A single fluorescent probe reveals changes in endoplasmic reticulum–mitochondria contact in hepatocytes during ferroptosis

Ferroptosis is a recently proposed cell death mechanism characterized by iron-dependent lipid peroxidation. Reactive oxygen/nitrogen species (ROS/RNS) are responsible for lipid peroxidation in the endoplasmic reticulum (ER) and mitochondria (Mito) and result in glutathione (GSH) depletion in ferroptosis. Peroxynitrite (ONOO–) is a highly reactive RNS, and its equilibrium with GSH is vital to maintain redox homeostasis in ER and Mito. The GSH-ONOO– crosstalk is thus essential to understand the changes of ER and Mito in cells during ferroptosis. Further, the interplay between ER and Mito, the ER–Mito contact, is expected to change during ferroptosis, which has been observed in other cell death forms but not in ferroptosis. Here, we report a new fluorescent probe (DPY) with a dual response to GSH and ONOO– in both the ER and Mito. Fluorescence of DPY can be triggered by GSH and ONOO– at 608 (red) and 456 nm (blue) excited at 561 and 405 nm, respectively. The structure of DPY contains both the ER- and Mito-targeting units, and simultaneous anchoring of the probe is allowed in duple organelles. Application of the probe in cell imaging firstly reveals GSH/ONOO– crosstalk in ER and Mito in erastin- and RSL3-induced cells during ferroptosis. And a closer ER–Mito contact is also observed in hepatic cells during ferroptosis. Further studies on the blue-red merged images disclose the ER-Mito contact change in cells during ferroptosis through the observed difference in Pearson correlation coefficients. A similar trend is also verified in acetaminophen-induced ferroptosis.