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F1FO ATP synthase, also known as complex V, is a key enzyme of mitochondrial energy metabolism that can synthesize and hydrolyze ATP. It is not known whether the ATP synthase and ATPase function are correlated with a different spatio-temporal organisation of the enzyme. In order to analyze this, we tracked and localized single ATP synthase molecules in situ using live cell microscopy. Under normal conditions, complex V was mainly restricted to cristae indicated by orthogonal trajectories along the cristae membranes. In addition confined trajectories that are quasi immobile exist. By inhibiting glycolysis with 2-DG, the activity and mobility of complex V was altered. The distinct cristae-related orthogonal trajectories of complex V were obliterated. Moreover, a mobile subpopulation of complex V was found in the inner boundary membrane. The observed changes in the ratio of dimeric/monomeric complex V, respectively less mobile/more mobile complex V and its activity changes were reversible. In IF1-KO cells, in which ATP hydrolysis is not inhibited by IF1, complex V was more mobile, while inhibition of ATP hydrolysis by BMS-199264 reduced the mobility of complex V. Taken together, these data support the existence of different subpopulations of complex V, ATP synthase and ATP hydrolase, the latter with higher mobility and probably not prevailing at the cristae edges. Obviously, complex V reacts quickly and reversibly to metabolic conditions, not only by functional, but also by spatial and structural reorganization., This work was supported by the DFG (INST 190/167-2).

• Mitochondrial F1FO ATP synthase is the key enzyme for mitochondrial bioenergetics. Dimeric F1FO-ATP synthase, is preferentially located at the edges of the cristae and its oligomerization state determines mitochondrial ultrastructure. The ATP synthase inhibitor protein IF1 modulates not only ATP synthase activity but also regulates both the structure and function of mitochondria. In order to understand this in more detail, we have investigated the effect of IF1 on the spatiotemporal organization of the ATP synthase. Stable cell lines were generated that overexpressed IF1 and constitutively active IF1-H49K. The expression of IF1-H49K induced a change in the localization and mobility of the ATP synthase as analyzed by single molecule tracking and localization microscopy (TALM). In addition, the ultrastructure and function of mitochondria in cells with higher levels of active IF1 displayed a gradual alteration. In state III, cristae structures were significantly altered. The inhibition of the hydrolase activity of the F1FO-ATP synthase by IF1 together with altered inner mitochondrial membrane caused re-localization and altered mobility of the enzyme.

Mitochondria are known as dynamic organelles that fuse and divide under the control of certain proteins. These dynamics are important to shape mitochondria, maintain a healthy mitochondrial population, and enable physiological adaptations, to name just a few key processes. We are less aware that mitochondrial membrane lipids and proteins also exhibit dynamics in terms of lateral mobility and translocation. This single molecule dynamics is equally important for the above processes as it enables interaction with other proteins and complexes. Here we discuss some mitochondrial proteins and the role of their specific dynamic spatiotemporal organization for function and adaptation. For example, respiratory proteins are preferentially localized in cristae sheets, ATP synthase at the edges of cristae and compounds of the MICOS complex at cristae junctions. Trajectory patterns show how and whether molecules are restricted in their mobility and how this determines their distribution. The formation of supercomplexes has an influence on this. Recent studies have also shown that the distribution of proteins is not absolutely static. For example, the metabolic state of the cell obviously determines the activity of the mitochondria and finally the organization of the bioenergetic and structure-determining proteins inside. The ATP synthase has both classifications and additionally shows functional interactions with other cristae shaping proteins at cristae junctions. To understand the dynamics of mitochondria we have to consider all scales: from the dynamics of the molecular structure of the proteins to the dynamics of the molecules with respect to their localization and lateral mobility to the dynamics of the organelle structure.

