Université PSL



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Dansyl-peptides matrix-assisted laser desorption/ionization mass spectrometric (MALDI-MS) and tandem mass spectrometric (MS/MS) features improve the liquid chromatography/MALDI-MS/MS analysis of the proteome
Laboratoire Spectrométrie de masse biologique et protéomique - Chiappetta G, Ndiaye S, Demey E, Haddad I, Marino G, Amoresano A, Vinh J.
Rapid Commun Mass Spectrom - 24(20) :3021-32 - DOI:10.1002/rcm.4734 - 2010
Peptide tagging is a useful tool to improve matrix-assisted laser desorption/ionization tandem mass spectrometric (MALDI-MS/MS) analysis. We present a new application of the use of the dansyl chloride (DNS-Cl). DNS-Cl is a specific primary amine reagent widely used in protein biochemistry. It adds a fluorescent dimethylaminonaphthalene moiety to the molecule. The evaluation of MALDI-MS and MS/MS analyses of dansylated peptides shows that dansylation raises the ionization efficiency of the most hydrophilic species compared with the most hydrophobic ones. Consequently, higher Mascot scores and protein sequence coverage are obtained by combining MS and MS/MS data of native and tagged samples. The N-terminal DNS-Cl sulfonation improves the peptide fragmentation and promotes the generation of b-fragments allowing better peptide sequencing. In addition, we set up a labeling protocol based on the microwave chemistry. Peptide dansylation proved to be a rapid and cheap method to improve the performance of liquid chromatography (LC)/MALDI-MS/MS analysis at the proteomic scale in terms of peptide detection and sequence coverage.
Cell shape and cell division in fission yeast
Laboratoire Biologie cellulaire systémique de la polarité et de la division - Piel M, Tran PT
Curr Biol. - 17(19) :R823-7 - DOI:10.1016/j.cub.2009.08.012 - 2009
The fission yeast Schizosaccharomyces pombe has served as an important model organism for investigating cellular morphogenesis. This unicellular rod-shaped fission yeast grows by tip extension and divides by medial fission. In particular, microtubules appear to define sites of polarized cell growth by delivering cell polarity factors to the cell tips. Microtubules also position the cell nucleus at the cell middle, marking sites of cell division. Here, we review the microtubule-dependent mechanisms that regulate cell shape and cell division in fission yeast.
Simple and rapid process for single cell micro-patterning
Laboratoire Biologie cellulaire systémique de la polarité et de la division - Ammar Azioune, Marko Storch, Michel Bornens, Manuel Théry and Matthieu Piel
Lab. Chip - 9(11) :1640-2 - DOI:10.1039/b821581m - 2009
We present a simple and environmentally friendly process for cell patterning on glass covered with an ultrathin layer of poly-l-lysine-grafted-polyethylene glycol (PLL-g-PEG) by exposure to deep UV light. The patterned substrates are stable for months in the lab atmosphere before incubation with proteins. Incubation with proteins resulted in well defined patterns, with high feature resolution. RPE-1 cells seeded on fibronectin/fibrinogen-Alexa 488 patterns were constrained for days on the deep UV exposed regions. Finally, large glass plates were patterned with high homogeneity enabling the assembly of micro-patterned microplates in 96-well format.
Pushing off the walls: a mechanism of cell motility in confinement
Laboratoire Biologie cellulaire systémique de la polarité et de la division - Hawkins RJ, Piel M, Faure-Andre G, Lennon-Dumenil AM, Joanny JF, Prost J, Voituriez R
Phys. Rev. Lett. - 102(5) :58103 - DOI:10.1103/PhysRevLett.102.058103 - 2009
We propose a novel mechanism of cell motility, which relies on the coupling of actin polymerization at the cell membrane to geometric confinement. We consider a polymerizing viscoelastic cytoskeletal gel confined in a narrow channel, and show analytically that spontaneous motion occurs. Interestingly, this does not require specific adhesion with the channel walls, and yields velocities potentially larger than the polymerization velocity. The contractile activity of myosin motors is not necessary to trigger motility in this mechanism, but is shown quantitatively to increase the velocity. Our model qualitatively accounts for recent experiments which show that cells without specific adhesion proteins are motile only in confined environments while they are unable to move on a flat surface, and could help in understanding the mechanisms of cell migration in more complex confined geometries such as living tissues.
Diffusion through colloidal shells under stress
Laboratoire Colloïdes et Matériaux Divisés - J. Guery, J. Baudry, D. A. Weitz, P. M. Chaikin, J. Bibette
Phys. Rev. E - 79 :060402 - DOI:10.1103/PhysRevE.79.060402 - 2009
The permeability of solids has long been associated with a diffusive process involving activated mechanism as originally envisioned by Eyring. Tensile stress can affect the activation energy but definitive experiments of the diffusion rate of species through a stressed solid are lacking. Here we use core-shell (liquid core–solid shell) colloidal particles that are sensitive to osmotic pressure to follow the permeation of encapsulated probes at various stresses. We unambiguously show that the tensile stress applied on colloidal shells linearly reduces the local energy barrier for diffusion.
