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Mitochondrial supercomplexes form around a conserved core of monomeric complex I and dimeric complex III; wherein a subunit of the former, NDUFA11, is conspicuously situated at the interface. We identified nduf-11 (B0491.5) as encoding the Caenorhabditis elegans homologue of NDUFA11. Animals homozygous for a CRISPR-Cas9-generated knockout allele of nduf-11 arrested at the second larval (L2) development stage. Reducing (but not eliminating) expression using RNAi allowed development to adulthood, enabling characterisation of the consequences: destabilisation of complex I and its supercomplexes and perturbation of respiratory function. The loss of NADH dehydrogenase activity was compensated by enhanced complex II activity, with the potential for detrimental reactive oxygen species (ROS) production. Cryo-electron tomography highlighted aberrant morphology of cristae and widening of both cristae junctions and the intermembrane space. The requirement of NDUF-11 for balanced respiration, mitochondrial morphology and development presumably arises due to its involvement in complex I and supercomplex maintenance. This highlights the importance of respiratory complex integrity for health and the potential for its perturbation to cause mitochondrial disease. This article has an associated First Person interview with Amber Knapp-Wilson, joint first author of the paper., Summary: Destabilisation of complex I and its supercomplexes by removal of the assembly factor NDUF-11 severely affects balanced respiration, mitochondrial morphology and, ultimately, whole animal physiology.

In this thesis, I detail the use of various forms of correlative microscopy to investigate reorganization of mitochondrial architecture during two processes, apoptosis and mitophagy, in an effort to better understand the contribution of membrane remodeling to these biological processes. During apoptosis, Bax/Bak oligomerize and accumulate in and adjacent to the outer mitochondrial membrane, resulting in outer membrane ruptures several hundred nanometers wide. Using correlative microscopy, it was found that Bax clusters accumulated next to outer membrane ruptures and appeared to be amorphous. Electron cryo-tomography of vitreous cells was used to discern a higher-order organization to cluster components. In these near- native conditions, Bax clusters appeared to contain a sponge-like meshwork, suggesting that components of the outer membrane may be bound by Bax and then sequestered in the cytosol. Mitochondria with outer membrane ruptures presented a diverse array of inner membrane morphologies, such as dilution of the matrix and cristae unfolding. This unfolding was found to be linked to a loss of typical ATP synthase dimer organization. A new, comprehensive model of Bax-mediated apoptosis is presented, linking molecular reorganizations of the inner membrane with sequestration of outer membrane components into Bax clusters in an interplay to drive rupture formation. In mitophagy, the PINK1 kinase is stabilized on the outer mitochondrial membrane, where it recruits Parkin. Parkin then ubiquitinates a host of outer membrane proteins, targeting a mitochondrion for autophagy. While most literature details a wholesale removal of all mitochondria from a cell, recent work describes a localized form of mitophagy that removes selective parts of the network. Mitochondria targeted by Parkin during localized mitophagy were observed, and this process was compared between cells intact for Drp1, the mitochondrial fission protein, and those with Drp1 knocked out. Fluorescently-tagged Parkin was found on mitochondrial fragments, mitochondria-containing autophagosomes, and mitochondria-containing lysosomes. In both lines, the fragmentation that led to isolation of a mitochondrion targeted for degradation occurred prior to the recruitment of autophagic machinery. In some cases, mitochondria surrounded by multiple autophagosomes were observed. These results reveal new details on the tight link between mitochondrial membrane dynamics and the functional state of the mitochondrion. Overall, I present a thesis that describes the insights into mitochondrial biology gained from various correlative microscopy approaches., Marshall Aid Commemoration Commission, The International Biomedical Research Alliance, the NIH OxCam Program, the MRC LMB, and Hughes Hall.

During apoptosis, Bcl-2 proteins such as Bax and Bak mediate the release of pro-apoptotic proteins from the mitochondria by clustering on the outer mitochondrial membrane and thereby permeabilizing it. However, it remains unclear how outer membrane openings form. Here, we combined different correlative microscopy and electron cryo-tomography approaches to visualize the effects of Bax activity on mitochondria in human cells. Our data show that Bax clusters localize near outer membrane ruptures of highly variable size. Bax clusters contain structural elements suggesting a higher order organization of their components. Furthermore, unfolding of inner membrane cristae is coupled to changes in the supramolecular assembly of ATP synthases, particularly pronounced at membrane segments exposed to the cytosol by ruptures. Based on our results, we propose a comprehensive model in which molecular reorganizations of the inner membrane and sequestration of outer membrane components into Bax clusters interplay in the formation of outer membrane ruptures.Editorial note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (see decision letter).

