Publications

2021

• Forces generated by lamellipodial actin filament elongation regulate the WAVE complex during cell migration
  
  • Mehidi Amine
  • Kage Frieda
  • Karatas Zeynep
  • Cercy Maureen
  • Schaks Matthias
  • Polesskaya Anna
  • Sainlos Matthieu
  • Gautreau Alexis
  • Rossier Olivier
  • Rottner Klemens
  • Giannone Grégory
  Nature Cell Biology, Nature Publishing Group, 2021, 23 (11), pp.1148-1162. Actin filaments generate mechanical forces that drive membrane movements during trafficking, endocytosis and cell migration. Reciprocally, adaptations of actin networks to forces regulate their assembly and architecture. Yet, a demonstration of forces acting on actin regulators at actin assembly sites in cells is missing. Here we show that local forces arising from actin filament elongation mechanically control WAVE regulatory complex (WRC) dynamics and function, that is, Arp2/3 complex activation in the lamellipodium. Single-protein tracking revealed WRC lateral movements along the lamellipodium tip, driven by elongation of actin filaments and correlating with WRC turnover. The use of optical tweezers to mechanically manipulate functional WRC showed that piconewton forces, as generated by single-filament elongation, dissociated WRC from the lamellipodium tip. WRC activation correlated with its trapping, dwell time and the binding strength at the lamellipodium tip. WRC crosslinking, hindering its mechanical dissociation, increased WRC dwell time and Arp2/3-dependent membrane protrusion. Thus, forces generated by individual actin filaments on their regulators can mechanically tune their turnover and hence activity during cell migration. (10.1038/s41556-021-00786-8)
  DOI : 10.1038/s41556-021-00786-8
• A millisecond passive micromixer with low flow rate, low sample consumption and easy fabrication
  
  • Liao Yuanyuan
  • Mechulam Yves
  • Lassalle-Kaiser Benedikt
  Scientific Reports, Nature Publishing Group, 2021, 11. (10.1038/s41598-021-99471-x)
  DOI : 10.1038/s41598-021-99471-x
• Rôles d’Arpin dans la migration cellulaire et la réparation de l’ADN
  
  • Simanov Gleb
  , 2021. Au front de migration, la protrusion de la membrane appelée lamellipode est générée par la polymérisation de l'actine branchée par Arp2/3. Ce processus est orchestré par la petite GTPase Rac1 capable d'activer le complexe WAVE et d'exposer son domaine WCA nécessaire pour l'activation d'Arp2/3. Différentes boucles de rétroaction positive soutiennent l'activité de Rac1 et stabilisent le lamellipode. Rac1 active aussi l’inhibiteur d'Arp2/3, Arpin, créant une boucle de rétroaction négative. Comme WAVE, Arpin possède un motif A à son extrémité C-terminale, pour lequel il existe deux sites d’interaction à la surface d’Arp2/3. Au lieu d’empêcher la formation de lamellipode, Arpin régule sa durée de vie et, ainsi, contrôle la persistance de la migration cellulaire. Récemment, une nouvelle fonction d'Arp2/3 dans la recombinaison homologue (HDR) a été rapportée.Dans ma thèse, je me suis attaché à mieux comprendre les rôles et les régulations d'Arpin dans la cellule. Nous avons identifié de nouveaux partenaires d'interaction d'Arpin, les Tankyrases 1 et 2. En interrompant spécifiquement l’interaction des Tankyrases ou d’Arp2/3 avec Arpin, j’ai montré que ces deux interactions sont nécessaires pour l’activité d'Arpin dans la régulation de la migration cellulaire. En collaboration, nous avons étudié les mécanismes d'interaction d'Arpin avec le complexe Arp2/3 et montré qu’Arpin se lie à un seul site sur Arp2/3, au niveau de la sous-unité Arp3. Outre le motif A, l’interaction nécessite un motif C non identifié auparavant, similaire au domaine C des activateurs d’Arp2/3. Deuxièmement, j'ai montré que la déplétion d'Arpin stimulait l'efficacité de la HDR mais favorisait l'accumulation des sites de réparation d’ADN dans le noyau. Arpin, ainsi, pourrait jouer un rôle régulateur dans la réparation de l’ADN.
• Selenium metabolism and toxicity in the yeast Saccharomyces cerevisiae
  
