Fig. 1. eOS1, a calcium actuator with enhanced sensitivity.

a–d B3Z T cell hybridomas were transduced with the original OptoSTIM1 (OS1) actuator, a mutated version unable to aggregate (OS1-mut) or a mutated version with enhanced Cry2 aggregation potential (eOS1) and loaded with the Indo-1 calcium indicator. Cells were subjected to time-resolved flow cytometry and illuminated with an external LED (470 nm) during the acquisition at the indicated time points. Calcium responses shown as dot plots (left) and averaged kinetic curves (right) of bulk B3Z cells a or individual clones b transduced with the indicated actuators. PA indicates the timing of photoactivation during the experiment. Representative of two to four experiments. c eOS1 light-triggered calcium responses typically last 10 min. Time-resolved flow cytometry was performed for up to 20 min to assess the duration of calcium responses. Representative of two independent experiments. d Both eGFP-eOS1 and mScarlet-eOS1 actuators trigger robust calcium elevation upon light exposure. Representative of four independent experiments. e eOS1 actuators elicit enhanced NFAT activity in B3Z clones after photoactivation as measured by the production of β-galactosidase 3 h post photoactivation. Photoactivation was performed by LED illumination (12 photoactivations of 5 s every 5 min). The β-galactosidase activity observed in the absence of photoactivation was set to 1; NT, non-transduced B3Z. Results are compiled from four independent experiments (each dot represents one experiment, bars show mean ±SEM). Clones were compared using a two-tailed unpaired Student’s t-test (ns p = 0.8263, **p < 0.01, *p < 0.05). Source data are provided as a Source Data File.