pH-Regulated Nuclear F-Actin Assembly during Ferroptosis

Menghao Qiao¹Zhipeng Yan¹Jiewei Huang¹Yu Fang¹Kun Xu¹Lingbo Cao¹Haiying Mai¹Na Li¹Yanmei Li^1*Yunmiao Guo^2*Junqi Huang^1*

• ¹Jinan University, Guangzhou, China
• ²Central People's Hospital of Zhanjiang, Zhanjiang, China

Ferroptosis is an iron-dependent form of regulated cell death driven by lipid peroxidation and loss of membrane integrity, frequently modeled with small molecules such as RSL3 that inhibit Glutathione Peroxidase 4 (GPX4). Filamentous actin (F-actin) exists within the nucleus, modulating transcription, nuclear mechanics, and chromatin organization, yet its behavior during ferroptosis remain unexplored. Here, we show that nuclear F-actin assembles in HT-1080 cells undergoing RSL3-induced ferroptosis, visualized by phalloidin, SiR-actin, anti-actin staining, and live 3D/time-lapse imaging of a nuclear actin chromobody (nAC-TagGFP2). Mechanistically, nuclear G-actin increased during ferroptosis, and Importin-9 (IPO9) knockdown markedly reduced nuclear F-actin, indicating an import-dependent mechanism. Concurrently, cytoplasmic F-actin underwent substantial remodeling. Overexpression of a polymerization-defective cytoplasmic β-actin mutant (R62D) slightly delayed ferroptosis, whereas nuclear-targeted mutants had no effect, suggesting nuclear F-actin is a concomitant, not causative, feature. Notably, extracellular NaHCO3 or NaOH suppressed nuclear F-actin formation, while a pH-sensitive reporter revealed progressive intracellular acidification during ferroptosis, favoring nuclear F-actin assembly. These findings reveal nuclear F-actin assembly driven by cytoplasmic actin remodeling, nuclear import, and intracellular acidification, uncovering a previously unrecognized feature of ferroptotic cell death.