The N-terminal homology (ENTH) domain of Epsin 1 is a sensitive reporter of physiological PI(4,5)P dynamics.

Phospholipase Cβ (PLCβ)-induced depletion of phosphatidylinositol-(4,5)-bisphosphate (PI(4,5)P) transduces a plethora of signals into cellular responses. Importance and diversity of PI(4,5)P-dependent processes led to strong need for biosensors of physiological PI(4,5)P dynamics applicable in live-cell experiments. Membrane PI(4,5)P can be monitored with fluorescently-labelled phosphoinositide (PI) binding domains that associate to the membrane depending on PI(4,5)P levels. The pleckstrin homology domain of PLCδ1 (PLCδ1-PH) and the C-terminus of tubby protein (tubby) are two such sensors widely used to study PI(4,5)P signaling. However, certain limitations apply to both: PLCδ1-PH binds cytoplasmic inositol-1,4,5-trisphosphate (IP) produced from PI(4,5)P through PLCβ, and tubby responses do not faithfully report on PLCβ-dependent PI(4,5)P dynamics. In searching for an improved biosensor, we fused N-terminal homology domain of Epsin1 (ENTH) to GFP and examined use of this construct as genetically-encoded biosensor for PI(4,5)P dynamics in living cells. We utilized recombinant tools to manipulate PI or G protein-coupled receptors (GPCR) to stimulate PLCβ signaling and characterized PI binding properties of ENTH-GFP with total internal reflection (TIRF) and confocal microscopy. ENTH-GFP specifically recognized membrane PI(4,5)P without interacting with IP, as demonstrated by dialysis of cells with the messenger through a patch pipette. Utilizing Ci-VSP to titrate PI(4,5)P levels, we found that ENTH-GFP had low PI(4,5)P affinity. Accordingly, ENTH-GFP was highly sensitive to PLCβ-dependent PI(4,5)P depletion, and in contrast to PLCδ1-PH, overexpression of ENTH-GFP did not attenuate GPCR signaling. Taken together, ENTH-GFP detects minute changes of PI(4,5)P levels and provides an important complementation of experimentally useful reporters of PI(4,5)P dynamics in physiological pathways.