I2BC Paris-Saclay

For more than a year, the unprecedented context in which we live has prevented any meetings, a brand new format of physical event where Zeiss come to meet you, as close as possible to your place of work !

Tradeshows and conferences are ideal opportunities to discover new products and witness innovations firsthand. A real-time experience in an individual hands-on session will be offer to you to test new functionalities and give you the opportunity to talk to an expert about your needs. With Zeiss on your campus Truck,you can have the opportunity to experience microscopy workflows at first hand. Near your place of practice and taking into account the applicable hygiene measures, you can individually test the ZEISS instruments and solutions.

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Contact : Sandrine.LECART@i2bc.paris-saclay.fr

Membrane-less compartments are generated by viral infection of a bacterial cell.

Virus lytic infection imposes a major biosynthetic effort to the host cell and takes over significant cellular space. Viruses of prokaryotes must meet the challenge to restructure the cytoplasm open space of a small-sized cell. A study published in the Proceedings of the National Academy of Sciences USA reports the discovery that bacteriophage SPP1 infection leads to biogenesis of two types of membraneless compartments in the cytoplasm of the bacterium Bacillus subtilis. One is a single viral DNA compartment and others are warehouses for storage of viral particles. These compartments are temporal and spatially independent.
The DNA compartment sequesters machines operating viral DNA transactions. Multiple hybrid DNA replication centers, containing both phage replication proteins and hijacked bacterial replisomes, operate at different sub-locations within the compartment for parallelized synthesis of viral DNA. DNA is subsequently packaged in virion precursors without DNA (procapsids) at the compartment edges. Viral DNA-filled capsids then segregate from the DNA compartment, bind phage tails, and the resulting virions build warehouse compartments.
This spatial partition of the B subtilis cell responds to the requirements for exponential replication of SPP1 genomes and for the assembly of hundreds of viral particles. Its similarities to remodelling of the cell nucleus by herpesviruses led to the hypothesis that ancestral strategies used by viruses to invade the cell space were conserved to infect hosts of different Domains of Life.
Part of this work is from the PhD thesis of Audrey Labarde at Université Paris-Saclay.

More details here

Contact: paulo.tavares@i2bc.paris-saclay.fr

De CEA-Joliot.fr :

Researchers at I2BC, in collaboration with the RPBS platform, have developed the third version of their InterEvDock server for structural modelling of protein-protein interactions. The server integrates new algorithms for exploiting sequence evolution information. This greatly improves its performance for the generation of correct assembly models.

Predicting the structure of proteins and their interaction modes is a real challenge for structural biologists. In addition to the gap between the number of proteins whose sequence is known and the number of available structures, proteomics has recently revealed the previously hidden part of the iceberg: hundreds of thousands of physical interactions between proteins. Knowing the surfaces involved in these interactions is essential, not only for understanding the mechanisms that govern how cells and organisms function, but also for designing new therapeutic or enzymatic molecules for pharmaceutical and biotechnological purposes.
Jessica Andreani and Raphaël Guérois (Team “Molecular Assemblies and Genome Integrity”/LBSR/I2BC) have been working for several years on the modelling of protein-protein interactions. In particular, they contribute to the improvement of prediction methods by integrating an evolutionary dimension into molecular docking tools. Indeed, protein interfaces tend to be more conserved than other regions on the protein surface. Moreover, signs of co-evolution can be detected at interfaces, where potentially disruptive mutations are compensated for by mutations in contacting positions on the protein partner. The team has thus developed and released the InterEvDock server, in collaboration with the Ressource Parisienne en Bioinformatique Structurale (University of Paris). As in previous versions, the InterEvDock3 server offers a systematic search for possible interfaces between two partners (known as free docking) and generates numerous conformations that are then ranked, in particular by taking into account information on the evolution of protein sequences. This unique modelling server processes user requests in a variety of formats (structural or sequence data, on one or both partners).
The software is now in its third version (InterEvDock3). This version integrates three new prediction modes that are described in two articles published in NAR1 and Bioinformatics2. The first mode, which is not based on free docking, allows homology modelling of large complexes with potentially low sequence identity. It uses sequences (no structures) as input and runs a template-based modelling protocol by searching exhaustively for close and distant homologs to generate assembly models.
The second mode predicts the structure of complexes from contact maps resulting from methods combining covariation analysis and deep learning. This mode uses 3D structures of monomers or homomultimers (such as a helicase hexamer) to perform a free docking approach while trying to satisfy the contacts predicted in the contact map. It is able to handle some ambiguous information, especially if one of the two partners is a homomultimer (with its residues thus present several times in the structure) and a contact in the predicted map can thus materialise in different ways.
Finally, the third mode uses 3D structures of monomers or multimeric complexes (possibly modelled from sequences by mode 1) and implements a new strategy for evaluating interfaces with coevolutionary information. Ten to forty representative pairs of homologous sequences (i.e. ten to forty evolutionarily conserved interactions between homologs) are selected, modelled at the atomic scale and scored. This new algorithm, tested on a database of 752 complexes (see Bioinformatics2), increases the number of correctly predicted complexes by 30%.
It usually takes between 20 and 60 minutes for the server to propose an interaction model. Developed with funding from two national health and biology infrastructures (FRISBI and IFB), the server is accessible from the RPBS platform:
https://bioserv.rpbs.univ-paris-diderot.fr/services/InterEvDock3/

