Mitochondria in numerous cell types, especially in cultured cells, form a reticular network undergoing constant fusion and fission. The three dimensional (3D) morphology of these networks however has not been studied in detail to our knowledge. We have investigated insulinoma INS-1E and hepatocellular carcinoma HEP-G2 cells transfected with mitochondria-addressed GFP. Using 4Pi microscopy, 3D morphology changes responding to decreased oxidative phosphorylation and/or energetic status could be observed in these cells at an unprecedented 100 nm level of detail. In INS-1E cells cultivated at 11 mM glucose, the mitoreticulum appears predominantly as one interconnected mitochondrion with a nearly constant 262+/-26 nm tubule diameter. If cultured at 5 mM glucose, INS-1E cells show 311+/-36 nm tubules coexisting with numerous flat cisternae. Similar interconnected 284+/-38 nm and 417+/-110 nm tubules were found in HEP-G2 cells cultivated at 5 mM and hyperglycaemic 25 mM glucose, respectively. With rotenone inhibiting respiration to approximately 10%, disintegration into several reticula and numerous approximately 300 nm spheres or short tubules was observed. De-energization by uncoupling additionally led to formation of rings and bulky cisternae of 1.4+/-0.4 microm diameter. Rotenone and uncoupler acted synergically in INS-1E cells and increased fusion (ongoing with fission) forming bowl-like shapes. In HEP-G2 cells fission partially ceased with FCCP plus rotenone. Thus we have revealed previously undescribed details for shapes upon mitochondrial disintegration and clearly demonstrate that high resolution 3D microscopy is required for visualization of mitochondrial network. We recommend 4Pi microscopy as a new standard.

Department of Membrane Transport Biophysics No 75 Institute of Physiology Academy of Sciences of the Czech Republic Vídenská 1083 14220 Prague 4 Czech Republic

References provided by Crossref.org

Mitochondrial Physiology of Cellular Redox Regulations

Mitochondrial Cristae Morphology Reflecting Metabolism, Superoxide Formation, Redox Homeostasis, and Pathology

Pitfalls of Mitochondrial Redox Signaling Research

Mitochondrial Superoxide Production Decreases on Glucose-Stimulated Insulin Secretion in Pancreatic β Cells Due to Decreasing Mitochondrial Matrix NADH/NAD+ Ratio

Glucose-Induced Expression of DAPIT in Pancreatic β-Cells

Mitochondrial Uncoupling Proteins: Subtle Regulators of Cellular Redox Signaling

Delaunay algorithm and principal component analysis for 3D visualization of mitochondrial DNA nucleoids by Biplane FPALM/dSTORM

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