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Horseradish peroxidase nanopatterned electrodes by click chemistry: Application to the electrochemical detection of paracetamol
D. Quinton, A. Maringa, S. Griveau, T. Nyokong & F. Bedioui
Electrochemistry Communications - 31 :112-5 - https://doi.org/10.1002/elan.201300030 - 2013
Electrochemical DNA-biosensors : two-electrode set up well adapted for miniaturized devices
M. Lazerges, V. T. Tal, P. Bigey, D. Scherman & F. Bedioui
Sensors & Actuators - B 182 :510-513 - DOI:10.1016/j.snb.2013.02.098 - 2013
The proof of concept of a DNA-biosensor based on a two-electrode electrochemical setup and using a microelectrode as working electrode, well adapted for detection in microliter samples and miniaturization, is presented herein. A 23-base DNA-probe self-assembled monolayer was first formed onto a 50 μm-diameter gold surface. The microelectrode extremity was then immersed in a 50 μL DNA-target solution drop itself deposited onto a 2 mm-diameter gold counter electrode. Transduction occurs via long-range electron transfer, which is enhanced subsequently to hybridization, due to DNA-base π-stacking. Single mismatch detection of this first prototype was matched at room temperature in the nanomolar range without any optimization.
Mitotic Rounding Alters Cell Geometry to Ensure Efficient Bipolar Spindle Formation
Lancaster OM, Le Berre M, Dimitracopoulos A, Bonazzi D, Zlotek-Zlotkiewicz E, Picone R, Duke T, Piel M, Baum B
Dev Cell - 25 (3) :270-283 - DOI:10.1016/j.devcel.2013.03.014 - 2013
Accurate animal cell division requires precise coordination of changes in the structure of the microtubule-based spindle and the actin-based cell cortex. Here, we use a series of perturbation experiments to dissect the relative roles of actin, cortical mechanics, and cell shape in spindle formation. We find that, whereas the actin cortex is largely dispensable for rounding and timely mitotic progression in isolated cells, it is needed to drive rounding to enable unperturbed spindle morphogenesis under conditions of confinement. Using different methods to limit mitotic cell height, we show that a failure to round up causes defects in spindle assembly, pole splitting, and a delay in mitotic progression. These defects can be rescued by increasing microtubule lengths and therefore appear to be a direct consequence of the limited reach of mitotic centrosome-nucleated microtubules. These findings help to explain why most animal cells round up as they enter mitosis.
ESCRT-III assembly and cytokinetic abscission are induced by tension release in the intercellular bridge
Lafaurie-Janvore J, Maiuri P, Wang I, Pinot M, Manneville JB, Betz T, Balland M, Piel M
Science - 339(6127) :1625-9 - DOI:10.1126/science.1233866 - 2013
The last step of cell division, cytokinesis, produces two daughter cells that remain connected by an intercellular bridge. This state often represents the longest stage of the division process. Severing the bridge (abscission) requires a well-described series of molecular events, but the trigger for abscission remains unknown. We found that pulling forces exerted by daughter cells on the intercellular bridge appear to regulate abscission. Counterintuitively, these forces prolonged connection, whereas a release of tension induced abscission. Tension release triggered the assembly of ESCRT-III (endosomal sorting complex required for transport-III), which was followed by membrane fission. This mechanism may allow daughter cells to remain connected until they have settled in their final locations, a process potentially important for tissue organization and morphogenesis.
Triggering Cell Adhesion, Migration or Shape Change with a Dynamic Surface Coating
Van Dongen SF, Maiuri P, Marie E, Tribet C, Piel M
Adv Mater - 25(12) :1687-91 - DOI:10.1002/adma.201204474 - 2013
There's an APP for that: cell-repellent APP (azido-[polylysine-g-PEG]) is used to create substrates for spatially controlled dynamic cell adhesion. The simple addition of a functional peptide to the culture medium rapidly triggers cell adhesion. This highly accessible yet powerful technique allows diverse applications, demonstrated through tissue motility assays, patterned coculturing and triggered cell shape change.
Polymerase Exchange During Okazaki Fragment Synthesis Observed in Living Cells
G. Lia, B. Michel and J.-F. Allemand
Science - 335(6066) :328–31 - DOI:10.1126/science.1210400 - 2012
DNA replication machineries have been studied extensively, but the kinetics of action of their components remains largely unknown. We report a study of DNA synthesis during replication in living Escherichia coli cells. Using single-molecule microscopy, we observed repetitive fluorescence bursts of single polymerase IIIs (Pol IIIs), indicating polymerase exchange at the replication fork. Fluctuations in the amount of DNA-bound single-stranded DNA-binding protein (SSB) reflect different speeds for the leading- and lagging-strand DNA polymerases. Coincidence analyses of Pol III and SSB fluctuations show that they correspond to the lagging-strand synthesis and suggest the use of a new Pol III for each Okazaki fragment. Based on exchanges involving two Pol IIIs, we propose that the third polymerase in the replisome is involved in lagging-strand synthesis.
Single-molecule mechanical identification and sequencing
F. Ding, M. Manosas, M. M. Spiering, S. J. Benkovic, D. Bensimon, J.-F. Allemand and V. Croquette
Nat. Methods - 9(4) :367–72 - DOI:10.1038/nmeth.1925 - 2012
High-throughput, low-cost DNA sequencing has emerged as one of the challenges of the postgenomic era. Here we present the proof of concept for a single-molecule platform that allows DNA identification and sequencing. In contrast to most present methods, our scheme is not based on the detection of the fluorescent nucleotides but on DNA hairpin length. By pulling on magnetic beads tethered by a DNA hairpin to the surface, the molecule can be unzipped. In this open state it can hybridize with complementary oligonucleotides, which transiently block the hairpin rezipping when the pulling force is reduced. By measuring from the surface to the bead of a blocked hairpin, one can determine the position of the hybrid along the molecule with nearly single-base precision. Our approach can be used to identify a DNA fragment of known sequence in a mix of various fragments and to sequence an unknown DNA fragment by hybridization or ligation.
