Calcium regulation of CRACR2a subcellular localization in T cells. (A) Changes in CRACR2a localization before and after ionomycin treatment, as well as the method used for quantification of the distribution of CRACR2a adjacent to the MTOC. The same cell before and after ionomycin treatment is shown. MTOC is visualized by staining with 100 nM SiR-Tubulin for 4 h and adjusting the intensity scale of the image to show only high-intensity pixels (see Fig. S3 A). MTOC enrichment ratio is defined as the ratio of mean GFP intensity within a 0.5-µm region of the MTOC versus the mean GFP intensity within a 0.5-µm to ∼2.5-µm region of the MTOC. Scale bar: 5 µm. Inset scale bar: 1 µm. (B) Mutational study of CRACR2a clustering at the MTOC. EFmut, J.CRACR2a cell line expressing GFP-CRACR2a with mutated calcium-binding site in EF-hands (D63A, E65A, D97A, D99A). ΔTail, wild-type Jurkat line expressing GFP-CRACR2a with a deletion of C-terminal tail (Δ725–731). Scale bar: 10 µm. (C) Quantifications of relative enrichment of CRACR2a at the MTOC for experiments shown in B. Data are mean ± SEM of three independent experiments (n = 11–30 cells per group in each experiment). ****, P < 0.0001, two-way ANOVA with Sidak’s multiple comparisons test. (D) T cell activation induces clustering of CRACR2a compartments at the MTOC. Scale bar: 10 µm. (E) Quantifications of the levels of relative enrichment at MTOC for experiments shown in D. Data are mean ± SEM of three independent experiments (n = 10–30 cells per group in each experiment). **, P < 0.01, one-way ANOVA with Dunnett’s test comparing each sample with sample #1.