Structure of the mitochondrial ATP synthase dimer from Polytomella sp. Side view of the two mitochondrial ATP synthase in the V-shaped dimer. Adapted from [ 40 ]. The ubiquitous nature of the dimer rows raises the question as to the biological significance of this striking, conserved arrangement.
In yeast the two protomers are linked by the dimer-specific ATP synthase subunits e and g. If either subunit is knocked out, only monomeric ATP synthase is found in the inner membrane [ 30 ] and the usual lamellar or tubular cristae do not form [ 30 , 41 ].
ATP synthase dimers and dimer rows are thus a prerequisite for proper cristae formation. Although the loss of the dimer-specific subunits is not lethal, it is a serious disadvantage. This raises the further question about the role of the cristae, and hence the dimer rows, in cellular physiology and fitness.
Most likely the invaginations prevent protons that are pumped into the crista lumen by the respiratory chain from escaping rapidly to the inter-membrane space and the cytoplasm, so that they can be harnessed more efficiently for ATP production. In this way, the cristae, and hence the dimer rows, would contribute to effective ATP synthesis.
Prokaryotic ATP synthases lack the dimer-specific subunits, and ATP synthase dimers or dimer rows have not been found in bacterial or archaeal inner membranes, which also do not have cristae. The cristae and dimer rows may thus be an adaptation that enables mitochondria to satisfy the high energy demand of eukaryotic cells with the available, shallow proton gradient of around 0.
ATP synthase dimers have recently been implicated in the formation of the permeability transition pore [ 43 ] that triggers apoptosis. On the basis of the structure of the mitochondrial ATP synthase dimer [ 39 ] or the dimer rows [ 30 ], however, it is difficult to see how they might form a membrane pore. Complex I feeds electrons from the soluble carrier molecule NADH into the respiratory chain and transfers them to a quinol in the membrane.
The energy released in the electron transfer reaction is utilized for pumping four protons from the matrix into the crista lumen. Complex III takes the electrons from the reduced quinol and transfers them to the small, soluble electron carrier protein cytochrome c , pumping one proton in the process. Finally, complex IV transfers the electrons from cytochrome c to molecular oxygen and contributes to the proton gradient by using up four protons per consumed oxygen molecule to make water.
Complex II succinate dehydrogenase transfers electrons from succinate directly to quinol and does not contribute to the proton gradient. The respiratory chain complexes have been studied in great detail for decades. The mitochondrial complex has about three times as many protein subunits as its bacterial ancestor. Most functions of the extra subunits are unknown, but many of them are likely to work in assembly or the regulation of complex I function. However, the way in which electron transfer from NADH to ubiquinone in complex I is coupled to proton translocation is still unknown, and much else remains to be discovered.
Cryo-EM structure of bovine heart complex I. The matrix arm contains a row of eight iron-sulfur clusters red that conduct electrons from NADH to ubiquinol at the junction of the matrix and membrane arms Fig. The membrane arm consists of 78 trans-membrane helices, including three proton-pumping modules.
Adapted from [ 51 ]; EMDB code Respiratory chain supercomplexes were first postulated on the basis of blue-native gels of yeast and bovine heart mitochondria solubilized in the mild detergent digitonin [ 49 ]. Negative-stain electron microscopy [ 50 ] and single-particle cryo-EM [ 51 ] of the 1. X-ray structures of the component complexes were fitted to the 3D map Fig. Genetic evidence provides strong support for the existence of respirasomes in vivo [ 52 ], but they were long thought to be artifacts of detergent solubilization, notwithstanding their well defined structure.
Saccharomyces cerevisiae , which lacks complex I, nevertheless has a respiratory chain supercomplex consisting of complex III and IV [ 53 ]. Far from being randomly distributed in the membrane, the ATP synthase and electron transport complexes of the respiratory chain thus form supramolecular assemblies in the cristae, in a way that is essentially conserved from yeast to humans Fig.
A clear functional role of mitochondrial supercomplexes has not yet been established. They may make electron transfer to and from ubiquinone in complexes I and III more efficient, as the relative positions and orientations of the two complexes are precisely aligned rather than random. However, there is no direct evidence that this makes a difference. The supercomplexes may simply help to avoid random, unfavorable protein—protein interactions in the packed environment of the inner mitochondrial membrane [ 54 ].
