Fig. 4: Probing STIM1-mediated CRAC channel activation with PhoBIT2.

a Schematic demonstration of PhoBIT2 coupled with STIM1ct to photo-activate Ca²⁺ influx through endogenous ORAI channels. STIM1ct, stromal interaction molecule 1 cytoplasmic domain; CRY2, the photolyase homology domain of Arabidopsis cryptochrome 2 (residues 1–498). Created in BioRender. Lee, Y. (2025) https://BioRender.com/qnjigjh. b Domain architecture of human STIM1ct with ssrA2 insertion sites indicated by red triangles. CC1 coiled-coil domain 1, SOAR STIM-Orai activating region, P/S proline/serine-rich region, TRIP the S/TxIP microtubule-binding motif, PB polybasic tail. c Quantification of light-induced changes of intracellular Ca²⁺ levels (indicated by NEMOf) in HeLa cells co-expressing the indicated ssrA2-inserted STIM1ct variants and mCh-CRY2-sspB2. ssrA2-STIMct (site 685) showed the most notable changes and was used in experiments shown in (d–g). Ctrl: HeLa cells expressing only the calcium indicator NEMOf. WT: STIM1ct (233–685) without ssrA2 insertion. CRY2-STIM1ct: mCherry-CRY2-STIM1ct (233–685) construct previously reported by us. Data are shown as mean ± SD. n = 55, 59 or 60 cells from three independent biological replicates. Statistical significance was determined with two-way ANOVA followed by post-hoc Turkey’s test. d Confocal images of light-dependent Ca²⁺ influx at the indicated time points. HeLa cells were co-transfected with ssrA2-STIMct (site 685), mCh-CRY2-sspB2, and the NEMOf Ca²⁺ indicator. Scale bar, 10 µm. e Quantification of reversible light-induced cytosolic NEMOf signal changes. HeLa cells were subjected to three repeated dark-light cycles. n = 11 cells (mean ± s.e.m). f Confocal images showing light-triggered Ca²⁺-dependent subcellular localization of NFAT_1–460-GFP at the indicated time points. The nuclear shape is marked by red dashed lines. Scale bar, 10 µm. g Quantification of Ca²⁺/NFAT-dependent luciferase reporter gene expression in HEK293 cells expressing mCh-CRY2-sspB2 and STIM1ct without ssrA2 insertion as control or ssrA2 inserted at sites 463 and 685, in the absence (black) or presence (blue) of photostimulation. n = 3 independent biological replicates. *** P = 0.000420 (mean ± s.e.m, Two-sided unpaired Student’s t-test). (Source Data).