Mitofusin-mediated contacts between mitochondria and peroxisomes regulate mitochondrial fusion.

Mitofusins are large GTPases that trigger fusion of mitochondrial outer membranes. Similarly to the human mitofusin Mfn2, which also tethers mitochondria to the endoplasmic reticulum (ER), the yeast mitofusin Fzo1 stimulates contacts between Peroxisomes and Mitochondria when overexpressed. Yet, the physiological significance and function of these "PerMit" contacts remain unknown. Here, we demonstrate that Fzo1 naturally localizes to peroxisomes and promotes PerMit contacts in physiological conditions. These contacts are regulated through co-modulation of Fzo1 levels by the ubiquitin-proteasome system (UPS) and by the desaturation status of fatty acids (FAs). Contacts decrease under low FA desaturation but reach a maximum during high FA desaturation. High-throughput genetic screening combined with high-resolution cellular imaging reveal that Fzo1-mediated PerMit contacts favor the transit of peroxisomal citrate into mitochondria. In turn, citrate enters the TCA cycle to stimulate the mitochondrial membrane potential and maintain efficient mitochondrial fusion upon high FA desaturation. These findings thus unravel a mechanism by which inter-organelle contacts safeguard mitochondrial fusion.

Single-fluorophore biosensors based on conformation-sensitive GFP variants.

The ß-strands of GFP form a rigid barrel that protects the chromophore from external influence. Herein, we identified specific mutations in ß-strand 7 that render the chromophore sensitive to interactions of GFP with another protein domain. In the process of converting the FRET-based protein kinase A (PKA) sensor AKAR2 into a single-wavelength PKA sensor containing a GFP and a quencher, we discovered that the quencher was not required and that the sensor response relied on changes in GFP intrinsic fluorescence. The identified mutations in ß-strand 7 render GFP fluorescence intensity and lifetime sensitive to conformational changes of the PKA-sensing domain. In addition, sensors engineered from the GCaMP2 calcium indicator to incorporate a conformation-sensitive GFP (csGFP) exhibited calcium-dependent fluorescence changes. We further demonstrate that single GFP sensors report PKA dynamics in dendritic spines of neurons from brain slices on 2-photon imaging with a high signal-to-baseline ratio and minimal photobleaching. The susceptibility of GFP variants to dynamic interactions with other protein domains provides a new approach to generate single wavelength biosensors for high-resolution imaging.

Type 4 phosphodiesterase plays different integrating roles in different cellular domains in pyramidal cortical neurons.

We investigated the role of phosphodiesterases (PDEs) in the integration of cAMP signals and protein kinase A (PKA) activity following beta-adrenergic stimulation, by carrying out real-time imaging of male mouse pyramidal cortical neurons expressing biosensors to monitor cAMP levels (Epac1-camps and Epac2-camps300) or PKA activity (AKAR2). In the soma, isoproterenol (ISO) increased the PKA signal to approximately half the maximal response obtained with forskolin, with a characteristic beta(1) pharmacology and an EC(50) of 4.5 nm. This response was related to free cAMP levels in the submicromolar range. The specific type 4 PDE (PDE4) inhibitor rolipram had a very small effect alone, but strongly potentiated the PKA response to ISO. Blockers of other PDEs had no effect. PDE4 thus acts as a brake in the propagation of the beta(1)-adrenergic signal from the membrane to the bulk somatic cytosol. The results for a submembrane domain were markedly different, whether recorded with a PKA-sensitive potassium current related to the slow AHP or by two-photon imaging of small distal dendrites. The responses to ISO were stronger than in the bulk cytosol. This is consistent with the cAMP/PKA signal being strong at the membrane, as shown by electrophysiology, and favored in cellular domains with a high surface area to volume ratio, in which this signal was detected by imaging. Rolipram alone also produced a strong cAMP/PKA signal, revealing tonic cAMP production. PDE4 thus appears as a crucial integrator with different physiological implications in different subcellular domains.

Development and evaluation of robust molecular markers linked to disease resistance in tomato for distinctness, uniformity and stability testing.

