Structural insights into crista junction formation by the Mic60-Mic19 complex

Mitochondrial cristae membranes are the oxidative phosphorylation sites in cells. Crista junctions (CJs) form the highly curved neck regions of cristae and are thought to function as selective entry gates into the cristae space. Little is known about how CJs are generated and maintained. We show that the central coiled-coil (CC) domain of the mitochondrial contact site and cristae organizing system subunit Mic60 forms an elongated, bow tie–shaped tetrameric assembly. Mic19 promotes Mic60 tetramerization via a conserved interface between the Mic60 mitofilin and Mic19 CHCH (CC-helix-CC-helix) domains. Dimerization of mitofilin domains exposes a crescent-shaped membrane-binding site with convex curvature tailored to interact with the curved CJ neck. Our study suggests that the Mic60-Mic19 subcomplex traverses CJs as a molecular strut, thereby controlling CJ architecture and function

Dual role of Mic10 in mitochondrial cristae organization and ATP synthase-linked metabolic adaptation and respiratory growth

Invaginations of the mitochondrial inner membrane, termed cristae, are hubs for oxidative phosphorylation. The mitochondrial contact site and cristae organizing system (MICOS) and the dimeric F(1)F(o)-ATP synthase play important roles in controlling cristae architecture. A fraction of the MICOS core subunit Mic10 is found in association with the ATP synthase, yet it is unknown whether this interaction is of relevance for mitochondrial or cellular functions. Here, we established conditions to selectively study the role of Mic10 at the ATP synthase. Mic10 variants impaired in MICOS functions stimulate ATP synthase oligomerization like wild-type Mic10 and promote efficient inner membrane energization, adaptation to non-fermentable carbon sources, and respiratory growth. Mic10's functions in respiratory growth largely depend on Mic10(ATPsynthase), not on Mic10(MICOS). We conclude that Mic10 plays a dual role as core subunit of MICOS and as partner of the F(1)F(o)-ATP synthase, serving distinct functions in cristae shaping and respiratory adaptation and growth

A Negative Feedback Loop Regulates Integrin Inactivation and Promotes Neutrophil Recruitment to Inflammatory Sites

International audienc

Assembly, regulation and molecular architecture of mitochondrial cristae organising systems