  • Lazard Myriam
  Medical Research Archives, KEI Journals, 2021, 9 (9). Selenium (Se) is an essential trace element of considerable interest in humans from both a nutritional and a toxicological perspective because of the narrow margin between intakes that result in efficacy and toxicity. It is used as selenocysteine in a few selenoproteins with important physiological functions. Moreover, at supranutritional doses, Se-containing compounds have attracted interest as potential anticancer agents with high efficacy and selectivity against cancer cells. Thus, Se is becoming a widely used dietary supplement. However, accumulating evidence indicate that adverse health effects are associated with excess dietary supplementation. Therefore, characterizing the toxicity of Se metabolic intermediates are important steps to better understand both the beneficial and toxic mechanisms of Se. This review focuses on the metabolism of Se and the biological mechanisms explaining the toxicity of important Se-metabolites in the yeast Saccharomyces cerevisiae, which can be used as a model system to understand the mode of action and the biological effects of supranutritional Se in higher eukaryotes. (10.18103/mra.v9i9.2550)
  DOI : 10.18103/mra.v9i9.2550
• Nuclear Magnetic Resonance Spectroscopy: A Multifaceted Toolbox to Probe Structure, Dynamics, Interactions, and Real-Time In Situ Release Kinetics in Peptide-Liposome Formulations
  
  • Doyen Camille
  • Larquet Eric
  • Coureux Pierre-Damien
  • Frances Oriane
  • Herman Frédéric
  • Sablé Serge
  • Burnouf Jean-Pierre
  • Sizun Christina
  • Lescop Ewen
  Molecular Pharmaceutics, American Chemical Society, 2021, 18 (7), pp.2521-2539. Liposomal formulations represent attractive biocompatible and tunable drug delivery systems for peptide drugs. Among the tools to analyze their physicochemical properties, nuclear magnetic resonance (NMR) spectroscopy, despite being an obligatory technique to characterize molecular structure and dynamics in chemistry as well as in structural biology, yet appears to be rather sparsely used to study drug-liposome formulations. In this work, we exploited several facets of liquid-state NMR spectroscopy to characterize liposomal delivery systems for the apelinderived K14P peptide and K14P modified by Nα-fatty acylation. Various liposome compositions and preparation modes were analyzed. Using NMR, in combination with cryo-electron microscopy and dynamic light scattering, we determined structural, dynamic, and self-association properties of these peptides in solution and probed their interactions with liposomes. Using 31 P and 1 H NMR, we characterized membrane fluidity and thermotropic phase transitions in empty and loaded liposomes. Based on diffusion and 1 H NMR experiments, we localized and quantified peptides with respect to the interior/exterior of liposomes and changes over time and upon thermal treatments. Finally, we assessed the release kinetics of several solutes and compared various formulations. Taken together, this work shows that NMR has the potential to assist the design of peptide/liposome systems and more generally drug delivery systems. (10.1021/acs.molpharmaceut.1c00037)
  DOI : 10.1021/acs.molpharmaceut.1c00037
• Exposure to the Methylselenol Precursor Dimethyldiselenide Induces a Reductive Endoplasmic Reticulum Stress in Saccharomyces cerevisiae
  
  • Dauplais Marc
  • Mahou Pierre
  • Plateau Pierre
  • Lazard Myriam
  International Journal of Molecular Sciences, MDPI, 2021, 22 (11), pp.5467. Methylselenol (MeSeH) is a major cytotoxic metabolite of selenium, causing apoptosis in cancer cells through mechanisms that remain to be fully established. Previously, we demonstrated that, in Saccharomyces cerevisiae, MeSeH toxicity was mediated by its metabolization into selenomethionine by O-acetylhomoserine (OAH)-sulfhydrylase, an enzyme that is absent in higher eukaryotes. In this report, we used a mutant met17 yeast strain, devoid of OAH- sulfhydrylase activity, to identify alternative targets of MeSeH. Exposure to dimethyldiselenide (DMDSe), a direct precursor of MeSeH, caused an endoplasmic reticulum (ER) stress, as evidenced by increased expression of the ER chaperone Kar2p. Mutant strains (∆ire1 and ∆hac1) unable to activate the unfolded protein response were hypersensitive to MeSeH precursors but not to selenomethionine. In contrast, deletion of YAP1 or SKN7, required to activate the oxidative stress response, did not affect cell growth in the presence of DMDSe. ER maturation of newly synthesized carboxypeptidase Y was impaired, indicating that MeSeH/DMDSe caused protein misfolding in the ER. Exposure to DMDSe resulted in induction of the expression of the ER oxidoreductase Ero1p with concomitant reduction of its regulatory disulfide bonds. These results suggest that MeSeH disturbs protein folding in the ER by generating a reductive stress in this compartment. (10.3390/ijms22115467)
  DOI : 10.3390/ijms22115467
• Division of labor in epitranscriptomics: What have we learnt from the structures of eukaryotic and viral multimeric RNA methyltransferases?
  