Breaking of an Emulsion under an ac Electric Field
Laboratoire Colloïdes et Matériaux Divisés - A. R. Thiam, N. Bremond, J. Bibette
Phys. Rev. Lett. - 102(18) :18304 - DOI:10.1103/PhysRevLett.102.188304 - 2009
By using microfluidic chips, we investigate the stability regarding coalescence of droplet pairs under an electric field as a function of drop separation and ac field intensity. Three different regimes are found: stable, coalescence, and partial merging. From this, we identify the two breaking scenarios of a one dimensional train of droplets: in one case the coalescence front propagates; in the other case, in which pairs belong to the partial merging regime, the coalescence front can become heterogeneous. From these findings, we can propose a destruction mechanism for a macroscopic emulsion, which includes the packing condition for which total and immediate destruction is effective.
Direct observation of twisting steps during Rad51 polymerization on DNA
Laboratoire Macromolécules et Microsystèmes en Biologie et Médecine - Arata H, Dupont A, Miné-Hattab J, Disseau L, Renodon-Cornière A, Takahashi M, Viovy JL, Cappello G
Proc. Nat. Acad. Sci. USA - 106(46) :19239-44 - DOI:10.1073/pnas.0902234106 - 2009
The human recombinase hRad51 is a key protein for the maintenance of genome integrity and for cancer development. Polymerization and depolymerization of hRad51 on duplex DNA were studied here using a new generation of magnetic tweezers, measuring DNA twist in real time with a resolution of 5°. Our results combined with earlier structural information suggest that DNA is somewhat less extended by hRad51 than by RecA (4.5 vs. 5.1 Å per base pair) and untwisted by 18.2° per base pair. They also confirm a stoichiometry of 3–4 bp per protein in the hRad51-dsDNA nucleoprotein filament. At odds with earlier claims, we show that after initial deposition of a multimeric nucleus, nucleoprotein filament growth occurs by addition/release of single proteins, involving DNA twisting steps of 65° ± 5°. Simple numeric simulations show that this mechanism is an efficient way to minimize nucleoprotein filament defects. Nucleoprotein filament growth from a preformed nucleus was observed at hRad51 concentrations down to 10 nM, whereas nucleation was never observed below 100 nM in the same buffer. This behavior can be associated with the different stoichiometries of nucleation and growth. It may be instrumental in vivo to permit efficient continuation of strand exchange by hRad51 alone while requiring additional proteins such as Rad52 for its initiation, thus keeping the latter under the strict control of regulatory pathways.
Droplet breakup in microfluidic T-junctions at small capillary numbers
Laboratoire Microfluidique, MEMS et nanostructures - M. C Jullien, M. J Tsang Mui Ching, C. Cohen, L. Ménétrier, P. Tabeling
Phys. Fluids - 21( 7) :7200-61 - DOI:10.1063/1.3170983 - 2009
We perform experimental studies of droplet breakup in microfluidic T-junctions in a range of capillary numbers lying between 4×10-4 and 2×10-1 and for two viscosity ratios of the fluids forming the dispersed and continuous phases. The present paper extends the range of capillary numbers explored by previous investigators by two orders of magnitude. We single out two different regimes of breakup. In a first regime, a gap exists between the droplet and the wall before breakup occurs. In this case, the breakup process agrees well with the analytical theory of Leshansky and Pismen [Phys. Fluids 21, 023303 (2009) ]. In a second regime, droplets keep obstructing the T-junction before breakup. Using physical arguments, we introduce a critical droplet extension for describing the breakup process in this case.
T7 RNA polymerase studied by force measurements varying cofactor concentration
Laboratoire Nanobiophysiques - P. Thomen, P. J. Lopez, U. Bockelmann, J. Guillerez, M. Dreyfus, and F. Heslot
Biophys. J. - 95(5) :2423–33 - DOI:10.1529/biophysj.107.125096 - 2009
RNA polymerases carry out the synthesis of an RNA copy from a DNA template. They move along DNA, incorporate nucleotide triphosphate (NTP) at the end of the growing RNA chain, and consume chemical energy. In a single-molecule assay using the T7 RNA polymerase, we study how a mechanical force opposing the forward motion of the enzyme along DNA affects the translocation rate. We also study the influence of nucleotide and magnesium concentration on this process. The experiment shows that the opposing mechanical force is a competitive inhibitor of nucleotide binding. Also, the single-molecule data suggest that magnesium ions are involved in a step that does not depend on the external load force. These kinetic results associated with known biochemical and mutagenic data, along with the static information obtained from crystallographic structures, shape a very coherent view of the catalytic cycle of the enzyme: translocation does not take place upon NTP binding nor upon NTP cleavage, but rather occurs after PPi release and before the next nucleotide binding event. Furthermore, the energetic bias associated with the forward motion of the enzyme is close to kT and represents only a small fraction of the free energy of nucleotide incorporation and pyrophosphate hydrolysis.
Probing DNA base pairing energy profiles using a nanopore
Laboratoire Nanobiophysiques - Virgile Viasnoff, Nicolas Chiaruttini, and Ulrich Bockelmann
European Biophysics Journal - 38(2) :263–9 - PMID:18836709 - 2009
We experimentally show that the voltage driven unzipping of long DNA duplexes by an a-hemolysin pore is sensitive to the shape of the base pairing energy landscape. Two sequences of equal global stability were investigated. The sequence with an homogeneous base pairing profile translocates faster than the one with alternative weak and strong regions. We could qualitatively account for theses observations by theoretically describing the voltage driven translocation as a biased random walk of the unzipping fork in the sequence dependent energy landscape.

410 publications.