Université PSL

Publications

RECHERCHER

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Visualizing the dynamics of exported bacterial proteins with the chemogenetic fluorescent reporter FAST.
Laboratoire Physique des biomolécules - Chekli, Yankel; Peron-Cane, Caroline; Dell'Arciprete, Dario; Allemand, Jean-François; Li, Chenge; Ghigo, Jean-Marc; Gautier, Arnaud; Lebreton, Alice; Desprat, Nicolas; Beloin, Christophe
journal article - 10(1) 15791 - 10.1038/s41598-020-72498-2 - 2020
Bacterial proteins exported to the cell surface play key cellular functions. However, despite the interest to study the localisation of surface proteins such as adhesins, transporters or hydrolases, monitoring their dynamics in live imaging remains challenging, due to the limited availability of fluorescent probes remaining functional after secretion. In this work, we used the Escherichia coli intimin and the Listeria monocytogenes InlB invasin as surface exposed scaffolds fused with the recently developed chemogenetic fluorescent reporter protein FAST. Using both membrane permeant (HBR-3,5DM) and non-permeant (HBRAA-3E) fluorogens that fluoresce upon binding to FAST, we demonstrated that fully functional FAST can be exposed at the cell surface and used to specifically tag the external side of the bacterial envelop in both diderm and monoderm bacteria. Our work opens new avenues to study the organization and dynamics of the bacterial cell surface proteins.
Fluorescent secreted bacterial effectors reveal active intravacuolar proliferation of Listeria monocytogenes in epithelial cells.
Laboratoire Physique des biomolécules - Peron-Cane, Caroline; Fernandez, José-Carlos; Leblanc, Julien; Wingertsmann, Laure; Gautier, Arnaud; Desprat, Nicolas; Lebreton, Alice
journal article - 16(10) e1009001 - 10.1371/journal.ppat.1009001 - 2020
Real-time imaging of bacterial virulence factor dynamics is hampered by the limited number of fluorescent tools suitable for tagging secreted effectors. Here, we demonstrated that the fluorogenic reporter FAST could be used to tag secreted proteins, and we implemented it to monitor infection dynamics in epithelial cells exposed to the human pathogen Listeria monocytogenes (Lm). By tracking individual FAST-labelled vacuoles after Lm internalisation into cells, we unveiled the heterogeneity of residence time inside entry vacuoles. Although half of the bacterial population escaped within 13 minutes after entry, 12% of bacteria remained entrapped over an hour inside long term vacuoles, and sometimes much longer, regardless of the secretion of the pore-forming toxin listeriolysin O (LLO). We imaged LLO-FAST in these long-term vacuoles, and showed that LLO enabled Lm to proliferate inside these compartments, reminiscent of what had been previously observed for Spacious Listeria-containing phagosomes (SLAPs). Unexpectedly, inside epithelial SLAP-like vacuoles (eSLAPs), Lm proliferated as fast as in the host cytosol. eSLAPs thus constitute an alternative replication niche in epithelial cells that might promote the colonization of host tissues.s.
Fluorescence-free quantification of protein/nucleic-acid binding through single-molecule kinetic locking
Laboratoire Physique des biomolécules - Martin Rieu; Valle-Orero, Jessica; Ducos, Bertrand; Allemand, Jean-François; Croquette, Vincent
bioRxiv - - 10.1101/2020.09.30.321232 - 2020
Fluorescence-free micro-manipulation of nucleic acids (NA) allows the functional characterization of DNA/RNA processing proteins, without the interference of labels, but currently fails to detect and quantify their binding. To overcome this limitation, we developed a new method based on single-molecule force spectroscopy, called kinetic locking, that allows a direct in vitro visualization of protein binding while avoiding any kind of chemical disturbance of the protein’s natural function. We validate kinetic locking by measuring accurately the hybridization energy of ultrashort nucleotides (5,6,7 bases) and use it to measure the dynamical interactions of E. coli RecQ helicase with its DNA substrate.Competing Interest StatementThe authors have declared no competing interest.