We employed electron cryo‐tomography to visualize cytosolic ribosomes on the surface of mitochondria. Translation‐arrested ribosomes reveal the clustered organization of the TOM complex, corroborating earlier reports of localized translation. Ribosomes are shown to interact specifically with the TOM complex, and nascent chain binding is crucial for ribosome recruitment and stabilization. Ribosomes are bound to the membrane in discrete clusters, often in the vicinity of the crista junctions. This interaction highlights how protein synthesis may be coupled with transport. Our work provides unique insights into the spatial organization of cytosolic ribosomes on mitochondria.

We used electron cryotomography and subtomogram averaging to determine the in situ structures of mitochondrial ATP synthase dimers from two organisms belonging to the phylum euglenozoa: Trypanosoma brucei, a lethal human parasite, and Euglena gracilis, a photosynthetic protist. At a resolution of 32.5 Å and 27.5 Å, respectively, the two structures clearly exhibit a noncanonical F1 head, in which the catalytic (αβ)3 assembly forms a triangular pyramid rather than the pseudo-sixfold ring arrangement typical of all other ATP synthases investigated so far. Fitting of known X-ray structures reveals that this unusual geometry results from a phylum-specific cleavage of the α subunit, in which the C-terminal αC fragments are displaced by ∼20 Å and rotated by ∼30° from their expected positions. In this location, the αC fragment is unable to form the conserved catalytic interface that was thought to be essential for ATP synthesis, and cannot convert γ-subunit rotation into the conformational changes implicit in rotary catalysis. The new arrangement of catalytic subunits suggests that the mechanism of ATP generation by rotary ATPases is less strictly conserved than has been generally assumed. The ATP synthases of these organisms present a unique model system for discerning the individual contributions of the α and β subunits to the fundamental process of ATP synthesis.

Summary Lipid flow between cellular organelles occurs via membrane contact sites. Extended-synaptotagmins, known as tricalbins in yeast, mediate lipid transfer between the endoplasmic reticulum (ER) and plasma membrane (PM). How these proteins regulate membrane architecture to transport lipids across the aqueous space between bilayers remains unknown. Using correlative microscopy, electron cryo-tomography, and high-throughput genetics, we address the interplay of architecture and function in budding yeast. We find that ER-PM contacts differ in protein composition and membrane morphology, not in intermembrane distance. In situ electron cryo-tomography reveals the molecular organization of tricalbin-mediated contacts, suggesting a structural framework for putative lipid transfer. Genetic analysis uncovers functional overlap with cellular lipid routes, such as maintenance of PM asymmetry. Further redundancies are suggested for individual tricalbin protein domains. We propose a modularity of molecular and structural functions of tricalbins and of their roles within the cellular network of lipid distribution pathways., Graphical Abstract, Highlights • ER-PM proteins form contact sites with distinct ER shape but similar distance • Tricalbins localize to high ER curvature via their membrane domain • Genetic screens link tricalbin function to pathways for cellular lipid distribution • Cryo-ET reveals rod-shaped densities at tricalbin-mediated ER-PM contacts, Hoffmann et al. investigate the architecture and function of yeast ER-PM contact sites. They show how bridging proteins, particularly tricalbins, relate to membrane curvature and inter-organelle distance. Genetic screening points to functional overlap between tricalbins and multiple lipid distribution pathways. Cryo-ET visualizes rod-shaped linkers at tricalbin-mediated ER-PM contacts.

Electron cryo-tomography is a powerful tool in structural biology, capable of visualizing the three-dimensional structure of biological samples, such as cells, organelles, membrane vesicles, or viruses at molecular detail. To achieve this, the aqueous sample is rapidly vitrified in liquid ethane, which preserves it in a close-to-native, frozen-hydrated state. In the electron microscope, tilt series are recorded at liquid nitrogen temperature, from which 3D tomograms are reconstructed. The signal-to-noise ratio of the tomographic volume is inherently low. Recognizable, recurring features are enhanced by subtomogram averaging, by which individual subvolumes are cut out, aligned and averaged to reduce noise. In this way, 3D maps with a resolution of 2 nm or better can be obtained. A fit of available high-resolution structures to the 3D volume then produces atomic models of protein complexes in their native environment. Here we show how we use electron cryo-tomography to study the in situ organization of large membrane protein complexes in mitochondria. We find that ATP synthases are organized in rows of dimers along highly curved apices of the inner membrane cristae, whereas complex I is randomly distributed in the membrane regions on either side of the rows. By subtomogram averaging we obtained a structure of the mitochondrial ATP synthase dimer within the cristae membrane.