A Cell-Permeable Fluorescent Probe Reveals Temporally Diverse PI(4,5)P2 Dynamics Evoked by Distinct GPCR Agonists in Neurons

Lipids, key structural constituents of cell membranes, are amongst the first responders to cell signals. ¹ Bystander lipid molecules are also critical components of a signaling response when a chemical stimulus is received at a cell-surface receptor. ^2,3 At the crux of the spatiotemporal dynamics of lipid signaling response are phosphoinositides. ^4,5 Indeed, phosphoinositides like phosphatidylinositol-(4,5)-bisphosphate (PI(4,5)P2), present in the inner-leaflet of eukaryotic cell membranes, form the link between signal reception at a G-protein coupled receptor (GPCR) and downstream signal transmission. ⁶ The role of signaling lipids in driving the heterogeneity of signaling responses that ensue upon the binding of distinct ligands to the same GPCR remains unclear, and is central to understanding divergent physiological outcomes of ligand-binding. Different ligands/agonists upon binding to the same GPCR can invoke contrasting behavioral effects. Key examples are the divergent signaling signatures evoked by hallucinogenic versus non-hallucinogenic agonists at the serotonin2A (5-HT _2A ) receptor. ⁷ We asked whether probing PI(4,5)P2 dynamics could uncover the earliest of differences in signaling that arise upon distinct ligands binding to the 5-HT _2A receptor. The challenge was to track rapid < 1 min PI(4,5)P2 dynamics in living systems. ⁸ Here we report a computationally-designed, rapid-response, reversible, photo-stable, fluorescent probe that permeates living cells, neurons, and a multicellular organism within few min of direct incubation and distinctly visualizes PI(4,5)P2 pools, in an optical imaging setup. This novel probe facilitates rapid tracking of seconds to sub-minute PI(4,5)P2 dynamics that arise from ligand-binding at the serotonergic receptor. Our results reveal that a hallucinogenic ligand at the 5-HT _2A receptor leads to a slower rate of PI(4,5)P2 depletion when compared to a non-hallucinogenic ligand, but has a sustained longer effect. The ability of our designer chemical probe in timing early seconds-minute timescale lipid dynamics in living cells opens avenues for tracking early time-point molecular events in neuronal response to chemical and physical stimuli.