Parallelized DNA tethered bead measurements to scrutinize DNA mechanical structure
Laboratoire Physique des biomolécules - Allemand JF, Tardin C, Salomé L.
Nat. Methods - 1;169 46-56 - doi: 10.1016/j.ymeth.2019.07.020. - 2019
Tethering beads to DNA offers a panel of single molecule techniques for the refined analysis of the conformational dynamics of DNA and the elucidation of the mechanisms of enzyme activity. Recent developments include the massive parallelization of these techniques achieved by the fabrication of dedicated nanoarrays by soft nanolithography. We focus here on two of these techniques: the Tethered Particle motion and Magnetic Tweezers allowing analysis of the behavior of individual DNA molecules in the absence of force and under the application of a force and/or a torque, respectively. We introduce the experimental protocols for the parallelization and discuss the benefits already gained, and to come, for these single molecule investigations.
Anisotropic cellular forces support mechanical integrity of the Stratum Corneum barrier
Laboratoire Physique des biomolécules - Guo S, Domanov Y, Donovan M, Ducos B, Pomeau Y, Gourier C, Perez E, Luengo GS.
Chem. Mater - 92 45231 - doi: 10.1016/j.jmbbm.2018.12.027 - 2019
The protective function of biological surfaces that are exposed to the exterior of living organisms is the result of a complex arrangement and interaction of cellular components. This is the case for the most external cornified layer of skin, the stratum corneum (SC). This layer is made of corneocytes, the elementary 'flat bricks' that are held together through adhesive junctions. Despite the well-known protective role of the SC under high mechanical stresses and rapid cell turnover, the subtleties regarding the adhesion and mechanical interaction among the individual corneocytes are still poorly known. Here, we explore the adhesion of single corneocytes at different depths of the SC, by pulling them using glass microcantilevers, and measuring their detachment forces. We measured their interplanar adhesion between SC layers, and their peripheral adhesion among cells within a SC layer. Both adhesions increased considerably with depth. At the SC surface, with respect to adhesion, the corneocyte population exhibited a strong heterogeneity, where detachment forces differed by more than one order of magnitude for corneocytes located side by side. The measured detachment forces indicated that in the upper-middle layers of SC, the peripheral adhesion was stronger than the interplanar one. We conclude that the stronger peripheral adhesion of corneocytes in the SC favors an efficient barrier which would be able to resist strong stresses.
Anisotropic cellular forces support mechanical integrity of the Stratum Corneum barrier
Laboratoire Physique des biomolécules - Guo S, Domanov Y, Donovan M, Ducos B, Pomeau Y, Gourier C, Perez E, Luengo GS.
Chem. Mater - 92 45231 - doi: 10.1016/j.jmbbm.2018.12.027 - 2019
The protective function of biological surfaces that are exposed to the exterior of living organisms is the result of a complex arrangement and interaction of cellular components. This is the case for the most external cornified layer of skin, the stratum corneum (SC). This layer is made of corneocytes, the elementary 'flat bricks' that are held together through adhesive junctions. Despite the well-known protective role of the SC under high mechanical stresses and rapid cell turnover, the subtleties regarding the adhesion and mechanical interaction among the individual corneocytes are still poorly known. Here, we explore the adhesion of single corneocytes at different depths of the SC, by pulling them using glass microcantilevers, and measuring their detachment forces. We measured their interplanar adhesion between SC layers, and their peripheral adhesion among cells within a SC layer. Both adhesions increased considerably with depth. At the SC surface, with respect to adhesion, the corneocyte population exhibited a strong heterogeneity, where detachment forces differed by more than one order of magnitude for corneocytes located side by side. The measured detachment forces indicated that in the upper-middle layers of SC, the peripheral adhesion was stronger than the interplanar one. We conclude that the stronger peripheral adhesion of corneocytes in the SC favors an efficient barrier which would be able to resist strong stresses.