Alternatively, they may control the ratio of respiratory chain complexes in the membrane, or aid their long-term stability. Cryo-EM structure of the 1. Cofactors active in electron transport are marked in yellow FMN , orange iron—sulfur clusters , dark blue quinols , red hemes , and green copper atoms. Arrows indicate the electron path through the supercomplex. Adapted from [ 51 ]. ATP synthase dimer rows shape the mitochondrial cristae. At the cristae ridges, the ATP synthases yellow form a sink for protons red , while the proton pumps of the electron transport chain green are located in the membrane regions on either side of the dimer rows.
Guiding the protons from their source to the proton sink at the ATP synthase, the cristae may work as proton conduits that enable efficient ATP production with the shallow pH gradient between cytosol and matrix. Red arrows show the direction of the proton flow. A main protein component of the crista lumen is the small soluble electron carrier protein cytochrome c that shuttles electrons from complex III to complex IV.
If released into the cytoplasm, cytochrome c triggers apoptosis [ 55 ]. It is imperative, therefore, that cytochrome c does not leak from the cristae and that the outer membrane remains tightly sealed during mitochondrial fission and fusion.
Ageing is a fundamental yet poorly understood biological process that affects all eukaryotic life. Deterioration in mitochondria is clearly seen in ageing, but details of the underlying molecular events are largely unknown. In normal mitochondria of young cells, the cristae protrude deeply into the matrix. With increasing age, the cristae recede into the inner boundary membrane and the inter-membrane space widens. Eventually, the matrix breaks up into spherical vesicles within the outer membrane.
The ATP synthase dimer rows disperse and the dimers dissociate into monomers. As the inner membrane vesiculates, the sharp local curvature at the dimer rows inverts, so that the ATP synthase monomers are surrounded by a shallow concave membrane environment, rather than the sharply convex curvature at the crista ridges Fig.
Finally, the outer membrane ruptures, releasing the inner membrane vesicles, along with apoptogenic cytochrome c , into the cytoplasm. Cytochrome c activates a cascade of proteolytic caspases, which degrade cellular proteins [ 55 ]. The cell enters into apoptosis and dies. Changes of inner membrane morphology and ATP synthase dimers in ageing mitochondria.
Tomographic volumes of mitochondria isolated from young 6-day-old a and ageing day-old b cultures of the model organism Podospora anserina. In young mitochondria, the ATP synthase dimers are arranged in rows along highly curved inner membrane ridges Movie S2. In ageing mitochondria, the cristae recede into the boundary membrane, with ATP synthases dimer rows along the shallow inner membrane ridges.
Outer membrane, transparent grey ; inner membrane, light blue. ATP synthase F 1 heads are shown as yellow spheres. Right : subtomogram averages with fitted X-ray models. Red lines , convex membrane curvature as seen from the matrix ; blue lines , concave membrane curvature. Adapted from [ 56 ]. The observed morphological changes during ageing in P. The electron-transfer reactions in complexes I and III generate reactive superoxide radicals as side products [ 58 ], which cause damage to mitochondrial proteins and DNA, as well as to other cellular components.
Senescent mitochondria that lack cristae and ATP synthase dimers would not be able to provide sufficient ATP to maintain essential cellular functions.
Cells normally deal with oxidative damage by oxygen radical scavenging enzymes such as superoxide dismutase or catalase, as well as by mitochondrial fission and fusion. Damaged or dysfunctional mitochondria are either complemented with an undamaged part of the mitochondrial network by fusion or sorted out for mitophagy [ 59 ].
During ageing, fission overpowers fusion and the mitochondrial network fragments [ 60 ]. This prevents the complementation of damaged mitochondria by fusion and thus accelerates their deterioration. Even though mitochondria and their membrane protein complexes have been studied intensely for more than five decades, they remain a constant source of fascinating and unexpected new insights.