  • Graille Marc
  Wiley Interdisciplinary Reviews-RNA, Wiley, 2021. (10.1002/wrna.1673)
  DOI : 10.1002/wrna.1673
• Arpin Regulates Migration Persistence by Interacting with Both Tankyrases and the Arp2/3 Complex
  
  • Simanov Gleb
  • Dang Irene
  • Fokin Artem I
  • Oguievetskaia Ksenia
  • Campanacci Valérie
  • Cherfils Jacqueline
  • Gautreau Alexis
  International Journal of Molecular Sciences, MDPI, 2021, 22 (8). (10.3390/ijms22084115)
  DOI : 10.3390/ijms22084115
• Assembly and Activity of the WASH Molecular Machine: Distinctive Features at the Crossroads of the Actin and Microtubule Cytoskeletons
  
  • Fokin Artem I
  • Gautreau Alexis
  Frontiers in Cell and Developmental Biology, Frontiers media, 2021, 9, pp.658865. The Arp2/3 complex generates branched actin networks at different locations of the cell. The WASH and WAVE Nucleation Promoting Factors (NPFs) activate the Arp2/3 complex at the surface of endosomes or at the cell cortex, respectively. In this review, we will discuss how these two NPFs are controlled within distinct, yet related, multiprotein complexes. These complexes are not spontaneously assembled around WASH and WAVE, but require cellular assembly factors. The centrosome, which nucleates microtubules and branched actin, appears to be a privileged site for WASH complex assembly. The actin and microtubule cytoskeletons are both responsible for endosome shape and membrane remodeling. Motors, such as dynein, pull endosomes and extend membrane tubules along microtubule tracks, whereas branched actin pushes onto the endosomal membrane. It was recently uncovered that WASH assembles a super complex with dynactin, the major dynein activator, where the Capping Protein (CP) is exchanged from dynactin to the WASH complex. This CP swap initiates the first actin filament that primes the autocatalytic nucleation of branched actin at the surface of endosomes. Possible coordination between pushing and pulling forces in the remodeling of endosomal membranes is discussed. (10.3389/fcell.2021.658865)
  DOI : 10.3389/fcell.2021.658865
• A scaffold lncRNA shapes the mitosis to meiosis switch
  
  • Andric Vedrana
  • Nevers Alicia
  • Hazra Ditipriya
  • Auxilien Sylvie
  • Menant Alexandra
  • Graille Marc
  • Palancade Benoit
  • Rougemaille Mathieu
  Nature Communications, Nature Publishing Group, 2021, 12 (1), pp.770. Long non-coding RNAs (lncRNAs) contribute to the regulation of gene expression in response to intra- or extracellular signals but the underlying molecular mechanisms remain largely unexplored. Here, we identify an uncharacterized lncRNA as a central player in shaping the meiotic gene expression program in fission yeast. We report that this regulatory RNA, termed mamRNA, scaffolds the antagonistic RNA-binding proteins Mmi1 and Mei2 to ensure their reciprocal inhibition and fine tune meiotic mRNA degradation during mitotic growth. Mechanistically, mamRNA allows Mmi1 to target Mei2 for ubiquitin-mediated downregulation, and conversely enables accumulating Mei2 to impede Mmi1 activity, thereby reinforcing the mitosis to meiosis switch. These regulations also occur within a unique Mmi1-containing nuclear body, positioning mamRNA as a spatially-confined sensor of Mei2 levels. Our results thus provide a mechanistic basis for the mutual control of gametogenesis effectors and further expand our vision of the regulatory potential of lncRNAs. (10.1038/s41467-021-21032-7)
  DOI : 10.1038/s41467-021-21032-7
• The Arp1/11 minifilament of dynactin primes the endosomal Arp2/3 complex
  