PICH and TOP3A cooperate to induce positive DNA supercoiling
Laboratoire Physique des biomolécules - Anna Hélène Bizard, Jean-Francois Allemand, Tue Hassenkam, Manikandan Paramasivam
Nature - 26(4) 1 - DOI: 10.1038/s41594-019-0201-6 - 2019
All known eukaryotic topoisomerases are only able to relieve torsional stress in DNA. Nevertheless, it has been proposed that the introduction of positive DNA supercoiling is required for efficient sister-chromatid disjunction by Topoisomerase 2a during mitosis. Here we identify a eukaryotic enzymatic activity that introduces torsional stress into DNA. We show that the human Plk1-interacting checkpoint helicase (PICH) and Topoisomerase 3a proteins combine to create an extraordinarily high density of positive DNA supercoiling. This activity, which is analogous to that of a reverse-gyrase, is apparently driven by the ability of PICH to progressively extrude hypernegatively supercoiled DNA loops that are relaxed by Topoisomerase 3a. We propose that this positive supercoiling provides an optimal substrate for the rapid disjunction of sister centromeres by Topoisomerase 2a at the onset of anaphase in eukaryotic cells.
Mechanistic characterization of the DEAD-box RNA helicase Ded1 from yeast as revealed by a novel technique using single-molecule magnetic tweezers
Laboratoire Physique des biomolécules - Saurabh Raj, Debjani Bagchi, Jessica Valle Orero, Josette Banroques, N Kyle Tanner, Vincent Croquette
Nucleic Acids Res. - 47(7) 3699–3710 - doi.org/10.1093/nar/gkz057 - 2019
DEAD-box helicases are involved in all steps of RNA metabolism. They are ATP-dependent RNA binding proteins and RNA-dependent ATPases. They can displace short duplexes, but they lack processivity. Their mechanism and functioning are not clearly understood; classical or bulk biochemical assays are not sufficient to answer these questions. Single-molecule techniques provide useful tools, but they are limited in cases where the proteins are nonprocessive and give weak signals. We present here a new, magnetic-tweezers-based, single-molecule assay that is simple and that can sensitively measure the displacement time of a small, hybridized, RNA oligonucleotide. Tens of molecules can be analyzed at the same time. Comparing the displacement times with and without a helicase gives insights into the enzymatic activity of the protein. We used this assay to study yeast Ded1, which is orthologous to human DDX3. Although Ded1 acts on a variety of substrates, we find that Ded1 requires an RNA substrate for its ATP-dependent unwinding activity and that ATP hydrolysis is needed to see this activity. Further, we find that only intramolecular single-stranded RNA extensions enhance this activity. We propose a model where ATP-bound Ded1 stabilizes partially unwound duplexes and where multiple binding events may be needed to see displacement.
Longitudinal Analyses of Blood Transcriptome During Conversion to Psychosis
Laboratoire Physique des biomolécules - Saurabh Raj, Debjani Bagchi, Jessica Valle Orero, Josette Banroques, N Kyle Tanner, Vincent Croquette
Schizophr Bull - 45(1) 247-255 - doi: 10.1093/schbul/sby009 - 2019
The biological processes associated with the onset of schizophrenia remain largely unknown. Current hypotheses favor gene × environment interactions as supported by our recent report about DNA methylation changes during the onset of psychosis. Here, we conducted the first longitudinal transcriptomic analysis of blood samples from 31 at-risk individuals who later converted to psychosis and 63 at-risk individuals who did not. Individuals were followed for a maximum of 1 year. Blood samples were collected at baseline and at the end of follow-up and individuals served as their own controls. Differentially expressed genes between the 2 groups were identified using the RNA sequencing of an initial discovery subgroup (n = 15 individuals). The most promising results were replicated using high-throughput real-time qPCR in the whole cohort (n = 94 individuals). We identified longitudinal changes in 4 brain-expressed genes based on RNAseq analysis. One of these genes (CPT1A) was replicated in the whole cohort. The previously observed hypermethylation in NRP1 and GSTM5 during the onset of psychosis correlated with a decrease in corresponding gene expression. RNA sequencing also identified 2 co-expression networks that were impaired after conversion compared with baseline-the Wnt pathway including AKT1, CPT1A and semaphorins, and the Toll-like receptor pathway, related to innate immunity. This longitudinal study of transcriptomic changes in individuals with at-risk mental state revealed alterations during conversion to psychosis in pathways and genes relevant to schizophrenia. These results may be a first step toward better understanding psychosis onset. They may also help to identify new biomarkers and targets for disease-modifying therapeutic strategies
Longitudinal Analyses of Blood Transcriptome During Conversion to Psychosis