Encart sur Proteo3Dnet
Another server to analyze interaction data obtained by proteomics
The team also collaborates with the RPBS on the Proteo3Dnet3 server designed to analyze interactions identified by proteomic techniques by integrating structural information (in particular 3D structures of known complexes). Developed with the funding of three national infrastructures in health and biology (FRISBI, ProFI and IFB), it is accessible from the RPBS: https://bioserv.rpbs.univ-paris-diderot.fr/services/Proteo3Dnet/

More details:

https://doi.org/10.1093/nar/gkab358

https://doi.org/10.1093/bioinformatics/btab254

https://doi.org/10.1093/nar/gkab332

Contact: Jessica.ANDREANI@i2bc.paris-saclay.fr

De CEA-Joliot.fr :

Researchers from I2BC in collaboration with a team from Institut Curie and IRCM/CEA-Jacob, lay the molecular basis to explain the dual role of the Mlh1-Mlh3 complex in DNA mismatch repair and, quite uniquely, in one of the key steps of genetic mixing during meiosis. Their study has been published in PNAS.

During meiosis, recombination of genetic material between homologous chromosomes occurs, which contributes to genetic mixing. Recombination can only take place through fine mechanisms of breakage and subsequent repair of DNA molecules. These mechanisms are highly conserved in eukaryotes, from yeast to humans. In particular, cross-shaped structures (Holliday junctions) are formed between homologous chromosomes to make crossovers during recombination. These recombination mechanisms are essential to ensure correct segregation of chromosomes in gametes. Failure of these steps results in the generation of gametes with an abnormal number of chromosomes (trisomy, Turner syndrome, infertility).
The Mlh1-Mlh3 complex is a repair factor for DNA mismatches that are generated as a result of errors in replicative polymerases. Mlh1-Mlh3 has endonuclease activity, i.e. it can cut a DNA molecule between two successive nucleotides and not at its ends. Unlike other similar mismatch repair factors, Mlh1-Mlh3 also exerts its endonuclease activity during meiosis, at the Holliday junctions; it is essential for the exchange of chromosome portions. The Mlh1-Pms1 complex, which is the main factor for repairing DNA mismatches, is not involved in meiosis. However, the two complexes have many structural similarities. For example, they are formed by the interaction between the C-terminal domains of Mlh1 and of its partner Mlh3 or Pms1. What are the structural bases for the differences in specificity between Mlh1-Mlh3 and Mlh1-Pms1? Researchers from I2BC B3S department, Team Nuclear Enveloppe, Telomeres and DNA repair) and the Institut Curie, with the help of CEA-Jacob (IRCM department) and a Swiss team, have solved the three-dimensional structure of a complex formed by the interaction domains of Mlh1 and Mlh3 (from purified recombinant proteins) of the yeast S. cerevisiae by radiocrystallography, and have functionally characterized it. They compared it with the already known equivalent complex formed between Mlh1 and Pms1.
Their study, published in PNAS, reveals differences between the two complexes, particularly with regard to the size of the heterodimerisation interface. The regulatory domains of Pms1 and Mlh3 are oriented differently in the complex with Mlh1. In addition, the shape of the cavity surrounding the endonuclease site varies, which could lead to differences in specificity towards their DNA substrates. The last ten residues of Mlh1 are known to be essential for mismatch repair but not for interaction with Pms1 or Mlh3. Experiments with mutant S. cerevisiae strains - into which deletions of the last residues of Mlh1 have been introduced - indicate that only the last 3 residues are essential for the meiotic activity of Mlh1. Other (DNA binding) experiments allow the authors to conclude that Mlh1-Mlh3, but not Mlh1-Pms1, binds preferentially to Holliday junctions. Finally, in the crystal, the Mlh1-Mlh3 dimers associate with each other to form a filamentary structure. This conformation supports the hypothesis proposed in previous studies that Mlh1-Mlh3 is oligomerised along the DNA. Mutations in the corresponding interaction surfaces strongly decrease the formation of chromosome crossovers. The authors of the study propose that Mlh1-Mlh3 oligomerization starts at a Holliday junction before extending away along the DNA.
This first comparison at the structural level of the Mlh1-Mlh3 and Mlh1-Pms1 complexes strongly suggests an evolutionary specialization of each. To go further, it will be necessary to obtain cryo-electron microscopy structures of the two whole proteins, in interaction with their DNA substrate.

