Spatiotemporally Traceable Peptide Self-Assemblies for Mitochondrial-Specific Dysfunction

Peptide self-assembly has emerged as a highly regarded strategy for developing functional agents to control cellular fate. However, due to the complexity of intracellular environments, achieving spatiotemporally traceable organelle localization and functional perturbation through peptide self-assembly remains a challenge. In this study, we developed T-FFVLK, a fluorescent peptide probe that integrates a red-emitting mitochondria-targeted molecule with a self-assembling peptide for visual tracking. This probe sequentially enters cellular compartments, ultimately targeting mitochondria to form disruptive self-assembled structures while reporting mitochondrial dysfunction. Once endocytosed into the cell, T-FFVLK initially forms short fibers that promote lysosomal escape, subsequently entering the mitochondria, where it further forms long fibers, all accompanied by a gradual increase in fluorescence and self-reporting of probe localization and mitochondrial dysfunction. T-FFVLK achieves mitochondrial enrichment within 6 h and induces morphological and functional changes within 12 h, leading to microtubule damage and cell cycle arrest. Thus, T-FFVLK is an invaluable tool for the observation of mitochondrial dysfunction in living cells, offering great potential for subcellular imaging and disease treatment.