Laboratoire Physique des biomolécules - Saurabh Raj, Debjani Bagchi, Jessica Valle Orero, Josette Banroques, N Kyle Tanner, Vincent Croquette
Schizophr Bull - 45(1) 247-255 - doi: 10.1093/schbul/sby009 - 2019
The biological processes associated with the onset of schizophrenia remain largely unknown. Current hypotheses favor gene × environment interactions as supported by our recent report about DNA methylation changes during the onset of psychosis. Here, we conducted the first longitudinal transcriptomic analysis of blood samples from 31 at-risk individuals who later converted to psychosis and 63 at-risk individuals who did not. Individuals were followed for a maximum of 1 year. Blood samples were collected at baseline and at the end of follow-up and individuals served as their own controls. Differentially expressed genes between the 2 groups were identified using the RNA sequencing of an initial discovery subgroup (n = 15 individuals). The most promising results were replicated using high-throughput real-time qPCR in the whole cohort (n = 94 individuals). We identified longitudinal changes in 4 brain-expressed genes based on RNAseq analysis. One of these genes (CPT1A) was replicated in the whole cohort. The previously observed hypermethylation in NRP1 and GSTM5 during the onset of psychosis correlated with a decrease in corresponding gene expression. RNA sequencing also identified 2 co-expression networks that were impaired after conversion compared with baseline-the Wnt pathway including AKT1, CPT1A and semaphorins, and the Toll-like receptor pathway, related to innate immunity. This longitudinal study of transcriptomic changes in individuals with at-risk mental state revealed alterations during conversion to psychosis in pathways and genes relevant to schizophrenia. These results may be a first step toward better understanding psychosis onset. They may also help to identify new biomarkers and targets for disease-modifying therapeutic strategies
Optical control of protein activity and gene expression by photoactivation of caged cyclofen
Laboratoire Physique des biomolécules - Hamouri F, Zhang W, Aujard I, Le Saux T, Ducos B, Vriz S, Jullien L, Bensimon D
Methods Enzymol - 624 44927 - doi: 10.1016/bs.mie.2019.04.009 - 2019
The use of light to control the expression of genes and the activity of proteins is a rapidly expanding field. While many of these approaches use a fusion between a light activatable protein and the protein of interest to control the activity of the latter, it is also possible to control the activity of a protein by uncaging a specific ligand. In that context, controlling the activation of a protein fused to the modified estrogen receptor (ERT) by uncaging its ligand cyclofen-OH has emerged as a generic and versatile method to control the activation of proteins quantitatively, quickly and locally in a live organism. Here, we present the experimental details behind this approach.
Connexin 43 Controls the Astrocyte Immunoregulatory Phenotype.
Laboratoire Physique des biomolécules - Boulay, Anne-Cécile; Gilbert, Alice; Oliveira Moreira, Vanessa; Blugeon, Corinne; Perrin, Sandrine; Pouch, Juliette; Le Crom, Stéphane; Ducos, Bertrand; Cohen-Salmon, Martine
Brain sciences - 8(4) - 10.3390/brainsci8040050 - 2018
Astrocytes are the most abundant glial cells of the central nervous system and have recently been recognized as crucial in the regulation of brain immunity. In most neuropathological conditions, astrocytes are prone to a radical phenotypical change called reactivity, which plays a key role in astrocyte contribution to neuroinflammation. However, how astrocytes regulate brain immunity in healthy conditions is an understudied question. One of the astroglial molecule involved in these regulations might be Connexin 43 (Cx43), a gap junction protein highly enriched in astrocyte perivascular endfeet-terminated processes forming the glia limitans. Indeed, Cx43 deletion in astrocytes (Cx43KO) promotes a continuous immune recruitment and an autoimmune response against an astrocyte protein, without inducing any brain lesion. To investigate the molecular basis of this unique immune response, we characterized the polysomal transcriptome of hippocampal astrocytes deleted for Cx43. Our results demonstrate that, in the absence of Cx43, astrocytes adopt an atypical reactive status with no change in most canonical astrogliosis markers, but with an upregulation of molecules promoting immune recruitment, complement activation as well as anti-inflammatory processes. Intriguingly, while several of these upregulated transcriptional events suggested an activation of the γ-interferon pathway, no increase in this cytokine or activation of related signaling pathways were found in Cx43KO. Finally, deletion of astroglial Cx43 was associated with the upregulation of several angiogenic factors, consistent with an increase in microvascular density in Cx43KO brains. Collectively, these results strongly suggest that Cx43 controls immunoregulatory and angiogenic properties of astrocytes.
