Deciphering Spatiotemporal Control of Ferroptosis in HCC: A Bifunctional Probe for Lipid Droplet Viscosity Dynamics and Interorganelle Communication

Although lipid droplets (LDs) are established as metabolic regulators of iron-dependent ferroptosis in hepatocellular carcinoma (HCC), their biophysical remodeling and spatiotemporal interactions with other organelles during this cell death process have been incompletely characterized. In this study, we developed ZYB, a bifunctional molecular probe that combines lipophilicity-guided organelle targeting with viscosity-dependent fluorescence activation while exhibiting ideal spectral orthogonality to commercial fluorescent markers. Using ZYB, we detected elevated LD viscosity during erastin-induced ferroptosis and reduced viscosity upon ferrostatin-1-mediated inhibition. Time-lapse imaging of ZYB-labeled LDs revealed significant alterations in diameter, number, morphology, and spatial dynamics as ferroptosis progressed. Capitalizing on ZYB’s spectral properties (Ex/Em ∼350/500 nm) alongside organelle-specific dyes (lysosome red tracker: ∼577/590 nm; mitochondria red tracker: ∼579/599 nm), we uncovered a highly dynamic interorganelle communication network involving sequential contact and separation events between LDs and mitochondria, LDs and lysosomes, and neighboring LDs. Beyond providing a powerful LD-imaging tool, this work deciphers the role of organelle communication networks in ferroptosis, offering a mechanistic foundation for targeting HCC vulnerabilities.