Université PSL



Laboratoire :
Auteur :
Revue :
Année :


- Pour toute publication de résultats ayant reçu l’aide de l’IPGG (présence dans les locaux de l’IPGG, passage sur la plateforme technologique de l’IPGG, collaboration inter équipes IPGG, lié à une bourse doctorale ou postdoctorale IPGG, ou encore utilisation des espaces communs), il vous faut indiquer  cette phrase « Ce travail a été réalisé avec le soutien du laboratoire d’excellence Institut Pierre-Gilles de Gennes (programme Investissements d’avenir ANR-10-IDEX-0001-02 PSL et ANR-10-LABX-31). » / « This work has received the support of "Institut Pierre-Gilles de Gennes" (laboratoire d’excellence, “Investissements d’avenir” program ANR-10-IDEX-0001-02 PSL and ANR-10-LABX-31.). ».

- Pour toute publication de résultats obtenu via l'utilisation d’un équipement acheté par l’Equipex IPGG, il vous faut ajouter  la codification suivante : « ANR-10-EQPX-34 ».

Anthrax receptors position the spindle
Laboratoire Biologie cellulaire systémique de la polarité et de la division - Minc N, Piel M
Nat. Cell Biol. - 15(1) :11-3 - DOI:10.1038/ncb2664 - 2012
Oriented mitosis is essential during tissue morphogenesis. The Wnt/planar cell polarity (Wnt/PCP) pathway orients mitosis in a number of developmental systems, including dorsal epiblast cell divisions along the animal-vegetal (A-V) axis during zebrafish gastrulation. How Wnt signalling orients the mitotic plane is, however, unknown. Here we show that, in dorsal epiblast cells, anthrax toxin receptor 2a (Antxr2a) accumulates in a polarized cortical cap, which is aligned with the embryonic A-V axis and forecasts the division plane. Filamentous actin (F-actin) also forms an A-V polarized cap, which depends on Wnt/PCP and its effectors RhoA and Rock2. Antxr2a is recruited to the cap by interacting with actin. Antxr2a also interacts with RhoA and together they activate the diaphanous-related formin zDia2. Mechanistically, Antxr2a functions as a Wnt-dependent polarized determinant, which, through the action of RhoA and zDia2, exerts torque on the spindle to align it with the A-V axis.
Microfabricated devices for cell biology: all for one and one for all
Laboratoire Biologie cellulaire systémique de la polarité et de la division - Lautenschläger F, Piel M
Curr Opin Cell Biol - 25(1) :116-24 - DOI:10.1016/j.ceb.2012.10.017 - 2012
Individual cells in their native physiological states face a dynamic multi-factorial environment. This is true of both single-celled and multi-cellular organisms. A key challenge in cell biology is the design of experimental methods and specific assays to disentangle the contribution of each of the parameters governing cell behavior. After decades of studying cells cultured in Petri dishes or on glass coverslips, researchers can now benefit from a range of recent technological developments that allow them to study cells in a variety of contexts, with different levels of complexity and control over a range of environmental parameters. These technologies include new types of microscopy for detailed imaging of large cell aggregates or even whole tissues, and the development of cell culture substrates, such as 3D matrices. Here we will review the contribution of a third type of tool, collectively known as microfabricated tools. Derived from techniques originally developed for microelectronics, these tools range in size from hundreds of microns to hundreds of nanometers.
Monitoring Single Cell Bioenergetics via the Coarsening of Emulsion Droplets
Laboratoire Colloïdes et Matériaux Divisés - L. Boitard, D. Cottinet, C. Kleinschmitt, N. Bremond, J. Baudry, G. Yvert, J. Bibette
Proc. Nat. Acad. Sci. USA - 109(19) :7181-6 - DOI:10.1073/pnas.1200894109 - 2012
Microorganisms are widely used to generate valuable products, and their efficiency is a major industrial focus. Bioreactors are typically composed of billions of cells, and available measurements only reflect the overall performance of the population. However, cells do not equally contribute, and process optimization would therefore benefit from monitoring this intrapopulation diversity. Such monitoring has so far remained difficult because of the inability to probe concentration changes at the single-cell level. Here, we unlock this limitation by taking advantage of the osmotically driven water flux between a droplet containing a living cell toward surrounding empty droplets, within a concentrated inverse emulsion. With proper formulation, excreted products are far more soluble within the continuous hydrophobic phase compared to initial nutrients (carbohydrates and salts). Fast diffusion of products induces an osmotic mismatch, which further relaxes due to slower diffusion of water through hydrophobic interfaces. By measuring droplet volume variations, we can deduce the metabolic activity down to isolated single cells. As a proof of concept, we present the first direct measurement of the maintenance energy of individual yeast cells. This method does not require any added probes and can in principle apply to any osmotically sensitive bioactivity, opening new routes for screening, and sorting large libraries of microorganisms and biomolecules.