  • Fokin Artem I.
  • David Violaine
  • Oguievetskaia Ksenia
  • Derivery Emmanuel
  • Stone Caroline E.
  • Cao Luyan
  • Rocques Nathalie
  • Molinie Nicolas
  • Henriot Véronique
  • Aumont-Nicaise Magali
  • Hinckelmann Maria-Victoria
  • Saudou Frédéric
  • Le Clainche Christophe
  • Carter Andrew P.
  • Romet ‐ Lemonne Guillaume
  • Gautreau Alexis M.
  Science Advances, American Association for the Advancement of Science (AAAS), 2021. Dendritic actin networks develop from a first actin filament through branching by the Arp2/3 complex. At the surface of endosomes, the WASH complex activates the Arp2/3 complex and interacts with the capping protein for unclear reasons. Here, we show that the WASH complex interacts with dynactin and uncaps it through its FAM21 subunit. In vitro, the uncapped Arp1/11 minifilament elongates an actin filament, which then primes the WASH-induced Arp2/3 branching reaction. In dynactin-depleted cells or in cells where the WASH complex is reconstituted with a FAM21 mutant that cannot uncap dynactin, formation of branched actin at the endosomal surface is impaired. Our results reveal the importance of the WASH complex in coordinating two complexes containing actin-related proteins. (10.1126/sciadv.abd5956)
  DOI : 10.1126/sciadv.abd5956
• Bulges in left-handed G-quadruplexes
  
  • Das Poulomi
  • Ngo Khac Huy
  • Winnerdy Fernaldo richtia
  • Maity Arijit
  • Bakalar Blaž
  • Mechulam Yves
  • Schmitt Emmanuelle
  • Phan Anh Tuân
  Nucleic Acids Research, Oxford University Press, 2021, 49 (3), pp.1724-1736. G-quadruplex (G4) DNA structures with a left-handed backbone progression have unique and conserved structural features. Studies on sequence dependency of the structures revealed the prerequisites and some minimal motifs required for left-handed G4 formation. To extend the boundaries, we explore the adaptability of left-handed G4s towards the existence of bulges. Here we present two X-ray crystal structures and an NMR solution structure of left-handed G4s accommodating one, two and three bulges. Bulges in left-handed G4s show distinct characteristics as compared to those in right-handed G4s. The elucidation of intricate structural details will help in understanding the possible roles and limitations of these unique structures. (10.1093/nar/gkaa1259)
  DOI : 10.1093/nar/gkaa1259
• The Arp2/3 Inhibitory Protein Arpin Is Required for Intestinal Epithelial Barrier Integrity
  
