Radical Stress Is More Cytotoxic in the Nucleus than in Other Organelles

Abstract

Cells are exposed to reactive oxygen species (ROS) as a by-product of mitochondrial metabolism, especially under hypoxia. ROS are also enzymatically generated at the plasma membrane during inflammation. Radicals cause cellular damage leading to cell death, as they react indiscriminately with surrounding lipids, proteins, and nucleotides. However, ROS are also important for many physiological processes, including signaling, pathogen killing and chemotaxis. The sensitivity of cells to ROS therefore likely depends on the subcellular location of ROS production, but how this affects cell viability is poorly understood. As ROS generation consumes oxygen, and hypoxia-mediated signaling upregulates expression of antioxidant transcription factor Nrf2, it is difficult to discern hypoxic from radical stress. In this study, we developed an optogenetic toolbox for organelle-specific generation of ROS using the photosensitizer protein SuperNova which produces superoxide anion upon excitation with 590 nm light. We fused SuperNova to organelle specific localization signals to induce ROS with high precision. Selective ROS production did not affect cell viability in most organelles except for the nucleus. SuperNova is a promising tool to induce locally targeted ROS production, opening up new possibilities to investigate processes and organelles that are affected by localized ROS production.

Keywords: DNA damage; optogenetics; oxidative stress; reactive oxygen species.

Figure 1

Schematic overview of SuperNova fusion proteins. Myc: myc-tag; COX8: mitochondrial protein cytochrome c oxidase subunit 8A; VAMP8: endosomal vesicle associated membrane protein 8; TGON3: trans-Golgi network integral membrane protein 2; Calreticulin: endoplasmic reticulum (ER) protein; KDEL: ER-retention signal; NLS: nuclear localization signal. Molecular weights of the fusion constructs are indicated.

Figure 2

Localization of SuperNova fusion constructs. (A) Representative confocal images of HeLa cells transfected with constructs encoding for SuperNova fusion proteins with appropriate counter stains for the target organelles. DAPI is in blue. Cytosolic: SuperNova-N1. Scale bar: 15 µm. (B) Pearson’s colocalization coefficients of the SuperNova fusion proteins with the organellar markers from panel A. Three technical repeats, each dot represents a microscopy image.

Figure 3

SuperNova fusion construct expression levels and viability upon light exposure. (A) Representative scatter plot of COS-7 cells. Polygon indicates gating to exclude debris from cells. (B) Fluorescent intensity histograms of SuperNova in COS-7 cells expression SuperNova fusion proteins after gating as shown in A. Dotted line indicates gating for SuperNova-positive cell population based on background fluorescence of untransfected cells. At least 20,000 cells were included in each measurement. (C) Percentage of COS-7 cells positive for SuperNova as shown in B, each dot represents a technical repeat. Analyzed with paired one-way ANOVA with Tukey post-hoc test (** p < 0.01; *** p < 0.001). (D) Representative fluorescent intensity histograms of COS-7 cells expressing SuperNova fusion protein (cytosolic) after stimulation with 590 nm excitation light for 1, 4, or 24 h and stained with the Zombie Violet cell viability dye. Dotted line indicates gating for Zombie Violet-positive cell population based on background fluorescence of unstained cells (not shown). (E) Percentage of cell death in COS-7 after stimulation with 590 nm excitation light for 24 h, each dot represents a technical repeat. Control cells were kept in the dark for 24 h. At least 20,000 cells were included in each measurement. Analyzed with unpaired one-way ANOVA with Dunnett post-hoc test comparing all conditions to the untransfected cells and to mCherry-N1 (* p < 0.05; *** p < 0.001). (F) Table showing analysis of data from panel E. Confidence intervals shown are 95% of average difference in cell death (%) between dark and 590 nm light exposure. Analyzed with unpaired one-way ANOVA with Dunnett post-hoc test (ns: not significant; * p < 0.05; ** p < 0.01; *** p < 0.001).