Open questions abound, many of them of a fundamental nature and of direct relevance to human health [ 61 ]. Concerning macromolecular structure and function, we do not yet understand the precise role of the highly conserved feature of ATP synthase dimers and dimer rows in the cristae and the interplay between the MICOS complex and the dimer rows in cristae formation. Are there other factors involved in determining crista size and shape? We still do not know how complex I works, especially how electron transfer is coupled to proton translocation.
What is the role of respiratory chain supercomplexes? Do they help to prevent oxidative damage to mitochondria, and if so, how? And how does this affect ageing and senescence?
How does it anchor the cristae to the outer membrane, and how does it separate the cristae form the contiguous boundary membrane? Similarly, the mechanisms of mitochondrial fission and fusion and the precise involvement and coordination of the various protein complexes in this intricate process is a fascinating area of discovery.
The biogenesis and assembly of large membrane protein complexes in mitochondria is largely unexplored. Where and exactly how do the respiratory chain complexes and the ATP synthase assemble? How is their assembly from mitochondrial and nuclear gene products coordinated? Does this involve feedback from the mitochondrion to the cytoplasm or the nucleus, and what is it?
And finally, how exactly are mitochondria implicated in ageing? Why do some cells and organisms live only for days, while others have lifespans of years or decades?
Is this genetically programmed or simply a consequence of different levels of oxidative damage? How is this damage prevented or controlled, and how does it affect the function of mitochondrial complexes? Is the breakdown of ATP synthase dimers also an effect of oxidative damage, and is it a cause of ageing?
It will be challenging to find answers to these questions because many of the protein complexes involved are sparse, fragile and dynamic, and they do not lend themselves easily to well established methods, such as protein crystallography. Cryo-EM, which is currently undergoing rapid development in terms of high-resolution detail, will have a major impact but is limited to molecules above about kDa [ 62 ].
Even better, more sensitive electron detectors than the ones that have precipitated the recent resolution revolution, in combination with innovative image processing software, will yield more structures at higher resolution. However, small, rare and dynamic complexes will remain difficult to deal with. New labeling strategies in combination with other biophysical and genetic techniques are needed.
Cloneable labels for electron microscopy, equivalent to green fluorescent protein in fluorescence microscopy, would be a great help; first steps in this direction look promising [ 26 ]. Once the structures and locations of the participating complexes have been determined, molecular dynamics simulations, which can analyze increasingly large systems, can help to understand their molecular mechanisms.
Without any doubt, mitochondria and their membrane protein complexes will remain an attractive research area in biology for many years to come. Mitochondria as sensors and regulators of calcium signalling. Nat Rev Mol Cell Biol. Mitophagy and the mitochondrial unfolded protein response in neurodegeneration and bacterial infection. BMC Biol. Chandel NS. Mitochondria as signaling organelles. Bratic A, Larsson NG. The role of mitochondria in aging. J Clin Invest.
Genome structure and gene content in protist mitochondrial DNAs. Nucleic Acids Res. Germain, M. EMBO J. Gilkerson, R. The cristal membrane of mitochondria is the principal site of oxidative phosphorylation.
FEBS Lett. Hackenbrock, C. Ultrastructural bases for metabolically linked mechanical activity in mitochondria. Reversible ultrastructural changes with change in metabolic steady state in isolated liver mitochondria.
Cell Biol. Hahn, A. Structure of a Complete ATP synthase dimer reveals the molecular basis of inner mitochondrial membrane morphology. Cell 63, — Harner, M. The mitochondrial contact site complex, a determinant of mitochondrial architecture. PubMed Abstract Google Scholar. An evidence based hypothesis on the existence of two pathways of mitochondrial crista formation.
Khalifat, N. Membrane deformation under local pH gradient: mimicking mitochondrial cristae dynamics. Lane, N. London: W. Lenaz, G. Structural and functional organization of the mitochondrial respiratory chain: a dynamic super-assembly. Li, W. Elasticity of synthetic phospholipid vesicles and submitochondrial particles during osmotic swelling. Biochemistry 25, — Mannella, C. Structure of the outer mitochondrial membrane: ordered arrays of porelike subunits in outer-membrane fractions from Neurospora crassa mitochondria.