Control of protein activity and gene expression by cyclofen-OH uncaging.
Laboratoire Physique des biomolécules - Zhang, Weiting; Hamouri, Fatima; Feng, Zhiping; Aujard, Isabelle; Ducos, Bertrand; Ye, Shixin; Weiss, Shimon; Volovitch, Michel; Vriz, Sophie; Jullien, Ludovic; Bensimon, David
Chembiochem - - 10.1002/cbic.201700630 - 2018
The use of light to control the expression of genes and the activity of proteins is a rapidly expanding field. While many of these approaches use a fusion between a light activable protein and the protein of interest to control the activity of the later, it is also possible to control the activity of a protein by uncaging a specific ligand. In that context, controlling the activation of a protein fused to the modified estrogen receptor (ERT) by uncaging its ligand cyclofen-OH has emerged as a generic and versatile method to control the activation of proteins quantitatively, quickly and locally in a live organism. We here present that approach and its uses in a variety of physiological contexts.
Rolling and aging in temperature-ramp soft adhesion.
Laboratoire Physique des biomolécules - Boniello, Giuseppe; Tribet, Christophe; Marie, Emmanuelle; Croquette, Vincent; Zanchi, Dražen
Phys. Rev. E - 97 1-1 - 10.1103/PhysRevE.97.012609 - 2018
Immediately before adsorption to a horizontal substrate, sinking polymer-coated colloids can undergo a complex sequence of landing, jumping, crawling, and rolling events. Using video tracking, we studied the soft adhesion to a horizontal flat plate of micron-size colloids coated by a controlled molar fraction f of the poly(lysine)-grafted-poly(N-isopropylacrylamide) (PLL-g-PNIPAM) which is a temperature-sensitive polymer. We ramp the temperature from below to above T_{c}=32±1^{∘}C, at which the PNIPAM polymer undergoes a transition, triggering attractive interaction between microparticles and surface. The adsorption rate, the effective in-plane (x-y) diffusion constant, and the average residence time distribution over z were extracted from the Brownian motion records during last seconds before immobilization. Experimental data are understood within a rate-equations-based model that includes aging effects and includes three populations: the untethered, the rolling, and the arrested colloids. We show that preadsorption dynamics casts a characteristic scaling function α(f) proportional to the number of available PNIPAM patches met by soft contact during Brownian rolling. In particular, the increase of in-plane diffusivity with increasing f is understood: The stickiest particles have the shortest rolling regime prior to arrest, so that their motion is dominated by the untethered phase.