  • Chánez-Paredes Sandra
  • Montoya-García Armando
  • Castro-Ochoa Karla F
  • García-Cordero Julio
  • Cedillo-Barrón Leticia
  • Shibayama Mineko
  • Nava Porfirio
  • Flemming Sven
  • Schlegel Nicolas
  • Gautreau Alexis
  • Vargas-Robles Hilda
  • Mondragón-Flores Ricardo
  • Schnoor Michael
  Frontiers in Cell and Developmental Biology, Frontiers media, 2021, 9, pp.625719. The intestinal epithelial barrier (IEB) depends on stable interepithelial protein complexes such as tight junctions (TJ), adherens junctions (AJ), and the actin cytoskeleton. During inflammation, the IEB is compromised due to TJ protein internalization and actin remodeling. An important actin regulator is the actin-related protein 2/3 (Arp2/3) complex, which induces actin branching. Activation of Arp2/3 by nucleation-promoting factors is required for the formation of epithelial monolayers, but little is known about the relevance of Arp2/3 inhibition and endogenous Arp2/3 inhibitory proteins for IEB regulation. We found that the recently identified Arp2/3 inhibitory protein arpin was strongly expressed in intestinal epithelial cells. Arpin expression decreased in response to tumor necrosis factor (TNF)α and interferon (IFN)γ treatment, whereas the expression of gadkin and protein interacting with protein C-kinase α-subunit 1 (PICK1), other Arp2/3 inhibitors, remained unchanged. Of note, arpin coprecipitated with the TJ proteins occludin and claudin-1 and the AJ protein E-cadherin. Arpin depletion altered the architecture of both AJ and TJ, increased actin filament content and actomyosin contractility, and significantly increased epithelial permeability, demonstrating that arpin is indeed required for maintaining IEB integrity. During experimental colitis in mice, arpin expression was also decreased. Analyzing colon tissues from ulcerative colitis patients by Western blot, we found different arpin levels with overall no significant changes. However, in acutely inflamed areas, arpin was significantly reduced compared to non-inflamed areas. Importantly, patients receiving mesalazine had significantly higher arpin levels than untreated patients. As arpin depletion (theoretically meaning more active Arp2/3) increased permeability, we wanted to know whether Arp2/3 inhibition would show the opposite. Indeed, the specific Arp2/3 inhibitor CK666 ameliorated TNFα/IFNγ-induced permeability in established Caco-2 monolayers by preventing TJ disruption. CK666 treatment also attenuated colitis development, colon tissue damage, TJ disruption, and permeability in dextran sulphate sodium (DSS)-treated mice. Our results demonstrate that loss of arpin triggers IEB dysfunction during inflammation and that low arpin levels can be considered a novel hallmark of acute inflammation. (10.3389/fcell.2021.625719)
  DOI : 10.3389/fcell.2021.625719
• A novel minimal motif for left-handed G-quadruplex formation
  
  • Das Poulomi
  • Winnerdy Fernaldo Richtia
  • Maity Arijit
  • Mechulam Yves
  • Phan Anh Tuân
  Chemical Communications, Royal Society of Chemistry, 2021, 57 (20), pp.2527-2530. A recent study on the left-handed G-quadruplex (LHG4) DNA revealed a 12-nt minimal motif GTGGTGGTGGTG with the ability to independently form an LHG4 and to drive an adjacent sequence to LHG4 formation. Here we have identified a second LHG4-forming motif, GGTGGTGGTGTG, and determined the X-ray crystal structure of an LHG4 involving this motif. Our structural analysis indicated the role of split guanines and single thymine loops in promoting LHG4 formation. (10.1039/d0cc08146a)
  DOI : 10.1039/d0cc08146a
• Methylselenol Produced In Vivo from Methylseleninic Acid or Dimethyl Diselenide Induces Toxic Protein Aggregation in Saccharomyces cerevisiae
  
  • Dauplais Marc
  • Bierla Katarzyna
  • Maizeray Coralie
  • Lestini Roxane
  • Lobinski Ryszard
  • Plateau Pierre
  • Szpunar Joanna
  • Lazard Myriam
  International Journal of Molecular Sciences, MDPI, 2021, 22 (5), pp.2241. Methylselenol (MeSeH) has been suggested to be a critical metabolite for anticancer activity of selenium, although the mechanisms underlying its activity remain to be fully established. The aim of this study was to identify metabolic pathways of MeSeH in Saccharomyces cerevisiae to decipher the mechanism of its toxicity. We first investigated in vitro the formation of MeSeH from methylseleninic acid (MSeA) or dimethyldiselenide. Determination of the equilibrium and rate constants of the reactions between glutathione (GSH) and these MeSeH precursors indicates that in the conditions that prevail in vivo, GSH can reduce the major part of MSeA or dimethyldiselenide into MeSeH. MeSeH can also be enzymatically produced by glutathione reductase or thioredoxin/thioredoxin reductase. Studies on the toxicity of MeSeH precursors (MSeA, dimethyldiselenide or a mixture of MSeA and GSH) in S.cerevisiae revealed that cytotoxicity and selenomethionine content were severely reduced in a met17 mutant devoid of O-acetylhomoserine sulfhydrylase. This suggests conversion of MeSeH into selenomethionine by this enzyme. Protein aggregation was observed in wild-type but not in met17 cells. Altogether, our findings support the view that MeSeH is toxic in S. cerevisiae because it is metabolized into selenomethionine which, in turn, induces toxic protein aggregation. (10.3390/ijms22052241)
  DOI : 10.3390/ijms22052241