More details here

Contact : jb.charbonnier@i2bc.paris-saclay.fr

What happens when the two subunits of the ribosome are attached together?

Ribosomes universally consist of two independent subunits that assemble on the mRNA during translation initiation. Surprisingly, it was shown that it was possible by synthetic biology to covalently link the two subunits together. Although this is consistent with E. coli growth, the molecular consequences for translation fidelity were unknown. We therefore tested the functioning of this tethered ribosome, and were able to demonstrate that it is strongly impacted in its ability to recognise the SD sequence. In consequence, initiation and SD-dependent frame shifting are both impacted. We were also able to show that the translation termination step was less efficient. It is precisely at this step that the two subunits are physically dissociated. The fact that this is no longer possible in the tethered ribosome clearly disrupts the release efficiency of the ribosome.This work reveals the subtle perturbations linked to the attachment of the two ribosome subunits and should be taken into account in the biotechnological developments associated with the use of these tethered ribosomes

https://academic.oup.com/nar/advance-article/doi/10.1093/nar/gkab259/6266428#.YJLH79KSbnM.twitter

Contact: olivier.namy@i2bc.paris-saclay.fr

An NMR and X-ray cristallography study shows that mitotic phosphorylation of the BAF dimer considerably stiffens its structure, leads to its dissociation from DNA, but unexpectedly does not modify its binding to the nuclear envelope proteins emerin and lamin.

In human cell nuclei, double-stranded DNA interacts with many proteins to form chromatin. Other proteins anchor chromatin to the nuclear membrane, which is important for genome stability. In 2018, our team elucidated the three-dimensional structure of a complex formed by the DNA-binding protein BAF, its inner nuclear membrane partner emerin, and a protein from the nucleoskeleton, lamin A/C. We showed that this complex is defective in patients with premature aging syndromes due to mutations in BAF or lamin A/C. It was reported that this complex must disassemble when cells divide (in mitosis), because then, the whole chromatin is reorganized. The present work aims at better understanding the mechanisms that regulate the assembly and disassembly of this complex during the cell cycle. Using biochemical, biophysical and structural biology experiments, we were able to reproduce in vitro the phosphorylation of the BAF protein by the mitotic kinase VRK1. We confirmed that BAF is doubly phosphorylated by VRK1 kinase, first on its serine 4, then on its threonine 3. These phosphorylation events significantly stiffen the N-terminal region of BAF, disrupt DNA binding, but unexpectedly, phosphorylated BAF remains bound to emerin and lamin A/C. This study paves the way for a broader exploration of the impact of mitotic phosphorylations on genome organization.