Electron microscopic tomography of rat-liver mitochondria and their interaction with the endoplasmic reticulum. Biofactors 8, — The connection between inner membrane topology and mitochondrial function. Cell Cardiol. Reconsidering mitochondrial structure: new views of an old organelle.
The internal compartmentation of rat-liver mitochondria: tomographic study using the high-voltage transmission electron microscope. Topology of the mitochondrial inner membrane: dynamics and bioenergetic implications.
Martinou, I. The release of cytochrome c from mitochondria during apoptosis of NGF-deprived sympathetic neurons is a reversible event. McArthur, K. Science eaao McCommis, K. The role of VDAC in cell death: friend or foe? Meeusen, S. Mitochondrial inner-membrane fusion and crista maintenance requires the dynamin-related GTPase Mgm1. Milenkovic, D. The enigma of the respiratory chain supercomplex. Cell Metab. Mootha, V. A reversible component of mitochondrial respiratory dysfunction in apoptosis can be rescued by exogenous cytochrome c.
Patten, D. OPA1-dependent cristae modulation is essential for cellular adaptation to metabolic demand. Paumard, P. The ATP synthase is involved in generating mitochondrial cristae morphology. Perkins, G. Electron tomography of neuronal mitochondria: three-dimensional structure and organization of cristae and membrane contacts.
Picas, L. Direct measurement of the mechanical properties of lipid phases in supported bilayers. Plecita-Hlavata, L. Integration of superoxide formation and cristae morphology for mitochondrial redox signaling. Rasola, A. Cell Calcium 50, — Renken, C. A thermodynamic model describing the nature of the crista junction: a structural motif in the mitochondrion. Rieger, B. Rostovtseva, T. VDAC inhibition by tubulin and its physiological implications. Scorrano, L. A distinct pathway remodels mitochondrial cristae and mobilizes cytochrome c during apoptosis.
Cell 2, 55— Strauss, M. Dimer ribbons of ATP synthase shape the inner mitochondrial membrane. Tait, S. Mitochondrial regulation of cell death. Cold Spring Harb. Perspect Biol. Toth, A. Kinetic coupling of the respiratory chain with ATP synthase, but not proton gradients, drives ATP production in cristae membranes.
Tsai, P. Cell Walker, D. Wolf, D. Individual cristae within the same mitochondrion display different membrane potentials and are functionally independent. Furthermore, most Yme1L KO mitochondrial cristae were crooked and arranged in a disordered manner Fig. In addition, Yme1L KO mitochondrial cristae, which were abnormal in shape, were also highly dynamic and moved quickly, similar to the control mitochondrial cristae, but the crista dynamic events of shortening and detachment from the IBM were markedly decreased Fig.
Moreover, in Mic10 KO, Mic19 KO, and Mic60 KD cells, mitochondrial crista dynamic events, including shortening, elongation, and crista—crista contact or fusion , were remarkably decreased Fig. The SamMicMic60 axis plays a key role in mitochondrial crista junction formation S2Q — S2R. Thus, we investigated the effect of Sam50 KD on mitochondrial cristae.
Sam50 KD reduced the number of mitochondrial cristae and resulted in a significant decrease in the number of CJs per mitochondrial crista but an increased number of abnormal mitochondrial cristae Fig. S2S and supplementary Table 1.
ATAD3A KO resulted in abnormal crista morphology and reduced the number of mitochondrial cristae but led to a significant increase in the number of CJs per mitochondrial crista Fig. These data indicate that ATAD3A negatively regulates the contact and subsequent fusion of mitochondrial cristae with the IBM or adjacent mitochondrial cristae.
Therefore, mitochondrial crista—IBM and crista—crista contacts play an important role in regulating mitochondrial crista morphology. We also used actinomycin D ActD , a potent inducer of apoptosis, for physiological stimulation to analyze mitochondrial crista morphology.
ActD treatment induced rapid mitochondrial fission in HeLa cells, leading to a dramatically increased number of fragmented mitochondria Figs.