Molecular markers linked to phenotypically important traits are of great interest especially when traits are difficult and/or costly to be observed. In tomato where a strong focus on resistance breeding has led to the introgression of several resistance genes, resistance traits have become important characteristics in distinctness, uniformity and stability (DUS) testing for Plant Breeders Rights (PBR) applications. Evaluation of disease traits in biological assays is not always straightforward because assays are often influenced by environmental factors, and difficulties in scoring exist. In this study, we describe the development and/or evaluation of molecular marker assays for the Verticillium genes Ve1 and Ve2, the tomato mosaic virus Tm1 (linked marker), the tomato mosaic virus Tm2 and Tm2 ( 2 ) genes, the Meloidogyne incognita Mi1-2 gene, the Fusarium I (linked marker) and I2 loci, which are obligatory traits in PBR testing. The marker assays were evaluated for their robustness in a ring test and then evaluated in a set of varieties. Although in general, results between biological assays and marker assays gave highly correlated results, marker assays showed an advantage over biological tests in that the results were clearer, i.e., homozygote/heterozygote presence of the resistance gene can be detected and heterogeneity in seed lots can be identified readily. Within the UPOV framework for granting of PBR, the markers have the potential to fulfil the requirements needed for implementation in DUS testing of candidate varieties and could complement or may be an alternative to the pathogenesis tests that are carried out at present.

The regulation of mitochondrial homeostasis by the ubiquitin proteasome system.

From mitochondrial quality control pathways to the regulation of specific functions, the Ubiquitin Proteasome System (UPS) could be compared to a Swiss knife without which mitochondria could not maintain its integrity in the cell. Here, we review the mechanisms that the UPS employs to regulate mitochondrial function and efficiency. For this purpose, we depict how Ubiquitin and the Proteasome participate in diverse quality control pathways that safeguard entry into the mitochondrial compartment. A focus is then achieved on the UPS-mediated control of the yeast mitofusin Fzo1 which provides insights into the complex regulation of this particular protein in mitochondrial fusion. We ultimately dissect the mechanisms by which the UPS controls the degradation of mitochondria by autophagy in both mammalian and yeast systems. This organization should offer a useful overview of this abundant but fascinating literature on the crosstalks between mitochondria and the UPS.

Use of a robotic RNA purification protocol based on the NucliSens easyMAG for real-time RT-PCR detection of hepatitis A virus in bottled water.

Hepatitis A virus (HAV) infection is the leading cause of acute viral hepatitis throughout the world. An important part of viral control is rapid detection of HAV in drinking water contaminated with feces. One critical step in HAV detection methods is sample preparation. The objective of this study was to test the efficacy of different approaches to extracting RNA from HAV-inoculated bottled water. The optimal method is based on viral concentration by filtration on membrane filters and elution of adsorbed viruses from filters before RNA extraction and RT-PCR amplification. In this approach, the commercially available NucliSens easyMAG bio-robot (Biomérieux) performs viral RNA purification with silica magnetic beads, which mediate purification of nucleic acids by binding them and allowing other substances to be washed away. A new rapid simplified NucliSens easyMAG-based approach is described and compared with the classical NucliSens easyMAG approach and with manual silica-based spin column purifications (Qiagen). The limit of detection (LOD) with the new rapid simplified NucliSens easyMAG approach was about 1PFU/1.5L against about 100PFU/1.5L using conventional sample treatments that included a concentration step using ultra-filtration.

Systematic mapping of contact sites reveals tethers and a function for the peroxisome-mitochondria contact.

The understanding that organelles are not floating in the cytosol, but rather held in an organized yet dynamic interplay through membrane contact sites, is altering the way we grasp cell biological phenomena. However, we still have not identified the entire repertoire of contact sites, their tethering molecules and functions. To systematically characterize contact sites and their tethering molecules here we employ a proximity detection method based on split fluorophores and discover four potential new yeast contact sites. We then focus on a little-studied yet highly disease-relevant contact, the Peroxisome-Mitochondria (PerMit) proximity, and uncover and characterize two tether proteins: Fzo1 and Pex34. We genetically expand the PerMit contact site and demonstrate a physiological function in ß-oxidation of fatty acids. Our work showcases how systematic analysis of contact site machinery and functions can deepen our understanding of these structures in health and disease.

Ubiquitination of ERMES components by the E3 ligase Rsp5 is involved in mitophagy.