Di-phosphorylated BAF shows altered structural dynamics and binding to DNA, but interacts with its nuclear envelope partners - PubMed (nih.gov)

Contact: sophie.zinn@i2bc.paris-saclay.fr

The RNA and protein products from C9orf72 mutations causing amyotrophic lateral sclerosis and frontotemporal dementia induce various and additive proteostasis impairments.

The most frequent genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia is a G4C2 repeat expansion in the C9orf72 gene. This expansion gives rise to translation of aggregating dipeptide repeat (DPR) proteins, including poly-GA as the most abundant species. However, gain of toxic function effects have been attributed to either the DPRs or the pathological G4C2 RNA. Here, we analyzed in a cellular model the relative toxicity of DPRs and RNA. Cytoplasmic poly-GA aggregates, generated in the absence of G4C2 RNA, interfered with nucleocytoplasmic protein transport, but had little effect on cell viability. In contrast, nuclear poly-GA was more toxic, impairing nucleolar protein quality control and protein biosynthesis. Production of the G4C2 RNA strongly reduced viability independent of DPR translation and caused pronounced inhibition of nuclear mRNA export and protein biogenesis. Thus, while the toxic effects of G4C2 RNA predominate in the cellular model used, DPRs exert additive effects that may contribute to pathology.

More details here

Contact: frederic.frottin@i2bc.paris-saclay.fr

The IMAPP group is prominently involved in the MéDynA second plenary meeting, 6-9 September, 2021 at Ile d'Oléron (organising committee: Sonia Fieulaine, Stéphane Bressanelli, Maïté Paternostre, Edith Godard, Yves Boulard).

MéDynA is a 'GdR' (coordination of labs all across France around a specific research topic) whose objectives are to: * Bring together French labs studying by diverse means the dynamical pathways leading to self-organised protein assemblies or protein-based assemblies (incl. peptidomimetics)
* Forge a common language so as to benefit from the multiple expertise stemming from B13https://medyna.cnrs.fr/les-actualites/deuxieme-pleniere/B14 biophysics, chemistry, physics and mathematics to understand and master mechanisms of protein assembly
* Foster emergence of interdisciplinary and novel research projects
* Set up a nationwide interdisciplinary network useful to all researchers, whether at an early career stage (such as graduate students and postdocs) or well established

More information here

Contact: gdr.medyna@ibpc.fr

De CEA-Joliot.fr :

Dans une étude publiée dans JBC, une équipe de l'I2BC, en collaboration avec l’Institut botanique de Beijing (Chine), décrit pour la première fois un état intermédiaire de l’antenne collectrice de lumière des plantes supérieures qui permet de mieux comprendre les changements qui s’opèrent au niveau moléculaire lors de l’activation d’un mécanisme de photoprotection de la plante.