DNA methylation and gene expression alterations in zebrafish early-life stages exposed to the antibacterial agent triclosan
Laboratoire Physique des biomolécules - Falisse, Elodie; Ducos, Bertrand; Stockwell, Peter A; Morison, Ian M; Chatterjee, Aniruddha; Silvestre, Frédéric
Environmental pollution - 243 1867-1877 - 10.1016/j.envpol.2018.10.004 - 2018
There is increasing evidence that toxicant exposure can alter DNA methylation profile, one of the main epigenetic mechanisms, particularly during embryogenesis when DNA methylation patterns are being established. In order to investigate the effects of the antibacterial agent Triclosan on DNA methylation and its correlation with gene expression, zebrafish embryos were exposed during 7 days post-fertilization (starting at maximum 8-cells stage) to 50 and 100 μg/l, two conditions for which increased sensitivity and acclimation have been respectively reported. Although global DNA methylation was not significantly affected, a total of 171 differentially methylated fragments were identified by Reduced Representation Bisulfite Sequencing. The majority of these fragments were found between the two exposed groups, reflecting dose-dependant specific responses. Gene ontology analysis revealed that pathways involved in TGF-β signaling were enriched in larvae exposed to 50 μg/l, while de novo pyrimidine biosynthesis functions were overrepresented in fish exposed to 100 μg/l. In addition, gene expression analysis revealed a positive correlation between mRNA levels and DNA methylation patterns in introns, together with significant alterations of the transcription of genes involved in nervous system development, transcriptional factors and histone methyltransferases. Overall this work provides evidence that Triclosan alters DNA methylation in zebrafish exposed during embryogenesis as well as related genes expression and proposes concentration specific modes of action. Further studies will investigate the possible long-term consequences of these alterations, i.e. latent defects associated with developmental exposure and transgenerational effects, and the possible implications in terms of fitness and adaptation to environmental pollutants.
Single molecule kinetics uncover roles for E. coli RecQ DNA helicase domains and interaction with SSB
Laboratoire Physique des biomolécules - Debjani Bagchi , Maria Manosas, Weiting Zhang, Kelly A Manthei , Samar Hodeib , Bertrand Ducos , James L Keck , Vincent Croquette
Nat. Methods - 46(16) 8500-8515 - DOI: 10.1093/nar/gky647 - 2018
Most RecQ DNA helicases share a conserved domain arrangement that mediates their activities in genomic stability. This arrangement comprises a helicase motor domain, a RecQ C-terminal (RecQ-C) region including a winged-helix (WH) domain, and a 'Helicase and RNase D C-terminal' (HRDC) domain. Single-molecule real-time translocation and DNA unwinding by full-length Escherichia coli RecQ and variants lacking either the HRDC or both the WH and HRDC domains was analyzed. RecQ operated under two interconvertible kinetic modes, 'slow' and 'normal', as it unwound duplex DNA and translocated on single-stranded (ss) DNA. Consistent with a crystal structure of bacterial RecQ bound to ssDNA by base stacking, abasic sites blocked RecQ unwinding. Removal of the HRDC domain eliminates the slow mode while preserving the normal mode of activity. Unexpectedly, a RecQ variant lacking both the WH and HRDC domains retains weak helicase activity. The inclusion of E. coli ssDNA-binding protein (SSB) induces a third 'fast' unwinding mode four times faster than the normal RecQ mode and enhances the overall helicase activity (affinity, rate, and processivity). SSB stimulation was, furthermore, observed in the RecQ deletion variants, including the variant missing the WH domain. Our results support a model in which RecQ and SSB have multiple interacting modes.
UPF1-like helicase grip on nucleic acids dictates processivity
Laboratoire Physique des biomolécules -
Nat Commun - 9 1 - 10.1038/s41467-018-06313-y - 2018
Helicases are molecular engines which translocate along nucleic acids (NA) to unwind double-strands or remodel NA-protein complexes. While they have an essential role in genome structure and expression, the rules dictating their processivity remain elusive. Here, we developed single-molecule methods to investigate helicase binding lifetime on DNA. We found that UPF1, a highly processive helicase central to nonsense-mediated mRNA decay (NMD), tightly holds onto NA, allowing long lasting action. Conversely, the structurally similar IGHMBP2 helicase has a short residence time. UPF1 mutants with variable grip on DNA show that grip tightness dictates helicase residence time and processivity. In addition, we discovered via functional studies that a decrease in UPF1 grip impairs NMD efficiency in vivo. Finally, we propose a three-state model with bound, sliding and unbound molecular clips, that can accurately predict the modulation of helicase processivity.
HTLV-1 Tax plugs and freezes UPF1 helicase leading to nonsense-mediated mRNA decay inhibition.