Mitochondria are dynamic organelles that undergo permanent fission and fusion events. These processes play an essential role in maintaining normal cellular function. In the yeast Saccharomyces cerevisiae, the endoplasmic reticulum-mitochondrial encounter structure (ERMES) is a marker of sites of mitochondrial division, but it is also involved in a plethora of other mitochondrial functions. However, it remains unclear how these different functions are regulated. We show here that Mdm34 and Mdm12, 2 components of ERMES, are ubiquitinated by the E3 ligase Rsp5. This ubiquitination is not involved in mitochondrial dynamics or in the distribution and turnover of ERMES. Nevertheless, the ubiquitination of Mdm34 and Mdm12 was required for efficient mitophagy. We thus report here the first identification of ubiquitinated substrates participating in yeast mitophagy.

Analysis of Mitochondrial Membrane Protein Complexes by Electron Cryo-tomography.

The visualization of membrane protein complexes in their natural membrane environment is a major goal in an emerging area of research termed structural cell biology. Such approaches provide important information on the spatial distribution of protein complexes in their resident cellular membrane systems and on the structural organization of multi-subunit membrane protein assemblies. We have developed a method to specifically label active membrane protein complexes in their native membrane environment with electron-dense nanoparticles coupled to an activating ligand, in order to visualize them by electron cryo-tomography. As an example, we describe here the depiction of preprotein import sites of mitochondria, formed by the translocase of the outer membrane (TOM complex) and the presequence translocase of the inner membrane (TIM23 complex). Active import sites are selectively labeled via a biotinylated, quantum dot-coupled preprotein that is arrested in translocation across the outer and inner mitochondrial membranes. Additionally, a related method is described for direct labeling of mitochondrial outer membrane proteins that does not depend on binding of a ligand.

A membrane-inserted structural model of the yeast mitofusin Fzo1.

Mitofusins are large transmembrane GTPases of the dynamin-related protein family, and are required for the tethering and fusion of mitochondrial outer membranes. Their full-length structures remain unknown, which is a limiting factor in the study of outer membrane fusion. We investigated the structure and dynamics of the yeast mitofusin Fzo1 through a hybrid computational and experimental approach, combining molecular modelling and all-atom molecular dynamics simulations in a lipid bilayer with site-directed mutagenesis and in vivo functional assays. The predicted architecture of Fzo1 improves upon the current domain annotation, with a precise description of the helical spans linked by flexible hinges, which are likely of functional significance. In vivo site-directed mutagenesis validates salient aspects of this model, notably, the long-distance contacts and residues participating in hinges. GDP is predicted to interact with Fzo1 through the G1 and G4 motifs of the GTPase domain. The model reveals structural determinants critical for protein function, including regions that may be involved in GTPase domain-dependent rearrangements.

An ubiquitin-dependent balance between mitofusin turnover and fatty acids desaturation regulates mitochondrial fusion.

Mitochondrial integrity relies on homotypic fusion between adjacent outer membranes, which is mediated by large GTPases called mitofusins. The regulation of this process remains nonetheless elusive. Here, we report a crosstalk between the ubiquitin protease Ubp2 and the ubiquitin ligases Mdm30 and Rsp5 that modulates mitochondrial fusion. Ubp2 is an antagonist of Rsp5, which promotes synthesis of the fatty acids desaturase Ole1. We show that Ubp2 also counteracts Mdm30-mediated turnover of the yeast mitofusin Fzo1 and that Mdm30 targets Ubp2 for degradation thereby inducing Rsp5-mediated desaturation of fatty acids. Exogenous desaturated fatty acids inhibit Ubp2 degradation resulting in higher levels of Fzo1 and maintenance of efficient mitochondrial fusion. Our results demonstrate that the Mdm30-Ubp2-Rsp5 crosstalk regulates mitochondrial fusion by coordinating an intricate balance between Fzo1 turnover and the status of fatty acids saturation. This pathway may link outer membrane fusion to lipids homeostasis.

A mitofusin-dependent docking ring complex triggers mitochondrial fusion in vitro.

Fusion of mitochondrial outer membranes is crucial for proper organelle function and involves large GTPases called mitofusins. The discrete steps that allow mitochondria to attach to one another and merge their outer membranes are unknown. By combining an in vitro mitochondrial fusion assay with electron cryo-tomography (cryo-ET), we visualize the junction between attached mitochondria isolated from Saccharomyces cerevisiae and observe complexes that mediate this attachment. We find that cycles of GTP hydrolysis induce progressive formation of a docking ring structure around extended areas of contact. Further GTP hydrolysis triggers local outer membrane fusion at the periphery of the contact region. These findings unravel key features of mitofusin-dependent fusion of outer membranes and constitute an important advance in our understanding of how mitochondria connect and merge.