La photosynthèse commence par l’absorption de l’énergie lumineuse par les pigments chlorophylles des antennes collectrices de la lumière (LHC- Light Harvesting Complex, complexes composés de protéines et de pigments chlorophylles et caroténoïdes). Cette absorption crée une énergie d’excitation (passage d’un état électronique fondamental à un état excité de la chlorophylle collectrice), énergie qui est transférée de proche en proche, d’une chlorophylle à un autre, jusqu’au centre réactionnel de la photosynthèse où elle est convertie en énergie de potentiel chimique (par séparation de charge). Ce processus de conversion de l’énergie est extrêmement efficace. Tellement efficace, qu’il peut provoquer une surexcitation du système potentiellement délétère. La plante met en place des mécanismes pour s’en prémunir : de l’échelle macroscopique, par le mouvement de ses feuilles, à l’échelle moléculaire, par un mécanisme qui permet la dissipation de l’énergie sous forme de chaleur. Ce dernier mécanisme, multifactoriel, est appelé extinction non photochimique de la fluorescence de la chlorophylle (non photochemical quenching).
Que ce soit in vivo ou in vitro, l’équipe Bioénergétique Membranaire et stress (département I2BC), dirigée par Bruno Robert, a montré que ce quenching est lié à un réarrangement des protéines et pigments qui composent le LHC qui crée des « pièges à « énergie ». Les pigments chlorophylles excités transfèrent leur énergie à des pigments caroténoïdes qui la dissipent immédiatement sous forme de chaleur. Dans le cas du LHCII, la principale antenne collectrice des plantes supérieures, des expériences de spectroscopie in vitro menées sur le complexe agrégé (en l’absence de détergent utilisé classiquement pour le solubiliser) établissent que ce transfert se fait entre la chlorophylle a et une lutéine (un caroténoïde). L'ampleur de l'extinction semble corrélée aux changements de conformation (torsion) affectant la lutéine et un autre caroténoïde, la néoxanthine.
Pour aller plus loin dans la description et la compréhension de ces modifications, l’équipe de Bruno Robert, a étudié la structure du LHCII dans différents environnements qui influencent ses propriétés électroniques. De manière remarquable, l’équipe a réussi à isoler pour la première fois un état de LHCII, obtenu en utilisant le détergent n-dodécyl-α-D-maltoside, et à caractériser les propriétés spectroscopiques de ses pigments. Leur étude, publiée dans JBC, montre que, dans cet état, sont présents tous les changements associés à l’extinction non photochimique (changements dans les interactions protéine-chlorophylle, torsion de la néoxanthine) à l’exception de la torsion de lutéine, alors qu’aucun quenching n’est associé à cet état. Cet état de LHCII serait en quelque sorte un état intermédiaire qui permettrait le passage d’un état « allumé », capable d’absorber et de convertir l’énergie lumineuse en énergie chimique, à un état « éteint » par l’extinction non photochimique. La torsion de la néoxanthine serait un indicateur de changements de conformation à grande échelle du LHCII qui précéderaient des changements à plus petite échelle directement à l’origine du quenching et revélés par la torsion de la lutéine. Cet intermédiaire LHCII non éteint, décrit ici pour la première fois, permet de mieux comprendre le mécanisme moléculaire de l'extinction.

More details here

Contact : Bruno.ROBERT@i2bc.paris-saclay.fr

Replicative helicases are essential proteins that unwind DNA in front of replication forks. Their loading depends on accessory proteins and in bacteria, DnaC and DnaI are well characterized loaders. However, most bacteria do not express either of these two proteins. Instead, they are proposed to rely on DciA, an ancestral protein unrelated to DnaC/I. While the DciA structure from Vibrio cholerae shares no homology with DnaC, it reveals similarities with DnaA and DnaX, two proteins involved during replication initiation. As other bacterial replicative helicases, VcDnaB adopts a toroid-shaped homo-hexameric structure, but with a slightly open dynamic conformation in the free state. We show that VcDnaB can load itself on DNA in vitro and that VcDciA stimulates this function, resulting in an increased DNA unwinding. VcDciA interacts with VcDnaB with a 3/6 stoichiometry and we show that a determinant residue, which discriminates DciA- and DnaC/I-helicases, is critical in vivo. Our work is the first step toward the understanding of the ancestral mode of loading of bacterial replicative helicases on DNA. It sheds light on the strategy employed by phage helicase loaders to hijack bacterial replicative helicases and may explain the recurrent domestication of dnaC/I through evolution in bacteria.

More details here

Contacts: Sophie Quevillon-Cheruel@i2bc.paris-saclay.fr and Jean-Luc.FERAT@i2bc.paris-saclay.fr

Motile kinesins are motor proteins that translocate along microtubules as they hydrolyze ATP. They share a conserved motor domain which harbors both ATPase and microtubule-binding activities. An ATP hydrolysis mechanism involving two water molecules has been proposed based on the structure of the kinesin-5 Eg5 bound to an ATP analog. Whether this mechanism is general in the kinesin superfamily remains uncertain. Here, we present structural snapshots of the motor domain of OSM-3 along its nucleotide cycle. OSM-3 belongs to the homodimeric kinesin-2 subfamily and is the Caenorhabditis elegans homologue of human KIF17. OSM-3 bound to ADP or devoid of a nucleotide shows features of ADP-kinesins with a docked neck linker. When bound to an ATP analog, OSM-3 adopts a conformation similar to those of several ATP-like kinesins, either isolated or bound to tubulin. Moreover, the OSM-3 nucleotide-binding site is virtually identical to that of ATP-like Eg5, demonstrating a shared ATPase mechanism. Therefore, our data extend to kinesin-2 the two-water ATP hydrolysis mechanism and further suggest that it is universal within the kinesin superfamily.

