Investigation of the mitochondrial contact site and cristae organizing system and its role in cristae formation

Mitochondria are essential organelles in eukaryotic cells that feature a unique doublemembrane architecture. The mitochondrial inner membrane forms numerous invaginations, named cristae, which can adopt different shapes and sizes. The cristae membrane harbors the protein machinery that facilitates the function of mitochondria as powerhouses of the cell. Small openings, referred to as crista junctions, connect the cristae membrane to the inner boundary membrane, which runs in parallel to the outer membrane. The intricate shape of the inner membrane is determined by a set of membrane-shaping proteins including the dimeric F1FO-ATP synthase, the dynamin-like GTPase optic atrophy 1 (OPA1) and the mitochondrial contact site and cristae organizing system (MICOS), a heterooligomeric protein complex that is crucial for the formation of crista junctions. In mammals, MICOS consists of seven different subunits that are organized within two distinct MICOS subcomplexes. Although several studies demonstrated physical or genetic interactions between the MICOS subcomplexes, OPA1 and the dimeric F1FO-ATP synthase, their precise interplay in cristae formation and maintenance is largely unknown. Consequently, the mechanism of cristae formation is still under debate and a variety of conflicting models describing the formation of cristae have been suggested. Examination of such models requires the analysis of morphological changes of the inner membrane architecture and the determination of the intramitochondrial localization of the involved membrane-shaping factors. As the diameter of mitochondria is close to the diffraction limit of optical microscopy, visualization of both aspects requires the application of diffractionunlimited fluorescence nanoscopy techniques. However, the lack of adequate labeling strategies for the mitochondrial inner membrane has prohibited the visualization of its dynamics by fluorescence nanoscopy. The first part of this thesis introduces a reliable labeling approach that enables time-lapse recordings of individual cristae with a resolution of about 50 nm using stimulated emission depletion (STED) nanoscopy. Live-cell recordings of mitochondria demonstrated that cristae constantly change their appearance on the timescale of seconds and form well-organized groups inside the mitochondrial tubules. The second part of this thesis investigates the role of the MICOS complex in the organization of cristae and the positioning of crista junctions in mitochondria from Saccharomyces cerevisiae and from different human cell types. STED nanoscopy of Mic60, a core subunit of the MICOS complex, revealed that Mic60 forms spatially coordinated protein clusters, which reflect the distribution of the crista junctions. Frequently, distinct Mic60 clusters are organized in two opposite distribution bands, which run along the mitochondrial tubules. These opposite bands can adopt a helically twisted arrangement, supporting the idea that individual crista junctions are physically coupled along and across the mitochondrial tubules. 3D electron microscopy and STED nanoscopy data indicated that this junction coupling is largelyindependent from the cristae, but is instead an intrinsic feature of the mitochondrial inner membrane. The central part of this thesis further investigates the mechanisms that control cristae formation in humans. The findings demonstrate that an intricate interplay between the two MICOS subcomplexes, OPA1, and the dimeric F1FO-ATP synthase controls inner membrane remodeling, the formation of cristae, and the coordinated positioning of the crista junctions. HeLa cells were individually depleted of all known MICOS subunits and were analyzed using protein-biochemistry, super-resolution imaging, and electron microscopy. The presented data revealed that the Mic60-subcomplex enables the formation of crista junctions, whereas the Mic10-subcomplex modulates the formation of lamellar cristae. The generation of inducible stable cell lines allowed for the restoration of lamellar cristae upon re-expression of the MICOS complex in MICOS-depleted cells. Reconstitution of the MICOS complex triggered fission of disordered cristae as well as the de novo formation of crista junctions on preexisting cristae. STED recordings further demonstrated that association of the two MICOS subcomplexes, along with the dimeric F1FO-ATP synthase, controls the width of the opposite Mic60 distribution bands and thereby the positioning of crista junctions around the mitochondria. Contradicting previous reports from yeast, the formation of lamellar cristae in humans was found to be largely independent of fusion and fission of mitochondrial tubules as demonstrated by transient depletion of several important fusion and fission factors. Nevertheless, knockdown experiments illustrated that the fusion protein OPA1 stabilizes tubular crista junctions and controls the formation of Mic60 assemblies together with Mic10. Therefore, OPA1, together with the dimeric F1FO-ATP synthase, influences the positioning of the MICOS complex in the inner membrane. Finally, the findings described in this work allowed for the development of a new model of cristae formation in which the interplay of the MICOS-subcomplexes with OPA1 and with the dimeric F1FO-ATP synthase controls the remodeling of the inner membrane and facilitates the segmentation of unstructured cristae membranes into multiple highly organized lamellar cristae