A cell-permeable fluorescent probe reveals temporally diverse PI(4,5)P2 dynamics evoked by distinct GPCR agonists in neurons.

Lipids, key constituents of cell-membranes, are the first responders to cell signals. At the crux of spatiotemporal dynamics of lipid-signaling responses are phosphoinositides. 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 and downstream signal-transmission. In this backdrop, reversible fluorescent probes that can track live PI(4,5)P2 dynamics on a seconds time-scale will afford key insights into lipid-mediated signaling. However, realizing cell-permeable PI(4,5)P2-selective sensors for imaging dynamics remains a challenge due to the presence of structurally similar lipids and low levels of PI(4,5)P2. We report a computationally-designed, rapid-response, reversible, photo-stable, fluorescent sensor that permeates living cells, neurons, and a multicellular organism within few min of direct incubation and distinctly visualizes PI(4,5)P2 pools. We used the sensor to interrogate the role of PI(4,5)P2 in driving the heterogeneity of signaling responses and contrasting behavioral effects that ensue upon binding of distinct ligands to the same G protein-coupled receptor. Specifically, we asked whether probing PI(4,5)P2 dynamics using our novel sensor could uncover the earliest of signaling differences evoked by hallucinogenic non-hallucinogenic ligands at the serotonin2A (5-HT) receptor. Our results reveal that a hallucinogenic ligand at the 5-HT receptor leads to a slower rate of PI(4,5)P2-depletion when compared to a non-hallucinogenic ligand, within the initial seconds of ligand addition, but has a sustained 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.