Demonstrating Fluorescent Peptidyl Inhibitor Targeting of Mitochondrial ClpXP by Fluorescence Microscopy in Mammalian...

Mitochondrial ATP-dependent proteases are essential for maintaining protein homeostasis through degradation of damaged or misfolded proteins. Among these, the ClpXP protease complex locates in mitochondrial matrix and contributes to mitochondrial quality control under physiological and stress conditions. This work demonstrates a quantitative fluorescence microscopy workflow to assess mitochondrial targeting of the fluorescent peptidyl inhibitor FAM-FAPAL-CMK and evaluate mitochondrial morphological changes associated with ClpXP inhibition in mammalian cells. HeLa cells were treated with FAM-FAPAL-CMK and analyzed using confocal microscopy combined with immunofluorescence staining of mitochondrial markers and quantitative image analysis. Colocalization analysis using Costes thresholding and Manders' overlap coefficients demonstrated mitochondrial enrichments of the inhibitor signal. As a consequence, inhibition of ClpP altered mitochondrial morphology. Immunoblot analysis showed no significant change in ClpP protein abundance upon inhibitor treatment. Taken together, this work describes a reproducible imaging-based workflow that will enable interrogation of mitochondrial ClpXP functions in intact cells in response to perturbations of homeostasis, such as oxidative stress.