Lee 2024 ACS Nano

Lee CH, Wallace DC, Burke PJ (2024) Super-resolution imaging of voltages in the interior of individual, vital mitochondria. ACS Nano 18:1345−56. https://doi.org/10.1021/acsnano.3c02768

» https://doi.org/10.1021/acsnano.3c02768

Lee CH, Wallace DC, Burke PJ (2024) ACS Nano

Abstract: We present super-resolution microscopy of isolated functional mitochondria, enabling real-time studies of structure and function (voltages) in response to pharmacological manipulation. Changes in mitochondrial membrane potential as a function of time and position can be imaged in different metabolic states (not possible in whole cells), created by the addition of substrates and inhibitors of the electron transport chain, enabled by the isolation of vital mitochondria. By careful analysis of structure dyes and voltage dyes (lipophilic cations), we demonstrate that most of the fluorescent signal seen from voltage dyes is due to membrane bound dyes, and develop a model for the membrane potential dependence of the fluorescence contrast for the case of super-resolution imaging, and how it relates to membrane potential. This permits direct analysis of mitochondrial structure and function (voltage) of isolated, individual mitochondria as well as submitochondrial structures in the functional, intact state, a major advance in super-resolution studies of living organelles.

• Bioblast editor: Gnaiger E

Selected quotes and comments

• OXPHOS consists of the electron transport chain (ETC) plus the ATP synthase.

Comment: This definition is too narrow: The ANT and the inorganic phosphate carrier are additional essential components of the OXPHOS system [1].

• The four multisubunit enzyme complexes of the mitochondrial inner membrane ETC (complexes I−IV) oxidize hydrogen derived from carbohydrates and fats with oxygen to generate H2O.

Comment: Another multisubunit enzyme complex, electron transferring flavoprotein dehydrogenase Complex (CETFDH), is involved in fatty acid oxidation [2].

• the electrochemical gradient ΔP consists of a membrane potential (ΔΨ_m), also called voltage, and a pH gradient (Δμ^H+) with the pH gradient typically less significant: ΔP = ΔΨ_m + Δμ^H+.

Comment: ΔP (delta P) indicates a difference. In contrast, a gradient has magnitude and direction, and would be indicated as dP/dz in the direction of z. The units of the voltage difference ΔΨ_m [V] and the chemical potential difference Δμ^H+ [kJ·mol^-1] do not match [3].

• NADH-linked substrates such as pyruvate and glutamate feed electrons through complex I while succinate feeds electrons into complex II. The addition of these substrates results in electron transport, increased ΔP, and oxygen consumption known as state II respiration.

Comment: Chance and Williams (1955) [4] defined 'state 2' as high ADP level and ~0 substrate level. The use of state II is ambiguous [1].

• In this study, we fed electrons into the ETC through complex II using succinate as the substrate to initiate state II respiration.

Comment: Addition of succinate without rotenone leads to accumulation of oxaloacetate in mitochondria without malate-linked anaplerotic capacty. Oxaloacetate is a potent inhibitor of Complex II, which together with high ROS production near air saturation represents a non-physiological state of isolated mitochondria. Rotenone should be added before succinate [3].

• The recognition of the few # of protons in a mitochondrion was also recently pointed out by Silverstein.⁴⁵ [5]

Comment: As reviewed in ref. [3], the low number of free H⁺ in a mitochondrion was a topic addressed in the early literature [6-8]. Because quantities are quantized, thermodynamic terms such as temperature, Gibbs energy, pH, and the protonmotive force pmF can be defined and accounted for only on the basis of large counts in ergodic systems [3].

• One might even speculate even further that these strong fields affect the spin polarization and may even give rise to quantum effects in the microscopic environmental chemistry or even the macroscopic phenotype. Hints of this have already been reported in the literature.⁵¹

References

1. Gnaiger E et al ― MitoEAGLE Task Group (2020) Mitochondrial physiology. Bioenerg Commun 2020.1. https://doi.org/10.26124/bec:2020-0001.v1

2. Gnaiger E (2024) Complex II ambiguities ― FADH₂ in the electron transfer system. J Biol Chem 300:105470. https://doi.org/10.1016/j.jbc.2023.105470

3. Gnaiger E (2020) Mitochondrial pathways and respiratory control. An introduction to OXPHOS analysis. 5th ed. Bioenerg Commun 2020.2. https://doi.org/10.26124/bec:2020-0002

4. Chance B, Williams GR (1955b) Respiratory enzymes in oxidative phosphorylation: III. The steady state. J Biol Chem 217:409-27.

5. Silverstein TP (2021) The proton in biochemistry: impacts on bioenergetics, biophysical chemistry, and bioorganic chemistry. Front Mol Biosci 8:764099.

6. Baum H (1967) Energetics of coupled events involving small compartments. Nature 214:1326–7.

7. Mitchell P (1967) Proton current flow in mitochondrial systems. Nature 214:1327–8.

8. Bal W, Kurowska E, Maret W (2012) The final frontier of pH and the undiscovered country beyond. PLOS ONE 7(9):e45832.

Labels: MiParea: Respiration, mt-Structure;fission;fusion, mt-Membrane 

Preparation: Isolated mitochondria 

Regulation: mt-Membrane potential  Coupling state: LEAK, OXPHOS, ET