Close proximities between organelles have been described for decades. However, only recently a specific field dealing with organelle communication at membrane contact sites has gained wide ...acceptance, attracting scientists from multiple areas of cell biology. The diversity of approaches warrants a unified vocabulary for the field. Such definitions would facilitate laying the foundations of this field, streamlining communication and resolving semantic controversies. This opinion, written by a panel of experts in the field, aims to provide this burgeoning area with guidelines for the experimental definition and analysis of contact sites. It also includes suggestions on how to operationally and tractably measure and analyze them with the hope of ultimately facilitating knowledge production and dissemination within and outside the field of contact-site research.
The studies addressing the molecular mechanisms governing mitochondrial fusion and fission have brought to light a small group of dynamin-like GTPases (Guanosine-Triphosphate hydrolase) as central ...regulators of mitochondrial morphology and cristae remodeling, apoptosis, calcium signaling, and metabolism. One of them is the mammalian OPA1 (Optic atrophy 1) protein, which resides inside the mitochondrion anchored to the inner membrane and, in a cleaved form, is associated to oligomeric complexes, in the intermembrane space of the organelle. Here, we review the studies that have made OPA1 emerge as the best understood regulator of mitochondrial inner membrane fusion and cristae remodeling. Further, we re-examine the findings behind the recent claim that OPA1 mediates adrenergic control of lipolysis by functioning as a cytosolic A-kinase anchoring protein (AKAP), on the hemimembrane that envelops the lipid droplet. This article is part of a Special Issue entitled: Mitochondrial dynamics and physiology.
► OPA1 is the best characterized mitochondrial fusion protein. ► Its structure and function are evolutionarily conserved. ► A complex proteolytic cascade governs its role in mitochondria dynamics, apoptosis, and quality control. ► The recently proposed localization of OPA1 on the lipid droplets, and function as an AKAP, remains controversial.
The BRCA2 tumour suppressor protein preserves genomic integrity via interactions with the DNA-strand exchange RAD51 protein in homology-directed repair. The RAD51-binding TR2 motif at the BRCA2 ...C-terminus is essential for protection and restart of stalled replication forks. Biochemical evidence shows that TR2 recognises filamentous RAD51, but existing models of TR2 binding to RAD51 lack a structural basis. Here we used cryo-electron microscopy and structure-guided mutagenesis to elucidate the mechanism of TR2 binding to nucleoprotein filaments of human RAD51. We find that TR2 binds across the protomer interface in the filament, acting as a brace for adjacent RAD51 molecules. TR2 targets an acidic-patch motif on human RAD51 that serves as a recruitment hub in fission yeast Rad51 for recombination mediators Rad52 and Rad55-Rad57. Our findings provide a structural rationale for RAD51 filament stabilisation by BRCA2 and reveal a common recruitment mechanism of recombination mediators to the RAD51 filament.
The mitochondrial contact site and cristae organizing system (MICOS) and Optic atrophy 1 (OPA1) control cristae shape, thus affecting mitochondrial function and apoptosis. Whether and how they ...physically and functionally interact is unclear. Here, we provide evidence that OPA1 is epistatic to MICOS in the regulation of cristae shape. Proteomic analysis identifies multiple MICOS components in native OPA1-containing high molecular weight complexes disrupted during cristae remodeling. MIC60, a core MICOS protein, physically interacts with OPA1, and together, they control cristae junction number and stability, OPA1 being epistatic to MIC60. OPA1 defines cristae width and junction diameter independently of MIC60. Our combination of proteomics, biochemistry, genetics, and electron tomography provides a unifying model for mammalian cristae biogenesis by OPA1 and MICOS.