Mitochondria are double-membrane bound organelles with a plethora of metabolic and signalling functions, which are facilitated by their complex micro-architecture. The mitochondrial inner membrane possesses an intricate ultrastructure and is divided into a boundary membrane and specialized membrane invaginations, termed cristae, which are optimised for oxidative phosphorylation and a universal structural feature of aerobic eukaryotes. Several protein machineries help to shape cristae. Dimers of the ATP synthase create membrane curvature to stabilise cristae tips and rims, while the mitochondrial contact site and cristae organising system (MICOS) stabilises crista junctions and links inner and outer membrane. In addition, dynamin-like GTPases of the OP A1/Mgm1 family dynamically remodel cristae shape by a poorly characterised mechanism. The work described in this thesis further elucidates the assembly mechanisms of MICOS and Mgm1 using a combination of genetic and biochemical approaches in yeast and human cell lines. Studies in yeast reveal that the MICOS core subunits Mic10 and Mic60 are essential for the efficient adaptation to respiratory metabolism. A moonlighting function of Mic10 in modulating ATP synthase assembly appears to be particularly important for this process. The assembly of Mic10 can be regulated by the two paralogous MICOS subunits Mic26 and Mic27 in an antagonistic manner, possibly to coordinate Mic10 functions. The second core subunit Mic60 forms stable supercomplexes with the β-barrel biogenesis machinery of the outer membrane in human cells. A detailed analysis of knockout cell lines revealed that a lack of Mic60 leads to protein biogenesis defects and that a J-protein of the outer membrane interacts with MICOS to spatially coordinate protein biogenesis at import sites. The dynamic remodelling of cristae membranes is mostly mediated by proteins of the Mgm1/OPA1 family. The crystal structure of Mgm1 described here revealed a tetrameric assembly and cryo-electron tomography reconstructions of membrane-bound Mgm1 showed that these tetramers assemble into helical filaments on the inside and outside of membrane tubes. Mgm1 yeast mutants demonstrated that the assembly of Mgm1 into dimers and tetramers is essential for the maintenance of cristae architecture and respiratory growth. The work described here shed light on the assembly of MICOS and Mgm1, which are two major cristae organising systems. A structural biology approach combined with data from yeast mutants revealed how Mgm1 filaments can remodel cristae membranes. A comparative approach in yeast and human cells showed that MICOS organisation is evolutionarily conserved and that both core subunits affect distinct mitochondrial pathways. While Mic60 is directly involved in protein biogenesis of outer membrane proteins, Mic10 affects the assembly of inner membrane complexes such as the ATP synthase. Future work will have to dissect how the membrane-shaping protein machineries of the inner membrane coordinate their activities to control cristae shape in a regulated manner.Mitochondrien sind von zwei Membranen umschlossene Organellen aller eukaryotischen Zellen mit einer Vielzahl an Funktionen im zellulären Metabolismus und in Signalwegen. Die innere Membran formt Einstülpungen oder Cristae mit einer für die zelluläre Atmung optimierten Form. Ein großer Proteinkomplex (MICOS; mitochondrial contact site and cristae organising system) formt die Verbindung zwischen Cristae und dem Rest der Innenmembrane und verbindet außerdem Außen- und Innenmembran. Dimere der ATP-Synthase stabilisieren die Membrankrümmung in Cristae. Zusätzlich können Cristae durch die Dynamin-ähnliche GTPase Mgm1 verformt werden. Mit Hilfe genetischer und biochemischer Ansätze in Hefen und humanen Zelllinien konnten in dieser Arbeit detaillierte Modelle der Assemblierung von MICOS und Mgm1 erstellt werden. Experimente in Hefe zeigen, dass die zentralen MICOS Untereinheiten Mic10 und Mic60 in der Anpassung an respiratorischen Metabolismus beteiligt sind. Eine Zusatzfunktion von Mic10, welches auch die Assemblierung der ATP-Synthase beeinflusst, scheint hier besonders wichtig zu sein. Die Assemblierung von Mic10 wird auch aus diesem Grund von zwei anderen MICOS Untereinheiten, den paralogen Proteinen Mic26 und Mic27, in einer antagonistischen Weise reguliert. Die zweite zentrale Untereinheit Mic60 bildet in humanen Zellen sehr stabile Kontakte mit der β-barrel Proteinbiogenesemaschinerie der Außenmembran und koordiniert, zusammen mit einem hier charakterisiertem J-Protein, die Proteinbiogenese auf der Mitochondrienoberfläche. Die dynamische Veränderung der Cristaestruktur wird hauptsächlich durch die Dynamin- ähnliche GTPase Mgm1 vollzogen. Basierend auf einer Mgm1 Kristallstruktur und Kryo- Elektronen-Tomographie Daten konnte ein modularer Aufbau in Dimere, Tetramere und helikale Filamente auf der Außen- und Innenseite von Membranröhrchen gezeigt werden. Die hier beschriebenen Experimente mit Hefe Mgm1 Mutanten konnten zeigen, dass dieser modulare Aufbau essenziell für die Aufrechterhaltung der Cristaestruktur ist. Die hier dargestellten Arbeiten beschreiben neue Details des Aufbaus von MICOS und Mgm1. Mittels einer Kombination aus strukturbiologischen Daten und genetischen Ansätzen in Hefe konnte gezeigt werden wie Mgm1-Filamente Cristae verformen können. Ein Vergleich des molekularen Aufbaus des MICOS Komplexes in Hefe- und Säugerzellen offenbarte, dass MICOS hochkonserviert ist und dass die zentralen MICOS Untereinheiten unterschiedliche Prozesse in Mitochondrien beeinflussen. Während Mic60 die Biogenese mitochondrialer Außenmembranproteine steuert, hat Mic10 einen Einfluss auf die Assemblierung von Innenmembrankomplexen, wie zum Beispiel der ATP-Synthase. Zukünftige Studien müssen versuchen, das Zusammenspiel der verschiedenen cristaeverformenden Proteinkomplexe besser zu verstehen

Actin cytoskeleton and complex cell architecture in an Asgard archaeon

Asgard archaea are considered to be the closest known relatives of eukaryotes. Their genomes contain hundreds of eukaryotic signature proteins (ESPs), which inspired hypotheses on the evolution of the eukaryotic cell. A role of ESPs in the formation of an elaborate cytoskeleton and complex cellular structures has been postulated, but never visualized. Here we describe a highly enriched culture of ‘Candidatus Lokiarchaeum ossiferum’, a member of the Asgard phylum, which thrives anaerobically at 20 °C on organic carbon sources. It divides every 7–14 days, reaches cell densities of up to 5 × 107 cells per ml and has a significantly larger genome compared with the single previously cultivated Asgard strain7. ESPs represent 5% of its protein-coding genes, including four actin homologues. We imaged the enrichment culture using cryo-electron tomography, identifying ‘Ca. L. ossiferum’ cells on the basis of characteristic expansion segments of their ribosomes. Cells exhibited coccoid cell bodies and a network of branched protrusions with frequent constrictions. The cell envelope consists of a single membrane and complex surface structures. A long-range cytoskeleton extends throughout the cell bodies, protrusions and constrictions. The twisted double-stranded architecture of the filaments is consistent with F-actin. Immunostaining indicates that the filaments comprise Lokiactin—one of the most highly conserved ESPs in Asgard archaea. We propose that a complex actin-based cytoskeleton predated the emergence of the first eukaryotes and was a crucial feature in the evolution of the Asgard phylum by scaffolding elaborate cellular structures.ISSN:0028-0836ISSN:1476-468

