Visualization and Quantification of Endogenous Intra-Organelle Protein Interactions at ER-Mitochondria Contact Sites by Proximity Ligation Assays.

Membrane contact sites (MCSs) are areas of close membrane proximity that allow and regulate the dynamic exchange of diverse biomolecules (i.e., calcium and lipids) between the juxtaposed organelles without involving membrane fusion. MCSs are essential for cellular homeostasis, and their functions are ensured by the resident components, which often exist as multimeric protein complexes. MCSs often involve the endoplasmic reticulum (ER), a major site of lipid synthesis and cellular calcium storage, and are particularly important for organelles, such as the mitochondria, which are excluded from the classical vesicular transport pathways. In the last years, MCSs between the ER and mitochondria have been extensively studied, as their functions strongly impact cellular metabolism/bioenergetics. Several proteins have started to be identified at these contact sites, including membrane tethers, calcium channels, and lipid transfer proteins, thus raising the need for new methodologies and technical approaches to study these MCS components. Here, we describe a protocol consisting of combined technical approaches, that include proximity ligation assay (PLA), mitochondria staining, and 3D imaging segmentation, that allows the detection of proteins that are physically close (>40 nm) to each other and that reside on the same membrane at ER-mitochondria MCSs. For instance, we used two ER-anchored lipid transfer proteins, ORP5 and ORP8, which have previously been shown to interact and localize at ER-mitochondria and ER-plasma membrane MCSs. By associating the ORP5-ORP8 PLA with cell imaging software analysis, it was possible to estimate the distance of the ORP5-ORP8 complex from the mitochondrial surface and determine that about 50% of ORP5-ORP8 PLA interaction occurs at ER subdomains in close proximity to mitochondria.

Rabies virus P protein binds to TBK1 and interferes with the formation of innate immunity-related liquid condensates.

Viruses must overcome the interferon-mediated antiviral response to replicate and propagate into their host. Rabies virus (RABV) phosphoprotein P is known to inhibit interferon induction. Here, using a global mass spectrometry approach, we show that RABV P binds to TBK1, a kinase located at the crossroads of many interferon induction pathways, resulting in innate immunity inhibition. Mutations of TBK1 phosphorylation sites abolish P binding. Importantly, we demonstrate that upon RABV infection or detection of dsRNA by innate immunity sensors, TBK1 and its adaptor proteins NAP1 and SINTBAD form dynamic cytoplasmic condensates that have liquid properties. These condensates can form larger aggregates having ring-like structures in which NAP1 and TBK1 exhibit locally restricted movement. P binding to TBK1 interferes with the formation of these structures. This work demonstrates that proteins of the signaling pathway leading to interferon induction transiently form liquid organelles that can be targeted by viruses.

Properties of rabies virus phosphoprotein and nucleoprotein biocondensates formed in vitro and in cellulo.

Rabies virus (RABV) transcription and replication take place within viral factories having liquid properties, called Negri bodies (NBs), that are formed by liquid-liquid phase separation (LLPS). The co-expression of RABV nucleoprotein (N) and phosphoprotein (P) in mammalian cells is sufficient to induce the formation of cytoplasmic biocondensates having properties that are like those of NBs. This cellular minimal system was previously used to identify P domains that are essential for biocondensates formation. Here, we constructed fluorescent versions of N and analyzed by FRAP their dynamics inside the biocondensates formed in this minimal system as well as in NBs of RABV-infected cells using FRAP. The behavior of N appears to be different of P as there was no fluorescence recovery of N proteins after photobleaching. We also identified arginine residues as well as two exposed loops of N involved in condensates formation. Corresponding N mutants exhibited distinct phenotypes in infected cells ranging from co-localization with NBs to exclusion from them associated with a dominant-negative effect on infection. We also demonstrated that in vitro, in crowded environments, purified P as well as purified N0-P complex (in which N is RNA-free) form liquid condensates. We identified P domains required for LLPS in this acellular system. P condensates were shown to associate with liposomes, concentrate RNA, and undergo a liquid-gel transition upon ageing. Conversely, N0-P droplets were disrupted upon incubation with RNA. Taken together, our data emphasize the central role of P in NBs formation and reveal some physicochemical features of P and N0-P droplets relevant for explaining NBs properties such as their envelopment by cellular membranes at late stages of infection and nucleocapsids ejections from the viral factories.