S3A — S3B. Moreover, ActD treatment reduced the number of mitochondrial cristae and significantly increased the number of abnormal mitochondrial cristae Figs. S3C — S3E. Additionally, the number of CJs per crista remained unchanged, but the number of crooked cristae and the cristae width were increased in ActD-treated cells Figs. These data suggest that ActD treatment induces changes in mitochondrial crista shape.
Mitochondrial cristae are tethered to the IBM by CJs, which are narrow and ring-like or slot-like structures 3 , 6. In HeLa cells, most mitochondrial cristae are lamellar Fig.
Therefore, we named this inner-membrane compartment a cut-through crista. Cut-through crista does not contain a crista tip, and its CJ surround the edge of crista in a long groove. We previously demonstrated that outer and inner mitochondrial membrane fusion are separate processes and that OPA1 mediates inner membrane fusion According to the morphological characteristics of cut-through crista, we hypothesized that the formation is probably due to the lack of inner mitochondrial membrane fusion after outer mitochondrial membrane fusion.
We then analyzed the inner mitochondrial membrane structure in OPA1 KO cells lacking inner mitochondrial membrane fusion. Overall, outer mitochondrial membrane fusion and the subsequent absence of inner mitochondrial membrane fusion induce the formation of cut-through crista.
White, mitochondrial outer membrane; cyan, mitochondrial IBM; purple red and yellow, lamellar crista without CJ. The green arrowhead indicates the contact and fusion site of two different mitochondria, the red arrowhead indicates the newly formed crista.
In Mic10 KO mitochondria, cut-through crista were not observed Fig. To directly study the process of cut-through crista formation, inner mitochondrial membrane remodeling during mitochondrial fusion in living cells was tracked by Hessian-SIM. Normally, mitochondrial fusion does not lead to the formation of new mitochondrial cristae in HeLa cells Fig. However, sometimes, two mitochondria contact and fuse to form a cut-through crista, probably due to loss of inner mitochondrial membrane fusion Fig.
Together, according to our findings, we propose a mode of cut-through crista formation due to incomplete mitochondrial fusion Fig. S4C — S4D. Mic10 is required for CJ formation. Only 20—30 percent of the onion-like cristae displayed spherical crista after 3D tomographic reconstruction. These results demonstrate that OPA1 or Mic10 depletion promotes the formation of spherical crista. S5C — S5D. These findings indicate that cut-through crista are probably preforms of spherical crista.
Because spherical crista is detected in Mic10 KO cells, which still display normal tubular mitochondria Fig. White, mitochondrial outer membrane; cyan, mitochondrial IBM; purple red, the spherical crista. White, mitochondrial outer membrane; cyan, IBM; purple red, the spherical crista. F The mode of the spherical crista formation. Two mitochondria contact and process mitochondrial outer membrane fusion, the small mitochondrion is then engulfed into the bigger mitochondria, and detaches from the IBM of the bigger mitochondrion to form the spherical crista.
Therefore, we propose the following mechanism of spherical crista formation: after outer membrane fusion between a large and a small mitochondrion, the small spherical membrane coming from the inner membrane of the small mitochondrion is sequestered by the IBM of the large mitochondrion and then detached from the IBM to form a spherical crista, whose membranes arise from two originally distinct inner mitochondrial membranes Fig.
During this process, the deficiency in inner mitochondrial membrane fusion and impaired MICOS activity mainly contribute to the formation of spherical crista. The continuous nm-thick EM sections from the cells were analyzed. S6 , but before or after these sections, the onion-like crista is completely detached from the IBM Fig.
Further 3D tomographic reconstruction showed that the outer membrane of the spherical crista partly connects to the IBM Fig. We then used time-lapse Hessian-SIM to track the process of spherical crista formation in living cells. Some small spherical mitochondria fused with and entered the large spherical mitochondrion to form spherical crista in Mic10 KO cells Fig.
These findings strongly confirm the mode of spherical crista formation Fig. The red arrowhead indicates the spherical crista. Time-lapse Hessian-SIM images reveal that two small mitochondria the green and red arrowheads indicated underwent the process of entering into a large mitochondrion.