Laboratoire Physique des biomolécules - Fiorini, Francesca; Robin, Jean-Philippe; Kanaan, Joanne; Borowiak, Malgorzata; Croquette, Vincent; Le Hir, Hervé; Jalinot, Pierre; Mocquet, Vincent
Nat Commun - 9 1 - 10.1038/s41467-017-02793-6 - 2018
Up-Frameshift Suppressor 1 Homolog (UPF1) is a key factor for nonsense-mediated mRNA decay (NMD), a cellular process that can actively degrade mRNAs. Here, we study NMD inhibition during infection by human T-cell lymphotropic virus type I (HTLV-1) and characterise the influence of the retroviral Tax factor on UPF1 activity. Tax interacts with the central helicase core domain of UPF1 and might plug the RNA channel of UPF1, reducing its affinity for nucleic acids. Furthermore, using a single-molecule approach, we show that the sequential interaction of Tax with a RNA-bound UPF1 freezes UPF1: this latter is less sensitive to the presence of ATP and shows translocation defects, highlighting the importance of this feature for NMD. These mechanistic insights reveal how HTLV-1 hijacks the central component of NMD to ensure expression of its own genome.
HTLV-1 Tax plugs and freezes UPF1 helicase leading to nonsense-mediated mRNA decay inhibition.
Laboratoire Physique des biomolécules - Fiorini, Francesca; Robin, Jean-Philippe; Kanaan, Joanne; Borowiak, Malgorzata; Croquette, Vincent; Le Hir, Hervé; Jalinot, Pierre; Mocquet, Vincent
Nat Commun - 9 1 - 10.1038/s41467-017-02793-6 - 2018
Up-Frameshift Suppressor 1 Homolog (UPF1) is a key factor for nonsense-mediated mRNA decay (NMD), a cellular process that can actively degrade mRNAs. Here, we study NMD inhibition during infection by human T-cell lymphotropic virus type I (HTLV-1) and characterise the influence of the retroviral Tax factor on UPF1 activity. Tax interacts with the central helicase core domain of UPF1 and might plug the RNA channel of UPF1, reducing its affinity for nucleic acids. Furthermore, using a single-molecule approach, we show that the sequential interaction of Tax with a RNA-bound UPF1 freezes UPF1: this latter is less sensitive to the presence of ATP and shows translocation defects, highlighting the importance of this feature for NMD. These mechanistic insights reveal how HTLV-1 hijacks the central component of NMD to ensure expression of its own genome.
Macroscale fluorescence imaging against autofluorescence under ambient light.
Laboratoire Physique des biomolécules - Zhang, Ruikang; Chouket, Raja; Plamont, Marie-Aude; Kelemen, Zsolt; Espagne, Agathe; Tebo, Alison G; Gautier, Arnaud; Gissot, Lionel; Faure, Jean-Denis; Jullien, Ludovic; Croquette, Vincent; Le Saux, Thomas
Light - 7 97 - 10.1038/s41377-018-0098-6 - 2018
Macroscale fluorescence imaging is increasingly used to observe biological samples. However, it may suffer from spectral interferences that originate from ambient light or autofluorescence of the sample or its support. In this manuscript, we built a simple and inexpensive fluorescence macroscope, which has been used to evaluate the performance of Speed OPIOM (Out of Phase Imaging after Optical Modulation), which is a reference-free dynamic contrast protocol, to selectively image reversibly photoswitchable fluorophores as labels against detrimental autofluorescence and ambient light. By tuning the intensity and radial frequency of the modulated illumination to the Speed OPIOM resonance and adopting a phase-sensitive detection scheme that ensures noise rejection, we enhanced the sensitivity and the signal-to-noise ratio for fluorescence detection in blot assays by factors of 50 and 10, respectively, over direct fluorescence observation under constant illumination. Then, we overcame the strong autofluorescence of growth media that are currently used in microbiology and realized multiplexed fluorescence observation of colonies of spectrally similar fluorescent bacteria with a unique configuration of excitation and emission wavelengths. Finally, we easily discriminated fluorescent labels from the autofluorescent and reflective background in labeled leaves, even under the interference of incident light at intensities that are comparable to sunlight. The proposed approach is expected to find multiple applications, from biological assays to outdoor observations, in fluorescence macroimaging.

36 publications.