ORP5/8 and MIB/MICOS link ER-mitochondria and intra-mitochondrial contacts for non-vesicular transport of phosphatidylserine.

Mitochondria are dynamic organelles essential for cell survival whose structural and functional integrity rely on selective and regulated transport of lipids from/to the endoplasmic reticulum (ER) and across the mitochondrial intermembrane space. As they are not connected by vesicular transport, the exchange of lipids between ER and mitochondria occurs at membrane contact sites. However, the mechanisms and proteins involved in these processes are only beginning to emerge. Here, we show that the main physiological localization of the lipid transfer proteins ORP5 and ORP8 is at mitochondria-associated ER membrane (MAM) subdomains, physically linked to the mitochondrial intermembrane space bridging (MIB)/mitochondrial contact sites and cristae junction organizing system (MICOS) complexes that bridge the two mitochondrial membranes. We also show that ORP5/ORP8 mediate non-vesicular transport of phosphatidylserine (PS) lipids from the ER to mitochondria by cooperating with the MIB/MICOS complexes. Overall our study reveals a physical and functional link between ER-mitochondria contacts involved in lipid transfer and intra-mitochondrial membrane contacts maintained by the MIB/MICOS complexes.

Phospho-Ku70 induced by DNA damage interacts with RNA Pol II and promotes the formation of phospho-53BP1 foci to ensure optimal cNHEJ.

Canonical non-homologous end-joining (cNHEJ) is the prominent mammalian DNA double-strand breaks (DSBs) repair pathway operative throughout the cell cycle. Phosphorylation of Ku70 at ser27-ser33 (pKu70) is induced by DNA DSBs and has been shown to regulate cNHEJ activity, but the underlying mechanism remained unknown. Here, we established that following DNA damage induction, Ku70 moves from nucleoli to the sites of damage, and once linked to DNA, it is phosphorylated. Notably, the novel emanating functions of pKu70 are evidenced through the recruitment of RNA Pol II and concomitant formation of phospho-53BP1 foci. Phosphorylation is also a prerequisite for the dynamic release of Ku70 from the repair complex through neddylation-dependent ubiquitylation. Although the non-phosphorylable ala-Ku70 form does not compromise the formation of the NHEJ core complex per se, cells expressing this form displayed constitutive and stress-inducible chromosomal instability. Consistently, upon targeted induction of DSBs by the I-SceI meganuclease into an intrachromosomal reporter substrate, cells expressing pKu70, rather than ala-Ku70, are protected against the joining of distal DNA ends. Collectively, our results underpin the essential role of pKu70 in the orchestration of DNA repair execution in living cells and substantiated the way it paves the maintenance of genome stability.

Sinorhizobium meliloti Functions Required for Resistance to Antimicrobial NCR Peptides and Bacteroid Differentiation.