Time-lapse Hessian-SIM images reveal that a small mitochondrion the red arrowhead indicated entered into a giant mitochondrion to form a spherical crista. Although few mitochondrial spherical cristae are observed in normal HeLa and HCT cells, onion-like mitochondrial cristae 2D form of spherical crista are frequently observed in the mitochondria of cells with mtDNA mutations, deletion or loss 38 , S8C — S8D , suggesting that spherical crista in Mic10 KO mitochondria maintain a normal mitochondrial membrane potential.
Mitochondrial cristae are the main sites of electron transfer and oxidative phosphorylation in mitochondria. The study of mitochondrial crista dynamics and remodeling has been a focus and a challenge in the field of mitochondrial research. The traditional TEM technique can only display the crista morphology of a certain section of a mitochondrial sample and cannot reflect the dynamic changes of mitochondrial cristae in living cells.
However, due to the limitation of its optic resolution, Hessian-SIM cannot distinguish the fusion or contact between mitochondrial cristae and that between cristae and the IBM. These technologies complement each other functionally: Hessian-SIM enables visualization of the mitochondrial crista dynamic process in living cells, TEM has the advantage of optical resolution in visualizing the mitochondrial crista ultrastructure but can only capture fixed samples, and FIB-SEM and 3D tomographic reconstruction enables the stereoscopic presentation of mitochondrial cristae.
Therefore, the combination of these three techniques is the best choice for studying mitochondrial crista dynamics and will lead to a re-examination of previous concepts about mitochondrial membrane organization and biogenesis.
We discovered two novel types of mitochondrial cristae, cut-through crista and spherical crista Figs. Furthermore, we demonstrated the pathways of cut-through crista and spherical crista formation and proposed the modes of formation of these two types of mitochondrial cristae Fig. OPA1 dysfunction-mediated inhibition of inner mitochondrial membrane fusion contributes to the formation of these 2 types of cristae.
Spherical crista appear as onion-like structures in 2D EM imaging Figs. S5A and S5B. Onion-like cristae were largely formed in response to inhibition of oligomerization of F1FO ATP synthase 40 , but oligomerization of F1FO ATP synthase is required for the formation of tubular cristae 6 , indicating that the model of spherical crista formation is different from that of tubular cristae formation. Our findings reveal the diversity of mitochondrial crista morphology and multiple pathways for crista formation.
Mitochondrial crista remodeling is highly associated with mitochondrial functions. Mitochondrial crista shape regulates the organization and function of the oxidative phosphorylation system, directly affecting cellular energy metabolism 3 , 4. In addition, mitochondrial crista remodeling regulates cytochrome c release during apoptosis 10 , 13 , and we also observed that mitochondrial crista shape is remodeled in ActD-treated cells Fig.
S3C — S3D. Moreover, OPA1-dependent mitochondrial crista remodeling is required for cellular adaptation to metabolic demand and is critical for cell survival and growth 9. Lipofectamine Invitrogen were carried out as protocol present. Hessian-SIM analysis was performed as described previously Nine raw frames illuminated with a periodic pattern of parallel lines and shifted through three phases for each of three orientation angles were used to reconstruct one SR image.
All quantifications were analyzed by three independent experiments at least mitochondrial cristae in 10—30 mitochondria from 10—20 cells were analyzed for each experiment or each group. The procedure of TEM was performed according to the previous report HeLa and HCT cells were fixed, filtrated, and polymerized as described previously Cells were harvested by 0.
For statistical analysis, all cells and mitochondria were randomly selected. Mannella, C. Structure and dynamics of the mitochondrial inner membrane cristae. Biochim Biophys. Acta , — Fox, T. Mitochondrial protein synthesis, import, and assembly. Genetics , — Cogliati, S. Trends Biochem. Mitochondrial cristae shape determines respiratory chain supercomplexes assembly and respiratory efficiency. Cell , — Reconsidering mitochondrial structure: new views of an old organelle. Rabl, R.
Cell Biol. Harner, M. An evidence based hypothesis on the existence of two pathways of mitochondrial crista formation. Merkwirth, C. Prohibitins control cell proliferation and apoptosis by regulating OPA1-dependent cristae morphogenesis in mitochondria. Genes Dev. Patten, D. OPA1-dependent cristae modulation is essential for cellular adaptation to metabolic demand.
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