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•Complexes containing OPA1 and MIC60 are targeted during cristae remodeling•OPA1 lies upstream of MIC60 in regulating cristae junction number and stability•OPA1 is the sole regulator of cristae junction width
Mitochondrial ultrastructure dictates mitochondrial function and cell viability. Glytsou et al. find that Optic Atrophy 1 (OPA1) and the MICOS component MIC60 interact in cristae shape regulation and place OPA1 epistatic to MIC60 in cristae biogenesis and remodeling, providing a unifying molecular model of cristae morphology control.
Cell division cycle protein 45 (Cdc45) is required for DNA synthesis during genome duplication, as a component of the Cdc45-MCM-GINS (CMG) helicase. Despite its essential biological function, its ...biochemical role in DNA replication has remained elusive. Here we report the 2.1-Å crystal structure of human Cdc45, which confirms its evolutionary link with the bacterial RecJ nuclease and reveals several unexpected features that underpin its function in eukaryotic DNA replication. These include a long-range interaction between N- and C-terminal DHH domains, blocking access to the DNA-binding groove of its RecJ-like fold, and a helical insertion in its N-terminal DHH domain, which appears poised for replisome interactions. In combination with available electron microscopy data, we validate by mutational analysis the mechanism of Cdc45 association with the MCM ring and GINS co-activator, critical for CMG assembly. These findings provide an indispensable molecular basis to rationalize the essential role of Cdc45 in genomic duplication.
Eukaryotic DNA replication is performed by the replisome, a large and dynamic multi-protein machine endowed with the required enzymatic components for the synthesis of new DNA. Recent cryo-electron ...microscopy (cryoEM) analyses have revealed the conserved architecture of the core eukaryotic replisome, comprising the CMG (Cdc45-MCM-GINS) DNA helicase, the leading-strand DNA polymerase epsilon, the Timeless-Tipin heterodimer, the hub protein AND-1 and the checkpoint protein Claspin. These results bid well for arriving soon at an integrated understanding of the structural basis of semi-discontinuous DNA replication. They further set the scene for the characterisation of the mechanisms that interface DNA synthesis with concurrent processes such as DNA repair, propagation of chromatin structure and establishment of sister chromatid cohesion.
•The core architecture of the eukaryotic replisome is built around the CMG DNA helicase and is conserved from yeast to humans.•The CMG helicase adopts an asymmetric rotary mode of DNA translocation that reflects the heteromeric ring of MCM2-7 ATPases.•The CMG helicase separates the strands of the parental DNA by a version of the steric exclusion mechanism.•Pol epsilon C-end binds the CMG trailing edge and its N-end engages the emerging leading-strand template for DNA synthesis.•In the replisome, Timeless and AND-1 are poised to engage with parental DNA and other replication factors ahead of the fork.
Contacts between organelles create microdomains that play major roles in regulating key intracellular activities and signaling pathways, but whether they also regulate systemic functions remains ...unknown. Here, we report the ultrastructural organization and dynamics of the inter-organellar contact established by sheets of curved rough endoplasmic reticulum closely wrapped around the mitochondria (wrappER). To elucidate the in vivo function of this contact, mouse liver fractions enriched in wrappER-associated mitochondria are analyzed by transcriptomics, proteomics, and lipidomics. The biochemical signature of the wrappER points to a role in the biogenesis of very-low-density lipoproteins (VLDL). Altering wrappER-mitochondria contacts curtails VLDL secretion and increases hepatic fatty acids, lipid droplets, and neutral lipid content. Conversely, acute liver-specific ablation of Mttp, the most upstream regulator of VLDL biogenesis, recapitulates this hepatic dyslipidemia phenotype and promotes remodeling of the wrappER-mitochondria contact. The discovery that liver wrappER-mitochondria contacts participate in VLDL biology suggests an involvement of inter-organelle contacts in systemic lipid homeostasis.