Legumes of the Medicago genus have a symbiotic relationship with the bacterium Sinorhizobium meliloti and develop root nodules housing large numbers of intracellular symbionts. Members of the nodule-specific cysteine-rich peptide (NCR) family induce the endosymbionts into a terminal differentiated state. Individual cationic NCRs are antimicrobial peptides that have the capacity to kill the symbiont, but the nodule cell environment prevents killing. Moreover, the bacterial broad-specificity peptide uptake transporter BacA and exopolysaccharides contribute to protect the endosymbionts against the toxic activity of NCRs. Here, we show that other S. meliloti functions participate in the protection of the endosymbionts; these include an additional broad-specificity peptide uptake transporter encoded by the yejABEF genes and lipopolysaccharide modifications mediated by lpsB and lpxXL, as well as rpoH1, encoding a stress sigma factor. Strains with mutations in these genes show a strain-specific increased sensitivity profile against a panel of NCRs and form nodules in which bacteroid differentiation is affected. The lpsB mutant nodule bacteria do not differentiate, the lpxXL and rpoH1 mutants form some seemingly fully differentiated bacteroids, although most of the nodule bacteria are undifferentiated, while the yejABEF mutants form hypertrophied but nitrogen-fixing bacteroids. The nodule bacteria of all the mutants have a strongly enhanced membrane permeability, which is dependent on the transport of NCRs to the endosymbionts. Our results suggest that S. meliloti relies on a suite of functions, including peptide transporters, the bacterial envelope structures, and stress response regulators, to resist the aggressive assault of NCR peptides in the nodule cells. IMPORTANCE The nitrogen-fixing symbiosis of legumes with rhizobium bacteria has a predominant ecological role in the nitrogen cycle and has the potential to provide the nitrogen required for plant growth in agriculture. The host plants allow the rhizobia to colonize specific symbiotic organs, the nodules, in large numbers in order to produce sufficient reduced nitrogen for the plants' needs. Some legumes, including Medicago spp., produce massively antimicrobial peptides to keep this large bacterial population in check. These peptides, known as NCRs, have the potential to kill the rhizobia, but in nodules, they rather inhibit the division of the bacteria, which maintain a high nitrogen-fixing activity. In this study, we show that the tempering of the antimicrobial activity of the NCR peptides in the Medicago symbiont Sinorhizobium meliloti is multifactorial and requires the YejABEF peptide transporter, the lipopolysaccharide outer membrane, and the stress response regulator RpoH1.

Bradyrhizobium diazoefficiens USDA110 Nodulation of Aeschynomene afraspera Is Associated with Atypical Terminal Bacteroid Differentiation and Suboptimal Symbiotic Efficiency.

Legume plants can form root organs called nodules where they house intracellular symbiotic rhizobium bacteria. Within nodule cells, rhizobia differentiate into bacteroids, which fix nitrogen for the benefit of the plant. Depending on the combination of host plants and rhizobial strains, the output of rhizobium-legume interactions varies from nonfixing associations to symbioses that are highly beneficial for the plant. Bradyrhizobium diazoefficiens USDA110 was isolated as a soybean symbiont, but it can also establish a functional symbiotic interaction with Aeschynomene afraspera In contrast to soybean, A. afraspera triggers terminal bacteroid differentiation, a process involving bacterial cell elongation, polyploidy, and increased membrane permeability, leading to a loss of bacterial viability while plants increase their symbiotic benefit. A combination of plant metabolomics, bacterial proteomics, and transcriptomics along with cytological analyses were used to study the physiology of USDA110 bacteroids in these two host plants. We show that USDA110 establishes a poorly efficient symbiosis with A. afraspera despite the full activation of the bacterial symbiotic program. We found molecular signatures of high levels of stress in A. afraspera bacteroids, whereas those of terminal bacteroid differentiation were only partially activated. Finally, we show that in A. afraspera, USDA110 bacteroids undergo atypical terminal differentiation hallmarked by the disconnection of the canonical features of this process. This study pinpoints how a rhizobium strain can adapt its physiology to a new host and cope with terminal differentiation when it did not coevolve with such a host.IMPORTANCE Legume-rhizobium symbiosis is a major ecological process in the nitrogen cycle, responsible for the main input of fixed nitrogen into the biosphere. The efficiency of this symbiosis relies on the coevolution of the partners. Some, but not all, legume plants optimize their return on investment in the symbiosis by imposing on their microsymbionts a terminal differentiation program that increases their symbiotic efficiency but imposes a high level of stress and drastically reduces their viability. We combined multi-omics with physiological analyses to show that the symbiotic couple formed by Bradyrhizobium diazoefficiens USDA110 and Aeschynomene afraspera, in which the host and symbiont did not evolve together, is functional but displays a low symbiotic efficiency associated with a disconnection of terminal bacteroid differentiation features.