Assembly of the Mitochondrial Cristae Organizer Mic10 is Regulated by Mic26-Mic27 Antagonism and Cardiolipin

The multi-subunit mitochondrial contact site and cristae organizing system (MICOS) is a conserved protein complex of the inner mitochondrial membrane that is essential for maintenance of cristae architecture. The core subunit Mic10 forms large oligomers that build a scaffold and induce membrane curvature. The regulation of Mic10 oligomerization is poorly understood. We report that Mic26 exerts a destabilizing effect on Mic10 oligomers and thus functions in an antagonistic manner to the stabilizing subunit Mic27. The mitochondrial signature phospholipid cardiolipin shows a stabilizing function on Mic10 oligomers. Our findings indicate that the Mic10 core machinery of MICOS is regulated by several mechanisms, including interaction with cardiolipin and antagonistic actions of Mic26 and Mic27

Structural insights into crista junction formation by the Mic60-Mic19 complex

Mitochondrial cristae membranes are the oxidative phosphorylation sites in cells. Crista junctions (CJs) form the highly curved neck regions of cristae and are thought to function as selective entry gates into the cristae space. Little is known about how CJs are generated and maintained. We show that the central coiled-coil (CC) domain of the mitochondrial contact site and cristae organizing system subunit Mic60 forms an elongated, bow tie-shaped tetrameric assembly. Mic19 promotes Mic60 tetramerization via a conserved interface between the Mic60 mitofilin and Mic19 CHCH (CC-helix-CC-helix) domains. Dimerization of mitofilin domains exposes a crescent-shaped membrane-binding site with convex curvature tailored to interact with the curved CJ neck. Our study suggests that the Mic60-Mic19 subcomplex traverses CJs as a molecular strut, thereby controlling CJ architecture and function.ISSN:2375-254

Structural insights into crista junction formation by the Mic60-Mic19 complex

Mitochondrial cristae membranes are the oxidative phosphorylation sites in cells. Crista junctions (CJs) form the highly curved neck regions of cristae and are thought to function as selective entry gates into the cristae space. Little is known about how CJs are generated and maintained. We show that the central coiled-coil (CC) domain of the mitochondrial contact site and cristae organizing system subunit Mic60 forms an elongated, bow tie-shaped tetrameric assembly. Mic19 promotes Mic60 tetramerization via a conserved interface between the Mic60 mitofilin and Mic19 CHCH (CC-helix-CC-helix) domains. Dimerization of mitofilin domains exposes a crescent-shaped membrane-binding site with convex curvature tailored to interact with the curved CJ neck. Our study suggests that the Mic60-Mic19 subcomplex traverses CJs as a molecular strut, thereby controlling CJ architecture and function.ISSN:2375-254

Unsupervised learning approaches to characterizing heterogeneous samples using X-ray single-particle imaging

One of the outstanding analytical problems in X-ray single-particle imaging (SPI) is the classification of structural heterogeneity, which is especially difficult given the low signal-to-noise ratios of individual patterns and the fact that even identical objects can yield patterns that vary greatly when orientation is taken into consideration. Proposed here are two methods which explicitly account for this orientation-induced variation and can robustly determine the structural landscape of a sample ensemble. The first, termed common-line principal component analysis (PCA), provides a rough classification which is essentially parameter free and can be run automatically on any SPI dataset. The second method, utilizing variation auto-encoders (VAEs), can generate 3D structures of the objects at any point in the structural landscape. Both these methods are implemented in combination with the noise-tolerant expand-maximizecompress (EMC) algorithm and its utility is demonstrated by applying it to an experimental dataset from gold nanoparticles with only a few thousand photons per pattern. Both discrete structural classes and continuous deformations are recovered. These developments diverge from previous approaches of extracting reproducible subsets of patterns from a dataset and open up the possibility of moving beyond the study of homogeneous sample sets to addressing open questions on topics such as nanocrystal growth and dynamics, as well as phase transitions which have not been externally triggered