The N-terminal homology (ENTH) domain of Epsin 1 is a sensitive reporter of physiological PI(4,5)P2 dynamics

Abstract

Phospholipase Cβ (PLCβ)-induced depletion of phosphatidylinositol-(4,5)-bisphosphate (PI(4,5)P₂) transduces a plethora of signals into cellular responses. Importance and diversity of PI(4,5)P₂-dependent processes led to strong need for biosensors of physiological PI(4,5)P₂ dynamics applicable in live-cell experiments. Membrane PI(4,5)P₂ can be monitored with fluorescently-labelled phosphoinositide (PI) binding domains that associate to the membrane depending on PI(4,5)P₂ levels. The pleckstrin homology domain of PLCδ1 (PLCδ1-PH) and the C-terminus of tubby protein (tubby_CT) are two such sensors widely used to study PI(4,5)P₂ signaling. However, certain limitations apply to both: PLCδ1-PH binds cytoplasmic inositol-1,4,5-trisphosphate (IP₃) produced from PI(4,5)P₂ through PLCβ, and tubby_CT responses do not faithfully report on PLCβ-dependent PI(4,5)P₂ 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)P₂ dynamics in living cells. We utilized recombinant tools to manipulate PI or G_q protein-coupled receptors (G_qPCR) 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)P₂ without interacting with IP₃, as demonstrated by dialysis of cells with the messenger through a patch pipette. Utilizing Ci-VSP to titrate PI(4,5)P₂ levels, we found that ENTH-GFP had low PI(4,5)P₂ affinity. Accordingly, ENTH-GFP was highly sensitive to PLCβ-dependent PI(4,5)P₂ depletion, and in contrast to PLCδ1-PH, overexpression of ENTH-GFP did not attenuate G_qPCR signaling. Taken together, ENTH-GFP detects minute changes of PI(4,5)P₂ levels and provides an important complementation of experimentally useful reporters of PI(4,5)P₂ dynamics in physiological pathways.

Keywords: Ci-VSP; Epsin N-terminal homology; G(q) protein-coupled receptor; PLCδ1-PH; Phosphatidylinositol-(4,5)-bisphosphate; Phospholipase C; Phospholipids; Pleckstrin homology; Signal transduction; Tubby protein.