/2, as critical Ca2+ release channel for initial Ca2+ microdomains upon TCR stimulation.

Fig. 5.

(A) Characteristics of initial Ca2+ microdomains in primary wt (n=68) and Ryr1−/− (n=31) murine T cells in the first 15 s following bead contact. (B) Subcellular layers at the PM (as indicated) were analyzed in the first second before and post bead contact. Statistical analysis was performed with wt against RyR1−/−, Mann-Whitney-U-Test across all time points after activation (*p<0.05, **p<0.005, ***p<0.001). (C) Characteristics of initial Ca2+ microdomains in primary wt incubated over night with DMSO (n=13) and wt cells incubated over night with 500 μM BZ194 (n=34), in the first 15 s following bead contact. (C) Subcellular layers at the PM (as indicated) were analyzed in the first second before and after bead contact. (E, F) Characteristics of initial Ca2+ microdomains in (E): primary wt (n=30) and Tpc1-/−2−/− (n=33), and in (F): primary wt (n=15) and Ryr3−/− (n=19) in the first 15 s after bead contact. Comparison of the percentage of responding cells, the number of signals/cell/frame and the Ca2+ amplitude (data represent mean ± SEM). Statistically significant differences are marked by asterisks (* p<0.05, Mann-Whitney U). (G) Co-localization of RYR (green) and ORAI1 (red) in a representative primary wt T cell, showing single optical channels, merge of both optical channels and co-localization (white). Length scale bar corresponds to 2 μm. (H) Magnification of a high (1) and a low (2) colocalization area of the ROIs depicted in (G). Length scale bar is 100 nm. (I) RyR-ORAI1 plot profile of the arrow shown in H1. (J) Statistical co-localization analysis of RyR-ORAI1 (n=26). Data represent mean ± SEM. Statistically significant differences are marked by asterisks (****p<0.0001, unpaired t test).