Superresolution microscopy reveals spatial separation of UCP4 and F0F1-ATP synthase in neuronal mitochondria

Abstract

Because different proteins compete for the proton gradient across the inner mitochondrial membrane, an efficient mechanism is required for allocation of associated chemical potential to the distinct demands, such as ATP production, thermogenesis, regulation of reactive oxygen species (ROS), etc. Here, we used the superresolution technique dSTORM (direct stochastic optical reconstruction microscopy) to visualize several mitochondrial proteins in primary mouse neurons and test the hypothesis that uncoupling protein 4 (UCP4) and F0F1-ATP synthase are spatially separated to eliminate competition for the proton motive force. We found that UCP4, F0F1-ATP synthase, and the mitochondrial marker voltage-dependent anion channel (VDAC) have various expression levels in different mitochondria, supporting the hypothesis of mitochondrial heterogeneity. Our experimental results further revealed that UCP4 is preferentially localized in close vicinity to VDAC, presumably at the inner boundary membrane, whereas F0F1-ATP synthase is more centrally located at the cristae membrane. The data suggest that UCP4 cannot compete for protons because of its spatial separation from both the proton pumps and the ATP synthase. Thus, mitochondrial morphology precludes UCP4 from acting as an uncoupler of oxidative phosphorylation but is consistent with the view that UCP4 may dissipate the excessive proton gradient, which is usually associated with ROS production.

Keywords: direct stochastic optical reconstruction microscopy; mitochondrial membrane proteins; proton diffusion; reactive oxygen species; uncoupling.

Fig. 1.

Dual-color dSTORM images of F₀F₁-ATP synthase, VDAC, and UCP4. Superresolution images are shown pairwise for (A–C) UCP4 (stained with Alexa 488; green) and ATP synthase (stained with Alexa 647; red) and (D–F) UCP4 and VDAC (stained with Alexa 647; red). The different colored boxes represent magnified views. For each protein pair, at least three different images were further analyzed for the number of detected localizations per mitochondrion and shown as scatter plots for UCP/ATP synthase [(B) neuronal processes and (C) neuronal body] and UCP4/VDAC [(E) neuronal processes and (F) neuronal body]. The plots are segmented in three different sectors (I–III) covering angles of 0° to 30°, 30° to 60°, and 60° to 90°, respectively. The colored dots represent clusters of dominating protein: UCP4 (green dots), ATP synthase (red dots in B and C), and VDAC (red dots in E and F). Yellow dots indicate clusters with similar amounts of localizations within the clusters (sector II). Percentages refer to the fractions of detected localizations in the respective sectors, and tot specifies the total amount of mitochondria analyzed.

Fig. 2.

Spatial arrangement of F₀F₁-ATP synthase, COX, UCP4, and VDAC within single mitochondria. Superresolution images of (A) ATP synthase, (B) COX, (C) UCP4, and (D) VDAC stained with Alexa 647 and recorded with dSTORM are shown. For each protein, one example (white boxes in A–D) is shown as a magnified view in Center (green boxes in A–D). The areas in the green boxes were fitted with Gaussian intensity distributions and presented as cross-section plots with the obtained FWHM for each protein. (E) The scheme shows the different neuronal regions (gray) that were analyzed. In the magnified view of a mitochondrion, the colored ellipses represent CM (green), IBM (violet), and OMM (red). (F) Whisker–box plots represent the median and lower and upper quartile of the obtained widths (FWHM) for the different protein distributions; each box contains data of at least 50 mitochondria for neuronal processes (white boxes) and neuronal cell bodies (colored boxes).

Fig. 3.

Scheme of local proton gradients on the IMM at (A) low and (B) high potentials. Although the respiratory chain proteins (yellow) localized at the CM build up the proton gradient, ATP synthase (green), also found at the CM, is its main consumer. UCP4, localized at the IBM, depletes the proton gradient along the membrane only if it becomes excessive. Areas marked in blue indicate local proton concentration. MM, mitochondrial matrix.