Background

The endoplasmic reticulum (ER) plays key roles in essential functions including protein folding and maturation in the secretory pathway, lipid metabolism, hormone synthesis, and detoxification of reactive metabolites (Bock and Kohle, 2009; Chen and Cubillos-Ruiz, 2020; Erdbrugger and Frohlich, 2020; Morishita and Arvan, 2020). Research on the ER has attracted increasing interest in recent years, mainly due to the discovery of the unfolded protein response (UPR) signaling pathway, which is triggered by diverse forms of protein folding stress in the ER. The physical contact sites of the ER with other cell organelles and their involvement in cellular communication networks establish the ER as a multifaceted regulator of cell signaling. The relationship between ER stress and oxidative injury has been extensively investigated; however, the origin of ER stress-induced ROS production remains unclear (Appenzeller-Herzog, 2011), and tools to detect xenobiotics that enhance ROS in the ER are limited.

To understand the mechanisms of oxidative insults, specific tools are required to quantitate and describe ER redox conditions. Genetically encoded sensors to quantitate the oxidative status of the many redox couples present in the ER have proven useful. Changes in the amount of H₂O₂ in the cytoplasm can be monitored using the fluorescent probe, HyPer (Belousov et al., 2006). The HyPer sensor was constructed by inserting a circularly permutated yellow fluorescent protein (YFP) into the regulatory domain of the bacterial H₂O₂-sensing protein, OxyR. Importantly, HyPer was shown to selectively detect H₂O₂ over superoxide, peroxinitrite, nitric oxide, and oxidized glutathione in the cytosol. Upon oxidation of the cysteine corresponding to Cys199 of OxyR, the sensor protein HyPer undergoes a conformational change. HyPer has two excitation peaks at 420 nm and 500 nm, and one emission peak at 516 nm. Upon transition from the reduced to the oxidized state, the peak at 420 nm decreases and the peak at 500 nm increases, thus allowing ratiometric measurement of H₂O₂.

Here, we describe a detailed protocol for the real-time imaging and monitoring of the oxidizing milieu in the ER using the HyPerER sensor, which is targeted specifically to the ER lumen (Enyedi et al., 2010; Malinouski et al., 2011) by the addition of an N-terminal ER-targeting sequence and a C-terminal ER-retrieval signal (KDEL). It must be noted that the ER-targeted HyPerER, unlike cytosolic HyPer, is not specific for H₂O₂ but rather reflects the oxidative milieu within the ER (Mehmeti et al., 2012). The approach can be used in different cellular systems with basic understanding of live cell imaging and fluorescence microscopy (an example is shown in Figure 1), whereby data analysis is dependent on the available software. A limitation of the original HyPer sensors is their sensitivity to pH changes. To overcome this limitation, a very recent study introduced a new generation HyPer probe, HyPer7, which is pH-resistant yet remains ultra-sensitive to changes in H₂O₂ (Pak et al., 2020). Monitoring H₂O₂ specifically in the ER remains challenging, and most of the probes available to date have not been used in this very special cellular compartment.