CNRS Institute of Integrative Biology

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CNRS Institute of Integrative Biology

Paris, France
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Carlier M.-F.,CNRS Institute of Integrative Biology | Shekhar S.,CNRS Institute of Integrative Biology
Nature Reviews Molecular Cell Biology | Year: 2017

Various cellular processes (including cell motility) are driven by the regulated, polarized assembly of actin filaments into distinct force-producing arrays of defined size and architecture. Branched, linear, contractile and cytosolic arrays coexist in vivo, and cells intricately control the number, length and assembly rate of filaments in these arrays. Recent in vitro and in vivo studies have revealed novel molecular mechanisms that regulate the number of filament barbed and pointed ends and their respective assembly and disassembly rates, thus defining classes of dynamically different filaments, which coexist in the same cell. We propose that a global treadmilling process, in which a steady-state amount of polymerizable actin monomers is established by the dynamics of each network, is responsible for defining the size and turnover of coexisting actin networks. Furthermore, signal-induced changes in the partitioning of actin to distinct arrays (mediated by RHO GTPases) result in the establishment of various steady-state concentrations of polymerizable monomers, thereby globally influencing the growth rate of actin filaments. © 2017 Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved.


Midonet C.,CNRS Institute of Integrative Biology | Das B.,Translational Health Science and Technology Institute | Sherratt D.J.,University of Oxford
Proceedings of the National Academy of Sciences of the United States of America | Year: 2014

As in most bacteria, topological problems arising from the circularity of the two Vibrio cholerae chromosomes, chrI and chrII, are resolved by the addition of a crossover at a specific site of each chromosome, dif, by two tyrosine recombinases, XerC and XerD. The reaction is under the control of a cell division protein, FtsK, which activates the formation of a Holliday Junction (HJ) intermediate by XerD catalysis that is resolved into product by XerC catalysis. Many plasmids and phages exploit Xer recombination for dimer resolution and for integration, respectively. In all cases so far described, they rely on an alternative recombination pathway in which XerC catalyzes the formation of a HJ independently of FtsK. This is notably the case for CTXφ, the cholera toxin phage. Here, we show that in contrast, integration of TLCφ, a toxin-linked cryptic satellite phage that is almost always found integrated at the chrI dif site before CTXφ, depends on the formation of a HJ by XerD catalysis, which is then resolved by XerC catalysis. The reaction nevertheless escapes the normal cellular control exerted by FtsK on XerD. In addition, we show that the same reaction promotes the excision of TLCφ, along with any CTXφ copy present between dif and its left attachment site, providing a plausible mechanism for how chrI CTXφ copies can be eliminated, as occurred in the second wave of the current cholera pandemic.


Bossi L.,CNRS Institute of Integrative Biology | Figueroa-Bossi N.,CNRS Institute of Integrative Biology
Nature Reviews Microbiology | Year: 2016

Many bacterial regulatory small RNAs (sRNAs) have several mRNA targets, which places them at the centre of regulatory networks that help bacteria to adapt to environmental changes. However, different mRNA targets of any given sRNA compete with each other for binding to the sRNA; thus, depending on relative abundances and sRNA affinity, competition for regulatory sRNAs can mediate cross-regulation between bacterial mRNAs. This 'target-centric' perspective of sRNA regulation is reminiscent of the competing endogenous RNA (ceRNA) hypothesis, which posits that competition for a limited pool of microRNAs (miRNAs) in higher eukaryotes mediates cross-regulation of mRNAs. In this Opinion article, we discuss evidence that a similar network of RNA crosstalk operates in bacteria, and that this network also includes crosstalk between sRNAs and competition for RNA-binding proteins. © 2016 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.


Setif P.,CNRS Institute of Integrative Biology | Mutoh R.,Osaka University | Kurisu G.,Osaka University
Biochimica et Biophysica Acta - Bioenergetics | Year: 2017

Fast turnover of ferredoxin/Fd reduction by photosystem-I/PSI requires that it dissociates rapidly after it has been reduced by PSI:Fd intracomplex electron transfer. The rate constants of Fd dissociation from PSI have been determined by flash-absorption spectroscopy with different combinations of cyanobacterial PSIs and Fds, and different redox states of Fd and of the terminal PSI acceptor (FAFB). Newly obtained values were derived firstly from the fact that the dissociation constant between PSI and redox-inactive gallium-substituted Fd increases upon (FAFB) reduction and secondly from the characterization and elucidation of a kinetic phase following intracomplex Fd reduction to binding of oxidized Fd to PSI, a process which is rate-limited by the foregoing dissociation of reduced Fd from PSI. By reference to the complex with oxidized partners, dissociation rate constants were found to increase moderately with (FAFB) single reduction and by about one order of magnitude after electron transfer from (FAFB)− to Fd, therefore favoring turnover of Fd reduction by PSI. With Thermosynechococcus elongatus partners, values of 270, 730 and > 10000 s−1 were thus determined for (FAFB)Fdoxidized, (FAFB)− Fdoxidized and (FAFB)Fdreduced, respectively. Moreover, assuming a conservative upper limit for the association rate constant between reduced Fd and PSI, a significant negative shift of the Fd midpoint potential upon binding to PSI has been calculated (< −60 mV for Thermosynechococcus elongatus). From the present state of knowledge, the question is still open whether this redox shift is compatible with a large (> 10) equilibrium constant for intracomplex reduction of Fd from (FAFB)−. © 2017 Elsevier B.V.


