Cristae remodeling causes acidification detected by integrated graphene sensor during mitochondrial outer membrane permeabilization

Abstract

The intrinsic apoptotic pathway and the resultant mitochondrial outer membrane permeabilization (MOMP) via BAK and BAX oligomerization, cytochrome c (cytc) release, and caspase activation are well studied, but their effect on cytosolic pH is poorly understood. Using isolated mitochondria, we show that MOMP results in acidification of the surrounding medium. BAK conformational changes associated with MOMP activate the OMA1 protease to cleave OPA1 resulting in remodeling of the cristae and release of the highly concentrated protons within the cristae invaginations. This was revealed by utilizing a nanomaterial graphene as an optically clear and ultrasensitive pH sensor that can measure ionic changes induced by tethered mitochondria. With this platform, we have found that activation of mitochondrial apoptosis is accompanied by a gradual drop in extra-mitochondrial pH and a decline in membrane potential, both of which can be rescued by adding exogenous cytc. These findings have importance for potential pharmacological manipulation of apoptosis, in the treatment of cancer.

Figure 1. Overview of the experimental workflow.

(a) Mitochondria are isolated on the day of experimentation and loaded on (b) a pre-functionalized device. (c) After a brief incubation, mitochondrial functions can be probed via electrical and fluorescent methods.

Figure 2

(a) Overview of the functionalization scheme (Mitochondria not to scale); (b) Immobilized mitochondria (from HeLa cells) on functionalized graphene surface, false colored from 100 nM MitoTracker^® Green FM signals. A finished graphene-on-glass device; graphene is at the bottom of the chamber indicated by the red arrow; (c) I_ds vs V_g (electrolyte gate) characteristics with different pH KCl buffers; (b). Insignificant fluctuations of graphene conductance following the additions of various substrates.

Figure 3

(a) Illustrations of protons movement after CCCP addition; (b) Time-lapse of TMRE fluorescence, an indicator of the mitochondrial membrane potential, before and after the addition of CCCP; (c) The corresponding change in graphene conductance (measured simultaneously with the fluorescence) and the control; after CCCP, the concentration of protons changes dramatically in the extra-mitochondrial buffer.

Figure 4

(a,b) Addition of 100 μM BIM-BH3 causes graphene conductance and membrane potential to decrease. The fluorescence signal was defined as the average of three mitochondria. Results are representative of three independent experiments. (c,d) A similar experiment with 100 μM BIM-BH3 but with 10 μM cytc added to the buffer. The results show less reduction in both graphene conductance and membrane potential. Results are representative of two independent experiments.

Figure 5. BIM-BH3-induced MOMP in tethered, vital, isolated mitochondria.

The mitochondrion (0.5–1 μm in diameter) is drawn to scale with the antibody (~10 nm). (a) without BIM-BH3, BAK stays inactive; (b) with BIM-BH3, BAK is activated and oligomerize to form pores in the outer membrane, causing cytc release. This is observed to result in buffer acidification and membrane potential decline; (c) With exogenous cytc in the buffer, the mitochondrion maintains inner membrane potential with a reduced change in the buffer pH.

Figure 6. Schematic diagrams of membrane morphology and compartmental pHs for the three experiments performed.

The steady state of the mitochondria is shown at the top with buffer pH (indicated as measured) and the other pHs as inferred from the literature. In the case of CCCP, pH is equilibrated across the three compartments. The bottom shows the qualitative changes in pH vs. time (not to absolute scale) with the bolded line being the measured variable while the other lines are inferred. For the next two cases, the top two figures correspond to our proposal that the cristae remodel and release protons and cytc from the invaginations and the absence of such a mechanism when exogenous cytc is added. The bottom figures indicate again the qualitative assessment of the pHs.