Mitochondrial fission factor senses and governs ferroptosis.

Ferroptosis is a form of regulated cell death driven by iron-dependent lipid peroxidation and is implicated in diverse pathological states1-4. Although mitochondria and other organelles are increasingly being recognized as important modulators of ferroptosis5-7, a unifying mechanism that couples organelle dynamics to ferroptotic execution has remained elusive. Here using quantitative phosphoproteomics, we identified mitochondrial fission factor (MFF) as a key ferroptosis-selective regulator. Mechanistically, the lipid mediator 17-HETE promotes phosphorylation of MFF at Ser155, which triggers the coordinated fragmentation and dysfunction of mitochondria and peroxisomes. This remodelling intensifies interorganelle crosstalk, amplifies oxidative stress and accelerates ferroptotic death. To monitor this phosphorylation event in living cells, we developed MFF-SPARK, a phase-separation-based biosensor, for real-time tracking of ferroptosis through MFF activation. Using MFF-SPARK, we identified PKCβ and DUSP22 as a coordinated kinase-phosphatase pair that governs MFF phosphorylation. We also discovered avermectin B1 as a pharmacological activator of the PKCβ-MFF axis, which can sensitize tumours to ferroptosis in vivo. Together, our findings establish MFF phosphorylation as a central regulatory node in ferroptosis-associated organelle remodelling and provide a conceptual framework and toolbox for monitoring and pharmacologically interrogating ferroptosis.