Duigou S.,CNRS Institute of Integrative Biology | Boccard F.,CNRS Institute of Integrative Biology
PLoS Genetics | Year: 2017

The Escherichia coli chromosome is organized into four macrodomains (Ori, Ter, Right and Left) and two non-structured regions. This organization influences the segregation of sister chromatids, the mobility of chromosomal DNA, and the cellular localization of the chromosome. The organization of the Ter and Ori macrodomains relies on two specific systems, MatP/matS for the Ter domain and MaoP/maoS for the Ori domain, respectively. Here by constructing strains with chromosome rearrangements to reshuffle the distribution of chromosomal segments, we reveal that the difference between the non-structured regions and the Right and Left lateral macrodomains relies on their position on the chromosome. A change in the genetic location of oriC generated either by an inversion within the Ori macrodomain or by the insertion of a second oriC modifies the position of Right and Left macrodomains, as the chromosome region the closest to oriC are always non-structured while the regions further away behave as macrodomain regardless of their DNA sequence. Using fluorescent microscopy we estimated that loci belonging to a non-structured region are significantly closer to the Ori MD than loci belonging to a lateral MD. Altogether, our results suggest that the origin of replication plays a prominent role in chromosome organization in E. coli, as it determines structuring and localization of macrodomains in growing cell. © 2017 Duigou, Boccard.


Theillet F.-X.,Leibniz Institute for Molecular Pharmacology | Theillet F.-X.,CNRS Institute of Integrative Biology | Binolfi A.,Leibniz Institute for Molecular Pharmacology | Binolfi A.,Max Planck Institute of Biophysics | And 9 more authors.
Nature | Year: 2016

Intracellular aggregation of the human amyloid protein α-synuclein is causally linked to Parkinson's disease. While the isolated protein is intrinsically disordered, its native structure in mammalian cells is not known. Here we use nuclear magnetic resonance (NMR) and electron paramagnetic resonance (EPR) spectroscopy to derive atomic-resolution insights into the structure and dynamics of α-synuclein in different mammalian cell types. We show that the disordered nature of monomeric α-synuclein is stably preserved in non-neuronal and neuronal cells. Under physiological cell conditions, α-synuclein is amino-terminally acetylated and adopts conformations that are more compact than when in buffer, with residues of the aggregation-prone non-amyloid-β component (NAC) region shielded from exposure to the cytoplasm, which presumably counteracts spontaneous aggregation. These results establish that different types of crowded intracellular environments do not inherently promote α-synuclein oligomerization and, more generally, that intrinsic structural disorder is sustainable in mammalian cells. © 2016 Macmillan Publishers Limited. All rights reserved.


Valens M.,CNRS Institute of Integrative Biology | Thiel A.,CNRS Institute of Integrative Biology | Boccard F.,CNRS Institute of Integrative Biology
PLoS Genetics | Year: 2016

The Ori region of bacterial genomes is segregated early in the replication cycle of bacterial chromosomes. Consequently, Ori region positioning plays a pivotal role in chromosome dynamics. The Ori region of the E. coli chromosome is organized as a macrodomain with specific properties concerning DNA mobility, segregation of loci and long distance DNA interactions. Here, by using strains with chromosome rearrangements and DNA mobility as a read-out, we have identified the MaoP/maoS system responsible for constraining DNA mobility in the Ori region and limiting long distance DNA interactions with other regions of the chromosome. MaoP belongs to a group of proteins conserved in the Enterobacteria that coevolved with Dam methylase including SeqA, MukBEF and MatP that are all involved in the control of chromosome conformation and segregation. Analysis of DNA rings excised from the chromosome demonstrated that the single maoS site is required in cis on the chromosome to exert its effect while MaoP can act both in cis and in trans. The position of markers in the Ori region was affected by inactivating maoP. However, the MaoP/maoS system was not sufficient for positioning the Ori region at the ¼–¾ regions of the cell. We also demonstrate that the replication and the resulting expansion of bulk DNA are localized centrally in the cell. Implications of these results for chromosome positioning and segregation in E. coli are discussed. © 2016 Valens et al.