Molecular mobility in membranes of intracellular organelles is poorly understood, due to the lack of experimental tools applicable for a great diversity of shapes and sizes such organelles can acquire. Determinations of diffusion within the plasma membrane or cytosol are based mostly on the assumption of an infinite flat space, not valid for curved membranes of smaller organelles. Here we extend the application of FRAP to mitochondria of living cells by application of numerical analysis to data collected from a small region inside a single organelle. The spatiotemporal pattern of light pulses generated by the laser scanning microscope during the measurement is reconstructed in silico and consequently the values of diffusion parameters best suited to the particular organelle are found. The mobility of the outer membrane proteins hFis and Tom7, as well as oxidative phosphorylation complexes COX and F(1)F(0) ATPase located in the inner membrane is analyzed in detail. Several alternative models of diffusivity applied to these proteins provide insight into the mechanisms determining the rate of motion in each of the membranes. Tom7 and hFis move along the mitochondrial axis in the outer membrane with similar diffusion coefficients (D=0.7μm(2)/s and 0.6μm(2)/s respectively) and equal immobile fraction (7%). The notably slower motion of the inner membrane proteins is best represented by a dual-component model with approximately equal partitioning of the fractions (F(1)F(0) ATPase: 0.4μm(2)/s and 0.0005μm(2)/s; COX: 0.3μm(2)/s and 0.007μm(2)/s). The mobility patterns specific for the membranes of this organelle are unambiguously distinguishable from those of the plasma membrane or artificial lipid environments: The parameters of mitochondrial proteins indicate a distinct set of factors responsible for their diffusion characteristics.

Mitochondria are dynamic organelles on different levels. First, mitochondrial fusion and fission balance determines the morphology and dynamics of single organelles. Second, the internal structure can be re-organized following metabolic switches. Here, we investigate whether also on the protein level a dynamic adaption in terms of localization and dynamics occurs. By tracking and localization microscopy (TALM) we determine the dynamic organization of single F1FO ATP synthase in mitochondria under different conditions. It was shown before that mitochondria build stress induced hyperfused networks to escape mitophagy under starvation conditions. In parallel, β-oxidation of lipids in the matrix is enhanced. We asked, whether these changes are accompanied by a re-organization of mitochondrial substructures and whether and how this re-organization changes the spatio-temporal organization of proteins in the inner mitochondrial membrane. Indeed, we found that the spatio-temporal organization of mitochondrial F1FO ATP synthase was severely concerned. The mobility of F1FO ATP synthase was significantly enhanced and the localization more diffusive. We attribute this partially to ultrastructural changes since we found a strong reduction of the cristae membranes in starving cells. A switch towards higher OXPHOS rates by galactose supply had the same effect. Thus, the spatio-temporal organization of bioenergetics protein complexes on the molecular level is determined by metabolic conditions.

In this study we investigated the influence of adenine nucleotides and magnesium on ATP formation by oxidative phosphorylation and adenylate kinase (AK) and on ATP-export by the ADP/ATP translocator in mitochondria of potato (Solanum tuberosum L.). Our kinetics and inhibitor studies showed that ATP in mitochondria of potato tubers could be provided by two different enzymes, depending on the ADP concentration. ATP was primarily formed by oxidative phosphorylation at low ADP concentrations and by AK at high ADP concentrations. In addition, the extent of ATP formation and the fractions contributed by each of these two systems were controlled by the level of magnesium ions in the medium. At high magnesium concentrations ADP import, ATP-synthesis by ATP synthase and ATP export increased. The effect of increasing ADP (and ATP) and magnesium concentrations on respiratory rates, respiratory control values and P:O ratios were quantified. Respiration rates of mitochondria from potato tubers in state 3 [B. Chance, G.R. Williams, J. Biol Chem 217 (1955) 635–647] increased with rising ADP (and ATP) concentration, when magnesium was present (5 mM). The P:O ratio was a function of the added ADP but it was independent of the presence of magnesium. Applying the test suggested by Liu et al. [X.C. Liu, K. Jones, H.G. Dickinson, Theor. Appl. Genet. 76 (1988) 305–310] to measure ADP/ATP translocator activity we found some inconsistencies.

Mitochondria are cellular organelles that possess - due to their evolutionary origin - two functionally and biochemically distinct membranes. While the outer membrane encloses the organelle like a sausage casing, the inner mitochondrial membrane extrudes in the interior of mitochondrial. This cristal membrane is probably the site of oxidative phosphorylation (Gilkerson et al, 2003). Yet, the localization of respiratory complexes is not elusive to the cristal membrane (Vogel et al, 2006) as anticipated. On the other hand, cristae junctions were found to separate the two inner membrane compartments implicating a diffusion barrier for membrane proteins (Mannella et al, 2001). To reveal this discrepancy we investigated the spatio-temporal behavior of single respiratory complexes by means of superresolution imaging and single molecule tracking and found different mobilities. ATP synthase displayed the strongest confinement in the cristae when compared to other respiratory complexes. Complementary, outer membrane proteins showed an unrestricted diffusion alongside mitochondria. We thus can explain the patchy appearance of dynamic mitochondria as observed recently (Muster et al, 2010) as to be the result of hindered diffusion within the inner mitochondrial membrane.View Large Image | View Hi-Res Image | Download PowerPoint Slide