Fig. 1. Optogenetic activation of photoactivated adenylyl cyclase (PAC) and sensing of transient changes in cAMP levels by Pink Flamindo in live Neuro-2a cells. (A) Schematic representation of a cell expressing photoactivated adenylyl cyclase (PAC) and Flamindo for sensing the cAMP level. Photoactivation of PACs increases the cAMP level in the cells, which is reported as an increase in the fluorescence intensity of Flamindo. (B) Neuro-2a cells expressing GFP-P2A-PAC-T2A-Pink Flamindo imaged in the GFP channel indicate the expression of photoactivated adenylyl cyclase (PAC), where P2A and T2A represent self-cleaving sites (ESI†). Pseudocolour images indicate transient changes in the fluorescence of Flamindo imaged at 560 nm upon targeted photoactivation of PAC with 405 nm. Fluorescence intensity in the photoactivated region decayed rapidly with time. Scale bar: 10 μm. (C) Comparison of intensity fold change of Flamindo upon photoactivation of PACs with 405 nm (n = 10, mean ± s.e.m from 2 biological replicates, ***p < 0.001, Mann–Whitney test). Average intensity of GFP::PAC in the corresponding cells is depicted in Fig. S1C, ESI.† (D) Curves representing the temporal changes in fluorescence intensity upon photoactivation of the PACs (bPAC, TpPAC, NgPAC1, and NgPAC3) with 405 nm. Fold increase in fluorescence intensity is normalized to 1. (E) Table illustrating the biophysical properties of fluorescence decay computed from (D). A1 and A2 represent the fractional components corresponding to lifetimes τ1 and τ2, respectively. All PACs-Flamindo (bPAC, TpPAC, NgPAC1 and NgPAC3) exhibited an increase in average fold intensity upon targeted photoactivation, implying a surge in cAMP level in a localized illuminated region, which decayed exponentially with varying kinetics. The controls mApple and Flamindo did not show a similar increase upon photoactivation. Consistently, the decay kinetics of PACs-Flamindo were significantly slower compared to control Flamindo, whereas mApple did not display any difference in its kinetics.