A low-cost, label-free DNA detection method in lab-on-chip format based on electrohydrodynamic instabilities, with application to long-range PCR
Laboratoire Macromolécules et Microsystèmes en Biologie et Médecine - M. Lemine Youba Diakité, J. Champ, S. Descroix, L. Malaquin, F. Amblard and J.-L. Viovy
Lab. Chip - 12(22) :4738-47 - DOI:10.1039/C2LC40372B - 2012
In order to evolve from a "chip in the lab" to a "lab on a chip" paradigm, there is still a strong demand for low-cost, portable detection technologies, notably for analytes at low concentrations. Here we report a new label-free DNA detection method with direct electronic read, and apply it to long-range PCR. This method uses a nonlinear electrohydrodynamic phenomenon: when subjected to high electric fields (typically above 100 V cm(-1)), suspensions of large polyelectrolytes, such as long DNA molecules, create "giant" dynamic concentration fluctuations. These fluctuations are associated with large conductivity inhomogeneities, and we use here a contact-mode local conductivity detector to detect these fluctuations. In order to decouple the detection electronics from the high voltage excitation one, an original "doubly symmetric" floating mode battery-operated detection scheme was developed. A wavelet analysis was then applied, to unravel from the chaotic character of the electohydrodynamic instabilities a scalar signal robustly reflecting the amplification of DNA. As a first proof of concept, we measured the products of the off-chip amplification of 10 kbp DNA from lambda phage DNA, achieving a sensitivity better than 100 fg DNA in the original 50 µl sample. This corresponds to the amplification products of less than 100 initial copies of target DNA. The companion enabling technologies developed to implement this new concept, i.e. the doubly symmetric contact conductivity detection and wavelet analysis, may also find various other applications in lab-on-chips.
A programmable magnetic tweezers and droplet microfluidic platform for high throughput nanoliter multi-steps assays
Laboratoire Macromolécules et Microsystèmes en Biologie et Médecine - Ali-Cherif, S. Begolo, S. Descroix, J.L. Viovy, L.Malaquin
Angew Chem Int Ed Engl. - 51(43) :10765-9 - DOI:10.1002/anie.201203862 - 2012
Tweezing out the answer: A microfluidic device combining droplets (less than 100 nL) and magnetic particles was implemented for fast heterogeneous multiplexed assays. Magnetic tweezers can perform the manipulations required in an immunoassay (capture, extraction, mixing, and rinsing). This method was applied to the diagnosis of congenital hypothyroidism with 14 pM sensitivity. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
Obstructed breakup of slender drops in a microfluidic T-junction
Laboratoire Microfluidique MEMS et nanostructures - A.M. Leshansky, S. Afkhami, M.C Jullien, P. Tabeling
Phys. Rev. Lett. - 108(26) :4502 - DOI:10.1103/PhysRevLett.108.264502 - 2012
In this Letter we present a theoretical analysis of the droplet breakup with “permanent obstruction” in a microfluidic T junction [M.-C. Jullien et al., Phys. Fluids 21 072001 (2009)]. The proposed theory is based on a simple geometric construction for the interface shape combined with Tanner’s law for the local contact angle. The resulting scaling of the droplet deformation with time and capillary number is in excellent agreement with the results of direct numerical simulations and prior experiments. More rigorous analysis based on the lubrication approximation reveals a self-similar behavior analogous to the classical problem of a droplet spreading over a preexisting liquid film.
