Uncovering the onset and progression of ferroptosis via live-cell imaging

Ferroptosis is a form of non-apoptotic cell death involving formation of lipid peroxyl radicals with potential therapeutic applications. Intracellularly, sensitivity to ferroptosis is expected to differ based on the distribution of enzymes, metabolites, small molecule regulators and inducers. However, spatial-temporal information, necessary to understand the intricacies of subcellular membranes in ferroptosis, is currently missing. Here we designed organelle-targeting, lipophilic fluorogenic lipid peroxyl radical probes—radical trapping antioxidants (fluorogenic RTAs)—to monitor real-time the onset and progression of lipid peroxidation in ferroptosis. Working with HT-1080 cells and the canonical ferroptosis inducer RSL3, imaging peroxyl radicals, while simultaneously monitoring cell rounding and membrane permeability, indicate that lipid peroxyl radical production is characterized by an initial—organelle and RSL3 concentration dependent—induction or runaway period. This period is followed by a steady generation of lipid peroxyl radicals. The onset of lipid peroxidation occurs faster in the ER region than in the other organelles, yet the steady rate of progression is similar in all subcellular membranes explored. Here the ER- and lysosomes-embedding RTAs, and to a lesser extent the mitochondria, are more effective in protecting from cell rounding/death, compared to the PM-embedding RTA. This indicates that antioxidant protection, while vital at the internal membranes, plays a minor role at the PM. High-resolution single-cell imaging and colocalization studies show for the ER-embedding RTA, lipid hydroperoxide generation and/or accumulation initially in structures adjacent to the Golgi apparatus resembling the ER-Golgi intermediate compartment (ERGIC), followed by their late transport through the endomembrane system to plasma membrane. These results suggest Golgi-associated vesicles are initially acting as reservoirs of newly peroxidized lipids, which subsequently spread to other organelles effectively acting as “free radical embers.” In addition, single-cell imaging with the mitochondria, lysosomes, or PM-targeting RTA revealed that oxidized lipids are trafficked unidirectionally from internal membranes to plasma membrane, making the latter the ultimate sink for oxidized lipids. Lethal internal membrane damage occurs early in ferroptosis, where plasma membrane protection to lipid peroxidation shows little impact in overall cell survival. These results underscore Golgi-associated structures as the key organelle involved in accumulating lipid hydroperoxides early in ferroptosis, providing a putative target to regulate ferroptosis progression. The work further positions fluorogenic RTAs as valuable tools for unravelling the dynamic, subcellular progression of ferroptotic cell death for different cell lines and ferroptosis inhibitors and inducers.