Carlier M.-F.,CNRS Institute of Integrative Biology | Pernier J.,CNRS Institute of Integrative Biology | Montaville P.,CNRS Institute of Integrative Biology | Shekhar S.,CNRS Institute of Integrative Biology | Kuhn S.,CNRS Institute of Integrative Biology
Cellular and Molecular Life Sciences | Year: 2015

Actin cytoskeleton remodeling, which drives changes in cell shape and motility, is orchestrated by a coordinated control of polarized assembly of actin filaments. Signal responsive, membrane-bound protein machineries initiate and regulate polarized growth of actin filaments by mediating transient links with their barbed ends, which elongate from polymerizable actin monomers. The barbed end of an actin filament thus stands out as a hotspot of regulation of filament assembly. It is the target of both soluble and membrane-bound agonists as well as antagonists of filament assembly. Here, we review the molecular mechanisms by which various regulators of actin dynamics bind, synergize or compete at filament barbed ends. Two proteins can compete for the barbed end via a mutually exclusive binding scheme. Alternatively, two regulators acting individually at barbed ends may be bound together transiently to terminal actin subunits at barbed ends, leading to the displacement of one by the other. The kinetics of these reactions is a key in understanding how filament length and membrane-filament linkage are controlled. It is also essential for understanding how force is produced to shape membranes by mechano-sensitive, processive barbed end tracking machineries like formins and by WASP-Arp2/3 branched filament arrays. A combination of biochemical and biophysical approaches, including bulk solution assembly measurements using pyrenyl-actin fluorescence, single filament dynamics, single molecule fluorescence imaging and reconstituted self-organized filament assemblies, have provided mechanistic insight into the role of actin polymerization in motile processes. © 2015 The Author(s).


The natural history of tuberculosis may be tackled by various means, among which the record of molecular scars that have been registered by the Mycobacterium tuberculosis complex (MTBC) genomes transmitted from patient to patient for tens of thousands years and possibly more. Recently discovered polymorphic loci, the CRISPR sequences, are indirect witnesses of the historical phage-bacteria struggle, and may be related to the time when the ancestor of today's tubercle bacilli were environmental bacteria, i.e. before becoming intracellular parasites. In this article, we present what are CRISPRs and try to summarize almost 20 years of research results obtained using the genetic diversity of the CRISPR loci in MTBC as a perspective for studying new models. We show that the study of the diversity of CRISPR sequences, thanks to «spoligotyping», has played a great role in our global understanding of the population structure of MTBC. © 2015 Elsevier Ltd. All rights reserved.


Boussac A.,CNRS Institute of Integrative Biology | Rutherford A.W.,Imperial College London | Sugiura M.,Ehime University | Sugiura M.,Japan Science and Technology Agency
Biochimica et Biophysica Acta - Bioenergetics | Year: 2015

The site for water oxidation in Photosystem II (PSII) goes through five sequential oxidation states (S0 to S4) before O2 is evolved. It consists of a Mn4CaO5-cluster close to a redox-active tyrosine residue (YZ). Cl- is also required for enzyme activity. By using EPR spectroscopy it has been shown that both Ca2+/Sr2+ exchange and Cl-/I- exchange perturb the proportions of centers showing high (S = 5/2) and low spin (S = 1/2) forms of the S2-state. The S3-state was also found to be heterogeneous with: i) a S = 3 form that is detectable by EPR and not sensitive to near-infrared light; and ii) a form that is not EPR visible but in which Mn photochemistry occurs resulting in the formation of a (S2YZ)′ split EPR signal upon near-infrared illumination. In Sr/Cl-PSII, the high spin (S = 5/2) form of S2 shows a marked heterogeneity with a g = 4.3 form generated at low temperature that converts to a relaxed form at g = 4.9 at higher temperatures. The high spin g = 4.9 form can then progress to the EPR detectable form of S3 at temperatures as low as 180 K whereas the low spin (S = 1/2) S2-state can only advance to the S3 state at temperatures 235 K. Both of the two S2 configurations and the two S3 configurations are each shown to be in equilibrium at 235 K but not at 198 K. Since both S2 configurations are formed at 198 K, they likely arise from two specific populations of S1. The existence of heterogeneous populations in S1, S2 and S3 states may be related to the structural flexibility associated with the positioning of the oxygen O5 within the cluster highlighted in computational approaches and which has been linked to substrate exchange. These data are discussed in the context of recent in silico studies of the electron transfer pathways between the S2-state(s) and the S3-state(s). ©2015 Elsevier B.V. All rights reserved.

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