UV-Induced Bursting of Cell-Sized Multicomponent Lipid Vesicles in a Photosensitive Surfactant Solution
Laboratoire Nanobioscience et Microsystèmes group - A. Diguet, M. Yanagisawa, Y. J. Liu, E. Brun, S. Abadie, S. Rudiuk and D. Baigl
JACS - 134(10) :4898-904 - DOI:10.1021/ja211664f - 2012
We study the behavior of multicomponent giant unilamellar vesicles (GUVs) in the presence of AzoTAB, a photosensitive surfactant. GUVs are made of an equimolar ratio of dioleoylphosphatidylcholine (DOPC) and dipalmitoylphosphatidylcholine (DPPC) and various amounts of cholesterol (Chol), where the lipid membrane shows a phase separation into a DPPC-rich liquid-ordered (Lo) phase and a DOPC-rich liquid-disordered (Ld) phase. We find that UV illumination at 365 nm for 1 s induces the bursting of a significant fraction of the GUV population. The percentage of UV-induced disrupted vesicles, called bursting rate (Yburst), increases with an increase in [AzoTAB] and depends on [Chol] in a non-monotonous manner. Yburst decreases when [Chol] increases from 0 to 10 mol % and then increases with a further increase in [Chol], which can be correlated with the phase composition of the membrane. We show that Yburst increases with the appearance of solid domains ([Chol] = 0) or with an increase in area fraction of Lo phase (with increasing [Chol] = 10 mol %). Under our conditions (UV illumination at 365 nm for 1 s), maximal bursting efficiency (Yburst = 53%) is obtained for [AzoTAB] = 1 mM and [Chol] = 40 mol %. Finally, by restricting the illumination area, we demonstrate the first selective UV-induced bursting of individual target GUVs. These results show a new method to probe biomembrane mechanical properties using light as well as pave the way for novel strategies of light-induced drug delivery.
Polymerase Exchange During Okazaki Fragment Synthesis Observed in Living Cells
Laboratoire Physique des biomolécules - 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
Laboratoire Physique des biomolécules - 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.
Carbamazepine removal from water by dielectric barrier discharge : comparison of ex situ and in situ discharge on water
Laboratoire Procédés - Plasmas - Microsystèmes - Y. Liu, S. Mei, D. Iya-Sou, S. Cavadias, S. Ognier
Chemical Engineering And Processing - 56 :10-18 - DOI:10.1016/j.cep.2012.03.003 - 2012
Dielectric barrier discharges (DBD) were used for the degradation of carbamazepine (CBZ) in aqueous solution. The electric discharge was generated either ex situ or in situ directly on the water surface. To maintain the same ozone concentration of 40 ppm in both instances, the power injected was 0.7 W in the ex situ discharge and 12 W in the in situ discharge. The results showed 100% CBZ removal after 3 min of treatment with the ex situ discharge, while the in situ discharge only removed 81% of the CBZ after 60 min. According to measurements of UV absorbance at 285 nm and 254 nm, and of total organic carbon, the ex situ discharge system also proved to be more effective than the in situ system. The measurement of nitrogen oxides in both gaseous and liquid phases indicated that high energy in situ discharges produced a large amount of NOx. These species contributed to decreased pH and significantly slowed the CBZ oxidation rate, due to their competition with ozone. Production of NOx should be avoided when using the DBD technique for wastewater treatment.
Atmospheric Pressure Deposition of Thin Functional Coatings: Polymer Surface Patterning by DBD and Post-Discharge Polymerization of Liquid Vinyl Monomer from Surface Radicals
Laboratoire Procédés - Plasmas - Microsystèmes - J.P. Borra, A. Valt, F. Arefi-Khonsari, M. Tatoulian
Plasma Process - 9(11-12) :1104-15 - DOI:10.1002/ppap.201100210 - 2012
We present a route for grafting polyacid and polyether coatings on polymers by post-discharge polymerization of liquid vinyl monomer. Surface modifications of polymer films by Micro-Discharges in air Dielectric Barrier Discharge (MD, DBD) are depicted with sub-micrometer craters homogeneously distributed. Both the energy per MD and the power density are critical to avoid thermal film deformation. Homogeneous surface composition is related to the DBD energy density. The polymerization mechanism is depicted from yields versus DBD energy density and time of exposure to air between DBD and monomer deposition. Both parameters control the surface density of radicals and peroxides, triggering the post-DBD polymerization with 80 and 73% of monomer functionality remaining in acid and ether coatings, respectively. The effect of deposition conditions on coatings properties is shown as well as the stability of coatings upon washing.