Class I phosphoinositide 3-kinases control sustained NADPH oxidase activation in adherent neutrophils.

Phagocytes, especially neutrophils, can produce reactive oxygen species (ROS), through the activation of the NADPH oxidase (NOX2). Although this enzyme is crucial for host-pathogen defense, ROS production by neutrophils can be harmful in several pathologies such as cardiovascular diseases or chronic pulmonary diseases. The ROS production by NOX2 involves the assembly of the cytosolic subunits (p67phox, p47phox, and p40phox) and Rac with the membrane subunits (gp91phox and p22phox). Many studies are devoted to the activation of NOX2. However, the mechanisms that cause NADPH oxidase deactivation and thus terminate ROS production are not well known. Here we investigated the ability of class I phosphoinositide 3-kinases (PI3Ks) to sustain NADPH oxidase activation. The NADPH oxidase activation was triggered by seeding neutrophil-like PLB-985 cells, or human neutrophils on immobilized fibrinogen. Adhesion of the neutrophils, mediated by ß2 integrins, induced activation of the NADPH oxidase and translocation of the cytosolic subunits at the plasma membrane. Inhibition of class I PI3Ks, and especially PI3Kß, terminated ROS production. This deactivation of NOX2 is due to the release of the cytosolic subunits, p67phox and p47phox from the plasma membrane. Overexpression of an active form of Rac 1 did not prevent the drop of ROS production upon inhibition of class I PI3Ks. Moreover, the phosphorylation of p47phox at S328, a potential target of kinases activated by the PI3K pathway, was unchanged. Our results indicate that the experimental downregulation of class I PI3K products triggers the plasma membrane NADPH oxidase deactivation. Release of p47phox from the plasma membrane may involve its PX domains that bind PI3K products.

Functional diversification of Paramecium Ku80 paralogs safeguards genome integrity during precise programmed DNA elimination.

Gene duplication and diversification drive the emergence of novel functions during evolution. Because of whole genome duplications, ciliates from the Paramecium aurelia group constitute a remarkable system to study the evolutionary fate of duplicated genes. Paramecium species harbor two types of nuclei: a germline micronucleus (MIC) and a somatic macronucleus (MAC) that forms from the MIC at each sexual cycle. During MAC development, ~45,000 germline Internal Eliminated Sequences (IES) are excised precisely from the genome through a 'cut-and-close' mechanism. Here, we have studied the P. tetraurelia paralogs of KU80, which encode a key DNA double-strand break repair factor involved in non-homologous end joining. The three KU80 genes have different transcription patterns, KU80a and KU80b being constitutively expressed, while KU80c is specifically induced during MAC development. Immunofluorescence microscopy and high-throughput DNA sequencing revealed that Ku80c stably anchors the PiggyMac (Pgm) endonuclease in the developing MAC and is essential for IES excision genome-wide, providing a molecular explanation for the previously reported Ku-dependent licensing of DNA cleavage at IES ends. Expressing Ku80a under KU80c transcription signals failed to complement a depletion of endogenous Ku80c, indicating that the two paralogous proteins have distinct properties. Domain-swap experiments identified the α/ß domain of Ku80c as the major determinant for its specialized function, while its C-terminal part is required for excision of only a small subset of IESs located in IES-dense regions. We conclude that Ku80c has acquired the ability to license Pgm-dependent DNA cleavage, securing precise DNA elimination during programmed rearrangements. The present study thus provides novel evidence for functional diversification of genes issued from a whole-genome duplication.

Three-Dimensional Surface Rendering of ESCRT Proteins Microscopy Data Using UCSF Chimera Software.

