Dissection of Mitochondrial Function via Chemical Perturbation and Single‐Cell Profiling

Mitochondria play central roles in cellular energy metabolism and signal transduction, and maintenance of mitochondrial homeostasis is essential for proper cellular function. Rather than being regulated by individual genes alone, mitochondrial homeostasis is governed by coordinated functional modules, including glucose and lipid metabolism, the tricarboxylic acid (TCA) cycle, oxidative phosphorylation (OXPHOS), calcium handling, mitochondrial dynamics, mitochondrial reactive oxygen species (mtROS) regulation, and mitochondrial transcription and translation. However, how perturbation of these modules reshapes cellular states remains incompletely understood. Here, we combined targeted chemical perturbations with single-cell RNA sequencing (scRNA-seq) to systematically profile transcriptional responses to inhibition of core mitochondrial functional modules. Comparative analyses revealed both shared and module-specific transcriptional programs, including recurrent co-expression patterns across distinct perturbations. Analysis of mitochondrial gene expression across conditions implicated mtROS as an important regulator of mitochondrial respiratory chain (MRC) gene expression, potentially acting through activation of the mitochondrial integrated stress response (mtISR). Further comparative analysis of perturbations targeting individual MRC complexes uncovered distinct transcriptional and cellular consequences among complexes. Examination of cell-cycle dynamics showed that mitochondrial perturbations generally suppress cell proliferation; inhibition of most MRC complexes was associated with G1-phase arrest, whereas perturbation of complex III preferentially led to G2/M-phase arrest, potentially reflecting differential engagement of p53-associated signaling pathways. Finally, our analysis revealed both conserved and divergent transcriptional responses to mitochondrial perturbations between human and mouse cells. Together, these findings establish a systematic single-cell framework for dissecting mitochondrial functional modules and highlight both shared and function-specific principles by which mitochondrial perturbations influence cellular transcriptional states.