Fabrication Of Metallic Patterns On PDMS Using Transfer Technology: Application To MRI Microcoils
Laboratoire Procédés - Plasmas - Microsystèmes - M. Couty, S. Nazeer, C. Jelita, E. Martincic, M. Woytasik, J.C. Ginefri, L. Darrasse, M. Tatoulian, E. Dufour-Gergam
MICRO & NANO LETTERS - 7(6) :519-22 - DOI:10.1049/mnl.2012.0271 - 2012
Despite the large use of this material in the microsystem field, fabrication of metallic patterns on polydimethylsiloxane (PDMS) still remains a challenge. In this Letter, we present a new process based on the transfer principle and report its application to MRI microcoils. These double-side structures are well aligned and the transfer yield is higher than 90%. The limit of the working range for these flexible coils is a bending radius of 2 mm, similar to the radius of the coil. The developed process opens a wide range of further applications for flexible devices.
Stable modification of PDMS surface properties by plasma polymerization: Innovative process of allylamine PECVD deposition and microfluidic devices sealing
Laboratoire Procédés - Plasmas - Microsystèmes - S. Massey, A. Duboin, D. Mantovani, P. Tabeling, M. Tatoulian
Surface & Coatings Technology - 206(19-20) :4303-9 - DOI:10.1016/j.surfcoat.2012.04.047 - 2012
This paper presents a new and innovative process of modification of wetting of open micro-channels involving a method to seal the microfluidic devices. Allylamine was polymerized on poly(dimethylsiloxane) (PDMS) by plasma-enhanced chemical vapour deposition (PECVD) to modify the wetting properties of open micro-channels. The sealing of the devices was done by thermal pressing. All the steps of the process were characterized by different analysis techniques to understand the mechanisms of the process and to assess the performance of the technique. Physicochemical analysis of the polymerized allylamine coatings (X-ray photoelectron spectroscopy and static water contact angle) showed that the coatings were resistant to the thermal pressing and were stable in ambient air and underwater up to 14 days of ageing, even if the water contact angle decreased during the underwater ageing. Parallel tests were undergone in microfluidic devices and the stability of ageing was tested by the production of the simple oil-in-water emulsions. All the experiments showed that this new PECVD/thermal press process is an effective way to modify the wetting properties of an open microfluidic device and includes a technique to seal effectively the system afterwards.
Tau Pathology modulates Pin1 post-translational modifications and may be relevant as biomarker
Laboratoire Spectrométrie de masse biologique et protéomique - 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
Laboratoire Spectrométrie de masse biologique et protéomique - 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
Laboratoire Spectrométrie de masse biologique et protéomique - 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.
Electrografted nanostructured platforms for click chemistry
Laboratoire Synthèse Electrochimie Imagerie et Systèmes Analytiques - Cernat A., Griveau S., Martin S., Lacroix JC., Farcau M., Sandelescu R., Bedioui F.
Electrochemistry Communications - 23 :141-4 - DOI:10.1016/j.elecom.2012.07.014 - 2012
We report on the combination of nanosphere lithography, electrodeposition and click chemistry to produce nanostructured surfaces with improved number of anchored molecules. Our strategy was developed here for the immobilization of ferrocene at glassy carbon electrode, both used as models. The nanostructuration of the surface was obtained by adsorption of 900 nm-diameter polystyrene nanosphere followed either by electropolymerization of N-(10-azidodecyl)pyrrole or by electrografting of 4-azidobenzenediazonium. In the case of poly-N-(10-azidodecyl)pyrrole, the AFM analysis of the surface after electropolymerization and removal of the nanospheres show the formation of a patterned film with holes separated of ˜900 nm. In the case of 4-azidobenzenediazonium, the electrografting proceeds similarly to bare surfaces, but with a decrease of reduction peak intensity due to the partial coverage of the electrode surface with insulating nanospheres. For both modified surfaces, the immobilization of ferrocene by copper(I) catalyzed azide-alkyne cycloaddition was clearly evidenced by cyclic voltammetry. The evaluation of the surface coverage shows that the nanostructuration leads to larger specific area for the chemical anchorage of ferrocene. Finally this procedure produces versatile functionalization of conductive materials used in various applications.