Visualization of subcellular localization of ESCRT proteins and their interactions with different cellular compartments are critical to understand their function. This approach requires the generation of an important amount of 3D fluorescence microscopy data that is not always easy to visualize and apprehend.We describe a step-by-step protocol for 3D surface rendering of confocal microscopy acquisitions using the free software UCSF-Chimera, generating snapshots and animations to facilitate analysis and presentation of subcellular localization data.

Autophagy-related approaches for improving nutrient use efficiency and crop yield protection.

Autophagy is a eukaryotic catabolic pathway essential for growth and development. In plants, it is activated in response to environmental cues or developmental stimuli. However, in contrast to other eukaryotic systems, we know relatively little regarding the molecular players involved in autophagy and the regulation of this complex pathway. In the framework of the COST (European Cooperation in Science and Technology) action TRANSAUTOPHAGY (2016-2020), we decided to review our current knowledge of autophagy responses in higher plants, with emphasis on knowledge gaps. We also assess here the potential of translating the acquired knowledge to improve crop plant growth and development in a context of growing social and environmental challenges for agriculture in the near future.

Multiscale and Multimodal Approaches to Study Autophagy in Model Plants.

Autophagy is a catabolic process used by eukaryotic cells to maintain or restore cellular and organismal homeostasis. A better understanding of autophagy in plant biology could lead to an improvement of the recycling processes of plant cells and thus contribute, for example, towards reducing the negative ecological consequences of nitrogen-based fertilizers in agriculture. It may also help to optimize plant adaptation to adverse biotic and abiotic conditions through appropriate plant breeding or genetic engineering to incorporate useful traits in relation to this catabolic pathway. In this review, we describe useful protocols for studying autophagy in the plant cell, taking into account some specificities of the plant model.

Negri bodies are viral factories with properties of liquid organelles.

Replication of Mononegavirales occurs in viral factories which form inclusions in the host-cell cytoplasm. For rabies virus, those inclusions are called Negri bodies (NBs). We report that NBs have characteristics similar to those of liquid organelles: they are spherical, they fuse to form larger structures, and they disappear upon hypotonic shock. Their liquid phase is confirmed by FRAP experiments. Live-cell imaging indicates that viral nucleocapsids are ejected from NBs and transported along microtubules to form either new virions or secondary viral factories. Coexpression of rabies virus N and P proteins results in cytoplasmic inclusions recapitulating NBs properties. This minimal system reveals that an intrinsically disordered domain and the dimerization domain of P are essential for Negri bodies-like structures formation. We suggest that formation of liquid viral factories by phase separation is common among Mononegavirales and allows specific recruitment and concentration of viral proteins but also the escape to cellular antiviral response.Negative strand RNA viruses, such as rabies virus, induce formation of cytoplasmic inclusions for genome replication. Here, Nikolic et al. show that these so-called Negri bodies (NBs) have characteristics of liquid organelles and they identify the minimal protein domains required for NB formation.

Optimizing CLEM protocols for plants cells: GMA embedding and cryosections as alternatives for preservation of GFP fluorescence in Arabidopsis roots.

Recently, a number of diverse correlative light and electron microscopy (CLEM) protocols have been developed for several model organisms. However, these CLEM methods have largely bypassed plant cell research, with most protocols having little application to plants. Using autophagosome identification as a biological background, we propose and compare two CLEM protocols that can be performed in most plant research laboratories, providing a good compromise that preserves fluorescent signals as well as ultrastructural features. These protocols are based on either the adaptation of a high pressure fixation/GMA acrylic resin embedding method, or on the Tokuyasu approach. Both protocols suitably preserved GFP fluorescence while allowing the observation of cell ultrastructure in plants. Finally, the advantages and disadvantages of these protocols are discussed in the context of multiscale imaging of plant cells.

Phosphoinositol 3-phosphate acts as a timer for reactive oxygen species production in the phagosome.