More details here

contact: julie.menetrey@i2bc.paris-saclay.fr

While the ancestral role of the RNAi machinery in the eukaryotic ancestor(s) may have been geared toward targeting RNA transcript degradation and locus-specific histone modifications to promote gene silencing, findings in multiple organisms have now demonstrated the involvement of RNAi components in many other nuclear functions including centromere integrity, chromosome segregation and DNA repair. Previous findings implicated the RNAi machinery in the meiotic process, but left open the question of how these proteins impact the specific steps of prophase chromosome dynamics such as pairing, formation of the meiosis-specific structure called synaptonemal complex (SC), and homologous recombination. Here we exploited the power of Sordaria macrospora as a particularly attractive experimental system for examination of the roles of both Dicer and Argonaute proteins during its sexual cycle. We show that both Dicer proteins and both Argonautes present in Sordaria genome are dispensable for vegetative growth but required for the correct entry and progression into the sexual cycle, with a prominent role of one Dicer (Dcl1) and one Argonaute (Sms2). Moreover, we show that Dcl1, and in a lesser extent Dcl2 and the Argonaute Qde2, are involved in regulating chromosome axis length during meiosis, as well as crossover number and distribution along chromosomes. Taken together, these findings indicate clearly that the RNAi components play a central role in the meiotic process of Sordaria and extend our understanding of the different processes (e.g. axis length, crossover patterning) that are directly or indirectly dependent on the Dicer and/or the Argonaute proteins.

More details here

Contact: chloe.girard@i2bc.paris-saclay.fr

A posttranscriptional regulatory mechanism links the expression of the cyanobacterial Orange Carotenoid Protein related to photoprotection, directly and in an inverse fashion, to the synthesis of the phycobilisome, the cyanobacterial antenna via a 3’ end-derived sRNA.

The modular photoactive Orange Carotenoid Protein (OCP), which has a crucial role in cyanobacterial photoprotection, has gained a great interest in the recent years due to its ability to act as a molecular photoswitch and to its suppressive 12 Å translocation of the carotenoid upon photoactivation. Upon blue light absorption, the inactive orange form (OCPO) converts to the active red state (OCPR) which is able to interact with the phycobilisome, the cyanobacterial antenna, and to decrease the energy arriving at the photochemical centers. Due to its function in thermal dissipation of excess energy, the expression of OCP must be tightly controlled to avoid a loss of energy under non-stressing conditions and to increase this dissipation under stressing ones. A research group of the I2BC in collaboration of a group of the University of Freiburg discovered the first molecular factor involved in the regulation of OCP expression. Moreover, they show for the first time that a sRNA appended to a long operon mRNA functions in the regulatory network of cyanobacteria.

This factor is the first example in cyanobacteria of a sRNA derived from a polycistronic mRNA regulating at least one other mRNA in trans. The researchers demonstrated that ApcZ, a sRNA originating from the 3’end of the apcABC operon encoding the core phycobilisome proteins, is responsible for the repression of ocp translation under non-stress conditions.  The transcription of the apcABC operon decreases under most stress conditions and as a consequence ApcZ (free and as part of the entire operon transcript) concentration decreases leading to a de-repression of ocp mRNA translation. Thus, light harvesting and photoprotection are connected directly and in an inverse fashion by a single regulatory sRNA. If, under stress conditions, less energy must arrive at the photochemical centers, the transcription of phycobilisome genes decreases and synthesis of OCP increases. Hence, the OCP concentration is controlled in a simple and elegant way.

“Inverse Regulation of Light Harvesting and Photoprotection Is Mediated by a 3’ End-Derived sRNA in Cyanobacteria” Plant Cell (2021) 33, 358-380. Jiao Zhan, Claudia Steglich, Ingeborg Scholz, Wolfgang R. Hess and Diana Kirilovsky

Contact: diana.kirlovsky@cea.fr