Carbon nanotubes and metalloporphyrins and metallophthalocyanines-based materials for electroanalysis
Laboratoire Synthèse Electrochimie Imagerie et Systèmes Analytiques - Zagal JH., Griveau S., Santander-Nelli M., Gutierrez Granados S., Bedioui F.
J. Porph. Phthal. - 16 :713-40 - DOI:10.1142/S1088424612300054 - 2012
We discuss here the state of the art on hybrid materials made from single (SWCNT) or multi (MWCNT) walled carbon nanotubes and MN4 complexes such as metalloporphyrins and metallophthalocyanines. The hybrid materials have been characterized by several methods such as cyclic voltammetry (CV), scanning electron microscopy (SEM), transmission electron microscopy (TEM), X-ray photoelectron spectroscopy (XPS), atomic force microscopy (AFM) and scanning electrochemical microscropy (SECM). The materials are employed for electrocatalysis of reactions such as oxygen and hydrogen peroxide reduction, nitric oxide oxidation, oxidation of thiols and other pollutants.
Quantitative and sensitive detection of rare mutations using droplet microfluidics
Laboratoire Biochimie - D. Pekin, Y. Skhiri, J.-C. Baret, D. Le Corre, L. Mazutis, C. Ben Salem, F. Millot, A. El Harrak, J.B. Hutchison, J.W. Larson, D.R. Link, P. Laurent-Puig, A.D. Griffiths and V. Taly
Lab. Chip - 11(13) :2156-66 - DOI: 10.1039/c1lc20128j - 2011
Somatic mutations within tumoral DNA can be used as highly specific biomarkers to distinguish cancer cells from their normal counterparts. These DNA biomarkers are potentially useful for the diagnosis, prognosis, treatment and follow-up of patients. In order to have the required sensitivity and specificity to detect rare tumoral DNA in stool, blood, lymph and other patient samples, a simple, sensitive and quantitative procedure to measure the ratio of mutant to wild-type genes is required. However, techniques such as dual probe TaqMan(®) assays and pyrosequencing, while quantitative, cannot detect less than ∼1% mutant genes in a background of non-mutated DNA from normal cells. Here we describe a procedure allowing the highly sensitive detection of mutated DNA in a quantitative manner within complex mixtures of DNA. The method is based on using a droplet-based microfluidic system to perform digital PCR in millions of picolitre droplets. Genomic DNA (gDNA) is compartmentalized in droplets at a concentration of less than one genome equivalent per droplet together with two TaqMan(®) probes, one specific for the mutant and the other for the wild-type DNA, which generate green and red fluorescent signals, respectively. After thermocycling, the ratio of mutant to wild-type genes is determined by counting the ratio of green to red droplets. We demonstrate the accurate and sensitive quantification of mutated KRAS oncogene in gDNA. The technique enabled the determination of mutant allelic specific imbalance (MASI) in several cancer cell-lines and the precise quantification of a mutated KRAS gene in the presence of a 200,000-fold excess of unmutated KRAS genes. The sensitivity is only limited by the number of droplets analyzed. Furthermore, by one-to-one fusion of drops containing gDNA with any one of seven different types of droplets, each containing a TaqMan(®) probe specific for a different KRAS mutation, or wild-type KRAS, and an optical code, it was possible to screen the six common mutations in KRAS codon 12 in parallel in a single experiment.
External forces control mitotic spindle positioning
Laboratoire Biologie cellulaire systémique de la polarité et de la division - Fink J, Carpi N, Betz T, Bétard A, Chebah M, Azioune A, Bornens M, Sykes C,Fetler L, Cuvelier D, Piel M*. (*corresponding authors)
Nat. Cell Biol. - 13(7) :771-8 - DOI:10.1038/ncb2269 - 2011
The response of cells to forces is essential for tissue morphogenesis and homeostasis. This response has been extensively investigated in interphase cells, but it remains unclear how forces affect dividing cells. We used a combination of micro-manipulation tools on human dividing cells to address the role of physical parameters of the micro-environment in controlling the cell division axis, a key element of tissue morphogenesis. We found that forces applied on the cell body direct spindle orientation during mitosis. We further show that external constraints induce a polarization of dynamic subcortical actin structures that correlate with spindle movements. We propose that cells divide according to cues provided by their mechanical micro-environment, aligning daughter cells with the external force field.

638 publications.