Distribution of mitochondrial DNA nucleoids inside the linear tubules vs. bulk parts of mitochondrial network as visualized by 4Pi microscopy

Mitochondrial nucleoids are confined sites of mitochondrial DNA existing in complex clusters with the DNA–compacting mitochondrial (mt) transcription factor A (TFAM) and other accessory proteins and gene expression machinery proteins, such as a mt single–stranded–DNA–binding protein (mtSSB). To visualize nucleoid distribution within the mt reticular network, we have employed three–dimensional (3D) double–color 4Pi microscopy. The mt network was visualized in hepatocellular carcinoma HepG2 cells via mt–matrix–addressed GFP, while 3D immunocytochemistry of mtSSB was performed. Optimization of iso -surface computation threshold for nucleoid 4Pi images to 30 led to an average nucleoid diameter of 219 ± 110 and 224 ± 100 nm in glucose– and galactose–cultivated HepG2 cells (the latter with obligatory oxidative phosphorylation). We have positioned mtDNA nucleoids within the mt reticulum network and refined our model for nucleoid redistribution within the fragmented network –– clustering of up to ten nucleoids in 2 μm diameter mitochondrial spheroids of a fragmented mt network, arising from an original 10 μm mt tubule of a 400 nm diameter. However, the theoretically fragmented bulk parts were observed most frequently as being reintegrated into the continuous mt network in 4Pi images. Since the predicted nucleoid counts within the bulk parts corresponded to the model, we conclude that fragmentation/reintegration cycles are not accompanied by mtDNA degradation or that mtDNA degradation is equally balanced by mtDNA replication.