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•Liver mitochondria are extensively wrapped by curved sheets of rough ER (wrappER)•The wrappER is enriched in fatty acid and VLDL transcripts, proteins, and particles•WrappER-mitochondria contacts regulate VLDL synthesis and respond to lipid flux changes•WrappER-mitochondria contacts contain MAMs and sites of adhesion between organelles
Anastasia et al. characterize the contact established by a curved wrapping-type of rough ER (wrappER) with mitochondria and discover that this inter-organellar association is dynamic, regulated by lipid fluxes, and necessary for correct hepatic synthesis and secretion of very-low-density lipoproteins.
Hepatic metabolism requires mitochondria to adapt their bioenergetic and biosynthetic output to accompany the ever-changing anabolic/catabolic state of the liver cell, but the wiring of this process ...is still largely unknown. Using a postprandial mouse liver model and quantitative cryo-EM analysis, we show that when the hepatic mammalian target of rapamycin complex 1 (mTORCI) signaling pathway disengages, the mitochondria network fragments, cristae density drops by 30%, and mitochondrial respiratory capacity decreases by 20%. Instead, mitochondria-ER contacts (MERCs), which mediate calcium and phospholipid fluxes between these organelles, double in length. These events are associated with the transient expression of two previously unidentified C-terminal fragments (CTFs) of Optic atrophy 1 (Opa1), a mitochondrial GTPase that regulates cristae biogenesis and mitochondria dynamics. Expression of Opa1 CTFs in the intermembrane space has no effect on mitochondria morphology, supporting a model in which they are intermediates of an Opa1 degradation program. Using an in vitro assay, we show that these CTFs indeed originate from the cleavage of Opa1 at two evolutionarily conserved consensus sites that map within critical folds of the GTPase. This processing of Opa1, termed Gcleavage, is mediated by the activity of a cysteine protease whose activity is independent from that of Oma1 and presenilin-associated rhomboid-like (PARL), two known Opal regulators. However, C-deavage requires Mitofusin-2 (Mfn2), a key factor in mitochondria-ER tethering, thereby linking cristae remodeling to MERC assembly. Thus, in vivo, mitochondria adapt to metabolic shifts through the parallel remodeling of the cristae and of the MERCs via a mechanism that degrades Opa1 in an Mfn2-dependent pathway.
The synaptonemal complex (SC) is an evolutionarily-conserved protein assembly that holds together homologous chromosomes during prophase of the first meiotic division. Whilst essential for meiosis ...and fertility, the molecular structure of the SC has proved resistant to elucidation. The SC protein SYCP3 has a crucial but poorly understood role in establishing the architecture of the meiotic chromosome. Here we show that human SYCP3 forms a highly-elongated helical tetramer of 20 nm length. N-terminal sequences extending from each end of the rod-like structure bind double-stranded DNA, enabling SYCP3 to link distant sites along the sister chromatid. We further find that SYCP3 self-assembles into regular filamentous structures that resemble the known morphology of the SC lateral element. Together, our data form the basis for a model in which SYCP3 binding and assembly on meiotic chromosomes leads to their organisation into compact structures compatible with recombination and crossover formation.
Rhomboids, evolutionarily conserved integral membrane proteases, participate in crucial signaling pathways. Presenilin-associated rhomboid-like (PARL) is an inner mitochondrial membrane rhomboid of ...unknown function, whose yeast ortholog is involved in mitochondrial fusion.
Parl
−/− mice display normal intrauterine development but from the fourth postnatal week undergo progressive multisystemic atrophy leading to cachectic death. Atrophy is sustained by increased apoptosis, both in and ex vivo.
Parl
−/− cells display normal mitochondrial morphology and function but are no longer protected against intrinsic apoptotic death stimuli by the dynamin-related mitochondrial protein OPA1.
Parl
−/− mitochondria display reduced levels of a soluble, intermembrane space (IMS) form of OPA1, and OPA1 specifically targeted to IMS complements
Parl
−/− cells, substantiating the importance of PARL in OPA1 processing.
Parl
−/− mitochondria undergo faster apoptotic cristae remodeling and cytochrome c release. These findings implicate regulated intramembrane proteolysis in controlling apoptosis.
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