Mitochondrial oxidative phosphorylation and energetic status are reflected by morphology of mitochondrial network in INS-1E and HEP-G2 cells viewed by 4Pi microscopy

Abstract

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.

The mitochondrial dynamics in cancer and immune-surveillance

“…As previously stated, this switch frequently occurs along with fragmentation of the mitochondrial network [22,99]. For example, a correlation between mitochondrial fission and glycolysis is observed in Survivin-overexpressing neuroblastoma cells [100] and 3-BrPA-treated urinary bladder cancer cells [101].…”

Mitochondrial Dynamics in Diabetes

“…Recently, 4Pi microscopy, which has higher axial resolution, was employed to examine the mitochondrial morphology of pancreatic b-cells. Image analyses revealed that mitochondria of rat islet b-cells form highly interconnected reticulum similar to those observed in the insulinoma b-cell line (49,134). In contrast, disintegration of tubular mitochondrial network was observed in islet b-cells from diabetic Goto Kakizaki rats (49).…”

Effects of Chlorpyrifos and Chlorpyrifos-Oxon on the Dynamics and Movement of Mitochondria in Rat Cortical Neurons

“…In contrast, impaired mitochondrial fusion can result in the loss of ⌬⌿m and a reduction in oxidative phosphorylation (OXPHOS) (Olichon et al, 2003;Chen et al, 2005). It has been suggested that ATP production via OXPHOS results in the production of highly networked mitochondria, whereas glycolysis-dependent ATP production leads to the generation of more spherical mitochondria, although this has yet to be established in neuronal cells (Plecitá -Hlavatá et al, 2008). In the experiments reported here, we did not differentiate between OXPHOS-and glycolysis-related ATP production; however, our results would support the hypotheses that the concentrations of OPs evaluated did not compromise ⌬⌿m or OXPHOS-dependent ATP production or that they drove mitochondrial fusion to maintain OXPHOS-dependent ATP and preserve mitochondrial function.…”