Production of reactive oxygen species (ROS) in the phagosome by the NADPH oxidase is critical for mammalian immune defense against microbial infections and phosphoinositides are important regulators in this process. Phosphoinositol 3-phosphate (PI(3)P) regulates ROS production at the phagosome via p40phox by an unknown mechanism. This study tested the hypothesis that PI(3)P controls ROS production by regulating the presence of p40phox and p67phox at the phagosomal membrane. Pharmacologic inhibition of PI(3)P synthesis at the phagosome decreased the ROS production both in differentiated PLB-985 cells and human neutrophils. It also releases p67phox, the key cytosolic subunit of the oxidase, and p40phox from the phagosome. The knockdown of the PI(3)P phosphatase MTM1 or Rubicon or both increases the level of PI(3)P at the phagosome. That increase enhances ROS production inside the phagosome and triggers an extended accumulation of p67phox at the phagosome. Furthermore, the overexpression of MTM1 at the phagosomal membrane induces the disappearance of PI(3)P from the phagosome and prevents sustained ROS production. In conclusion, PI(3)P, indeed, regulates ROS production by maintaining p40phox and p67phox at the phagosomal membrane.

Cell division licensing in the multi-chromosomal Vibrio cholerae bacterium.

Cell division must be coordinated with chromosome replication and segregation to ensure the faithful transmission of genetic information during proliferation. In most bacteria, assembly of the division apparatus, the divisome, starts with the polymerization of a tubulin homologue, FtsZ, into a ring-like structure at mid-cell, the Z-ring(1). It typically occurs at half of the cell cycle when most of the replication and segregation cycle of the unique chromosome they generally harbour is achieved(2). The chromosome itself participates in the regulation of cell division, at least in part because it serves as a scaffold to position FtsZ polymerization antagonists(3). However, about 10% of bacteria have more than one chromosome(4), which raises questions about the way they license cell division(3). For instance, the genome of Vibrio cholerae, the agent of cholera, is divided between a 3 Mbp replicon that originates from the chromosome of its mono-chromosomal ancestor, Chr1, and a 1 Mbp plasmid-derived replicon, Chr2 (ref. 5). Here, we show that Chr2 harbours binding motifs for an inhibitor of Z-ring formation, which helps accurately position the V. cholerae divisome at mid-cell and postpones its assembly to the very end of the cell cycle.

Folding into an autophagosome: ATG5 sheds light on how plants do it.

Autophagosomes arise in yeast and animals from the sealing of a cup-shaped double-membrane precursor, the phagophore. The concerted action of about 30 evolutionarily conserved autophagy related (ATG) proteins lies at the core of this process. However, the mechanisms allowing phagophore generation and its differentiation into a sealed autophagosome are still not clear in detail, and very little is known in plants. This is due in part to the scarcity of structurally informative, real-time imaging data of ATG proteins at the phagophore site. Among these, the ATG5 complex directs anchoring of ATG8 to the phagophore, an event required for membrane expansion. Detailed real-time and 3D imaging of ATG5, ATG8, and an ER marker at the expanding phagophore allowed us to propose a model for autophagosome formation in plants. This model implies tight connections of the growing phagophore with the outer face of the cortical endoplasmic reticulum and prompts new questions on the mechanism of autophagosome biogenesis.

ATG5 defines a phagophore domain connected to the endoplasmic reticulum during autophagosome formation in plants.

Autophagosomes are the organelles responsible for macroautophagy and arise, in yeast and animals, from the sealing of a cup-shaped double-membrane precursor, the phagophore. How the phagophore is generated and grows into a sealed autophagosome is still not clear in detail, and unknown in plants. This is due, in part, to the scarcity of structurally informative, real-time imaging data of the required protein machinery at the phagophore formation site. Here we find that in intact living Arabidopsis tissue, autophagy-related protein ATG5, which is essential for autophagosome formation, is present at the phagophore site from early, sub-resolution stages and later defines a torus-shaped structure on a flat cisternal early phagophore. Movement and expansion of this structure are accompanied by the underlying endoplasmic reticulum, suggesting tight connections between the two compartments. Detailed real-time and 3D imaging of the growing phagophore are leveraged to propose a model for autophagosome formation in plants.