Tau Pathology modulates Pin1 post-translational modifications and may be relevant as biomarker
Ando K, Dourlen P, Sambo AV, Bretteville A, Bélarbi K, Vingtdeux V, Eddarkaoui S, Drobecq H, Ghestem A, Bégard S, Demey-Thomas E, Melnyk P, Smet C, Lippens G, Maurage CA, Caillet-Boudin ML, Verdier Y, Vinh J, Landrieu I, Galas MC, Blum D, Hamdane M, Serg
Neurobiol Aging - 34(3) :757-69 - DOI:10.1016/j.neurobiolaging.2012.08.004 - 2012
A prerequisite to dephosphorylation at Ser-Pro or Thr-Pro motifs is the isomerization of the imidic peptide bond preceding the proline. The peptidyl-prolyl cis/trans isomerase named Pin1 catalyzes this mechanism. Through isomerization, Pin1 regulates the function of a growing number of targets including the microtubule-associated tau protein and is supposed to be deregulated Alzheimer's disease (AD). Using proteomics, we showed that Pin1 is posttranslationally modified on more than 5 residues, comprising phosphorylation, N-acetylation, and oxidation. Although Pin1 expression remained constant, Pin1 posttranslational two-dimensional pattern was modified by tau overexpression in a tau-inducible neuroblastoma cell line, in our THY-Tau22 mouse model of tauopathy as well as in AD. Interestingly, in all of these systems, Pin1 modifications were very similar. In AD brain tissue when compared with control, Pin1 is hyperphosphorylated at serine 16 and found in the most insoluble hyperphosphorylated tau fraction of AD brain tissue. Furthermore, in all tau pathology conditions, acetylation of Pin1 may also contribute to the differences observed. In conclusion, Pin1 displays several posttranslational modifications, which are specific in tauopathies and may be useful as biomarker.
On-bead tryptic proteolysis: An attractive procedure for LC-MS/MS analysis of the Drosophila caspase 8 protein complex during immune response against bacteria
Fukuyama H & Ndiaye S, Hoffmann J, Rossier J, Liuu S, Vinh J, Verdier Y.
J Proteomics - 75(15) :4610-9 - DOI:10.1016/j.jprot.2012.03.003 - 2012
This study aims to characterize the immune response against bacteria in Drosophila melanogaster. Obtaining a description of the in vivo state of protein complexes requires their isolation as a snapshot of physiological conditions before their identification. Affinity purification with streptavidin-biotin system is widely used to address this issue. However, because of the extraordinary stability of the interaction between streptavidin and biotin, the release of biotin-labeled bait remains a challenge. We transfected Drosophila cells with a DNA construct encoding a biotin-tagged Dredd protein (ortholog of caspase 8). After affinity purification, different strategies were evaluated, and proteins analyzed by LC-MS/MS mass spectrometry. The on-bead digestion allowed the identification of more proteins associated to the Dredd complex than different protocols using competitive or acid elution. A functional assay showed that a large part of the proteins specifically identified in the Dredd sample are functionally involved in the activation of the Imd pathway. These proteins are immune response proteins (BG4, Q9VP57), stress response proteins (HSP7C, Q9VXQ5), structural proteins (TBB1, CP190), a protein biosynthesis protein (Q9W1B9) and an antioxidant system protein (SODC). Our results clearly show that on-bead digestion of proteins is an attractive procedure for the study of protein complexes by mass spectrometry. This article is part of a Special Issue entitled: Translational Proteomics.
Proteomics of cypress pollen allergens using double and triple one-dimensional electrophoresis
Shahali Y, Sutra JP, Haddad I, Vinh J, Guilloux L, Peltre G, Sénéchal H, Poncet P.
Electrophoresis - 33(3) :462-9 - DOI:10.1002/elps.20110032 - 2012
Italian cypress (Cupressus sempervirens, Cups) pollen causes allergic diseases in inhabitants of many of the cities surrounding the Mediterranean basin. However, allergens of Cups pollen are still poorly known. We introduce here a novel proteomic approach based on double one-dimensional gel electrophoresis (D1-DE) as an alternative to the 2-DE immunoblot, for the specific IgE screening of allergenic proteins from pollen extracts. The sequential one-dimensional combination of IEF and SDS-PAGE associated with IgE immunoblotting allows a versatile multiplexed immunochemical analysis of selected groups of allergens by converting a single protein spot into an extended protein band. Moreover, the method appears to be valuable for MS/MS identification, without protein purification, of a new Cups pollen allergen at 43?kDa. D1-DE immunoblotting revealed that the prevalence of IgE sensitization to this allergen belonging to the polygalacturonase (PG) family was 70% in tested French allergic patients. In subsequent triple one-dimensional gel electrophoresis, the Cups pollen PG was shown to promote lectin-based protein-protein interactions. Therefore, D1-DE could be used in routine work as a convenient alternative to 2-DE immunoblotting for the simultaneous screening of allergenic components under identical experimental conditions, thereby saving considerable amounts of sera and allergen extracts.

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579 publications.