Bilateral regulation of EGFR activity and local PI(4,5)P2 dynamics in mammalian cells observed with superresolution microscopy

Anionic lipid molecules, including phosphatidylinositol-4,5-bisphosphate (PI(4,5)P 2 ), are implicated in the regulation of epidermal growth factor receptor (EGFR). However, the role of the spatiotemporal dynamics of PI(4,5)P 2 in the regulation of EGFR activity in living cells is not fully understood, as it is difficult to visualize the local lipid domains around EGFR. Here, we visualized both EGFR and PI(4,5)P 2 nanodomains in the plasma membrane of HeLa cells using super-resolution single-molecule microscopy. The EGFR and PI(4,5)P 2 nanodomains aggregated before stimulation with epidermal growth factor (EGF) through transient visits of EGFR to the PI(4,5)P 2 nanodomains. The degree of coaggregation decreased after EGF stimulation and depended on phospholipase Cγ, the EGFR effector hydrolyzing PI(4,5)P 2 . Artificial reduction in the PI(4,5)P 2 content of the plasma membrane reduced both the dimerization and autophosphorylation of EGFR after stimulation with EGF. Inhibition of PI(4,5)P 2 hydrolysis after EGF stimulation decreased phosphorylation of EGFR-Thr654. Thus, EGFR kinase activity and the density of PI(4,5)P 2 around EGFR molecules were found to be mutually regulated. Residing on the surface of cells are proteins called receptors, which bind to external molecules. Once activated, receptors undergo various changes that allow them to relay the signal to other components inside the cell that can alter the cell’s behavior. One such protein is the epidermal growth factor receptor (or EGFR for short), which helps regulate cell division and development. When molecules bind to an EGFR, this causes the receptor to attach to another EGFR in the membrane to form a dimer. This dimerization is crucial as it allows the two receptors to add chemicals known as phosphates to each other, which recruit additional proteins that relay the activation signal to downstream targets inside the cell. Studies have shown that a lipid which sits within the cell membrane, called PI(4,5)P 2 , helps stabilize the EGFR dimer and aid its activation. However, it is not fully understood exactly how PI(4,5)P 2 achieves this. To investigate, Abe et al. used a super-resolution microscope that can visualize single molecules to examine how PI(4,5)P 2 lipids are distributed around the receptor. This revealed that EGFR and PI(4,5)P 2 overlap one another to form structures termed ‘nanodomains’ before the receptor is stimulated. Further experiments showed that the nanodomains promote dimeriza