Uncaging of IP ₃ and histamine-induced clustering. (A) HeLa cells were loaded with caged-IP₃ together with Calbryte 590. Ca²⁺ stores were depleted using CPA and the CPA was removed by a 20 min wash. SOCE was triggered by the re-addition of Ca²⁺. After 20 s a UV flash was triggered to induce IP₃ release (arrow). Average example traces are represented with uncaging of IP₃ (red circles, from 97 cells) and without (blue circles, from 29 cells). (B) Single traces illustrating the change in the Ca²⁺ signal during uncaging (arrow, red trace) compared with SOCE only (blue trace) over an expanded time course around the uncaging pulse. (C) Bar chart summarizing the signal amplitude during SOCE and when tunneling is induced by IP₃ uncaging (n = 274/105, P < 0.0001). (D) Pilot experiment showing inhibition of IP₃-induced tunneling by CatAr1 expression. The relative normalized expression levels of CatAr1 are indicated on the x-axis (n = 66/26, P = 0.0007). (E) Airyscan images of HeLa cells expressing STIM1-Ch and CaTAr1 before and after application of histamine (100 µM). (F) Intensities obtained from virtual line scans in the x/y plane (arrows) across CaTAr1 (green) and STIM1 (red) signals before and after the application of histamine. Both signals are colocalized at rest but the CatAr1 segregates away from STIM1 clusters following histamine application. (F) Bar chart summarizing the colocalization intensity between CaTAr1 and STIM1 before (Ctr) and during histamine application (His) (n = 24, paired-t test, P = 0.0009).