MitoFLAME: a split-GFP-based sensor for detecting cytosolic mitochondrial DNA release under inflammatory stress

Mitochondrial DNA (mtDNA) release into the cytosol has emerged as an important mechanism linking mitochondrial dysfunction to the activation of innate immune responses during cellular stress. However, currently available methods for detecting cytosolic mtDNA are often limited by destructive sample preparation, endpoint analysis, or insufficient temporal resolution. Therefore, the development of genetically encoded biosensors capable of monitoring mtDNA-associated release events in living cells is of considerable interest. The aim of this thesis was to evaluate the applicability of the split-GFP-based biosensor mitoFLAME for detecting cytosolic mtDNA-associated release under inflammatory stress. The sensor was tested in HeLa and BEAS-2B cells under basal conditions and following exposure to mitochondrial and inflammatory stressors, including carbonyl cyanide m-chlorophenyl hydrazone (CCCP), MITOK overexpression, diesel exhaust particles (DEP), and Poly I:C. Fluorescence microscopy was used to assess split-GFP reconstitution as an indicator of the redistribution of TFAM-associated mitochondrial material. Under basal conditions, no detectable cytosolic GFP fluorescence was observed. In contrast, all stress conditions induced a marked increase in cytosolic fluorescence, indicating split-GFP reconstitution outside mitochondria. Comparable results obtained using both separate plasmids and a bicistronic P2A-linked construct supported the robustness of the mitoFLAME system across different transfection strategies. Overall, these results suggest that mitoFLAME represents a promising genetically encoded tool for studying mitochondrial stress and mtDNA-associated inflammatory signaling in living cells, while complementing existing approaches for investigating mitochondrial dysfunction and innate immune activation.