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Single-cell analysis and sorting using droplet-based microfluidics
Laboratoire Biochimie - Linas Mazutis, John Gilbert, W Lloyd Ung, David A Weitz, Andrew D Griffiths and John A Heyman
Nature Protocols - -8 870–891 - DOI: 10.1038/nprot.2013.046 - 2013
We present a droplet-based microfluidics protocol for high-throughput analysis and sorting of single cells. Compartmentalization of single cells in droplets enables the analysis of proteins released from or secreted by cells, thereby overcoming one of the major limitations of traditional flow cytometry and fluorescence-activated cell sorting. As an example of this approach, we detail a binding assay for detecting antibodies secreted from single mouse hybridoma cells. Secreted antibodies are detected after only 15 min by co-compartmentalizing single mouse hybridoma cells, a fluorescent probe and single beads coated with anti-mouse IgG antibodies in 50-pl droplets. The beads capture the secreted antibodies and, when the captured antibodies bind to the probe, the fluorescence becomes localized on the beads, generating a clearly distinguishable fluorescence signal that enables droplet sorting at ∼200 Hz as well as cell enrichment. The microfluidic system described is easily adapted for screening other intracellular, cell-surface or secreted proteins and for quantifying catalytic or regulatory activities. In order to screen ∼1 million cells, the microfluidic operations require 2–6 h; the entire process, including preparation of microfluidic devices and mammalian cells, requires 5–7 d.
Multiplex Picodroplet Digital PCR to Detect KRAS Mutations in Circulating DNA from the Plasma of Colorectal Cancer Patients
Laboratoire Biochimie - Valerie Taly, Deniz Pekin, Leonor Benhaim, Steve K. Kotsopoulos, Delphine Le Corre, Xinyu Li, Ivan Atochin, Darren R. Link, Andrew D. Griffiths, Karine Pallier, Hélène Blons, Olivier Bouche, Bruno Landi, J. Brian Hutchison, and Pierre Laurent-Puig
Clin. Chem. - 59(12) 1722-31 - DOI: 10.1373/clinchem.2013.206359 - 2013
BACKGROUND: Multiplex digital PCR (dPCR) enables noninvasive and sensitive detection of circulating tumor DNA with performance unachievable by current molecular-detection approaches. Furthermore, picodroplet dPCR facilitates simultaneous screening for multiple mutations from the same sample.

METHODS: We investigated the utility of multiplex dPCR to screen for the 7 most common mutations in codons 12 and 13 of the KRAS (Kirsten rat sarcoma viral oncogene homolog) oncogene from plasma samples of patients with metastatic colorectal cancer. Fifty plasma samples were tested from patients for whom the primary tumor biopsy tissue DNA had been characterized by quantitative PCR.

RESULTS: Tumor characterization revealed that 19 patient tumors had KRAS mutations. Multiplex dPCR analysis of the plasma DNA prepared from these samples identified 14 samples that matched the mutation identified in the tumor, 1 sample contained a different KRAS mutation, and 4 samples had no detectable mutation. Among the tumor samples that were wild type for KRAS, 2 KRAS mutations were identified in the corresponding plasma samples. Duplex dPCR (i.e., wild-type and single-mutation assay) was also used to analyze plasma samples from patients with KRAS-mutated tumors and 5 samples expected to contain the BRAF (v-raf murine sarcoma viral oncogene homolog B) V600E mutation. The results for the duplex analysis matched those for the multiplex analysis for KRAS-mutated samples and, owing to its higher sensitivity, enabled detection of 2 additional samples with low levels of KRAS-mutated DNA. All 5 samples with BRAF mutations were detected.

CONCLUSIONS: This work demonstrates the clinical utility of multiplex dPCR to screen for multiple mutations simultaneously with a sensitivity sufficient to detect mutations in circulating DNA obtained by noninvasive blood collection.
Enhanced imine synthesis in water: from surfactantmediated catalysis to host–guest mechanisms
Laboratoire Biochimie - Kamel Meguellati, Ali Fallah-Araghi, Jean-Christophe Baret, Abdeslam El Harrak, Thomas Mangeat, Carlos M. Marques, Andrew D. Griffiths and Sylvain Ladame
Chem. Comm. - 49 11332-34 - DOI: 10.1039/C3CC46461J - 2013
An environment-responsive and fluorogenic reaction is reported and used as a model system to demonstrate experimentally three mechanisms of enhanced imine synthesis in water using either surfactants (below and above their CMC) or double-stranded DNA (acting as a reaction host).
New Glycosidase Substrates for Droplet-Based Microfluidic Screening
Laboratoire Biochimie - Majdi Najah, Estelle Mayot, Putu Mahendra-Wijaya, Andrew D. Griffiths, Sylvain Ladame, and Antoine Drevelle
Anal. Chem. - 85 (20) 9807–14 - DOI: 10.1021/ac4022709 - 2013
Droplet-based microfluidics is a powerful technique allowing ultra-high-throughput screening of large libraries of enzymes or microorganisms for the selection of the most efficient variants. Most applications in droplet microfluidic screening systems use fluorogenic substrates to measure enzymatic activities with fluorescence readout. It is important, however, that there is little or no fluorophore exchange between droplets, a condition not met with most commonly employed substrates. Here we report the synthesis of fluorogenic substrates for glycosidases based on a sulfonated 7-hydroxycoumarin scaffold. We found that the presence of the sulfonate group effectively prevents leakage of the coumarin from droplets, no exchange of the sulfonated coumarins being detected over 24 h at 30 °C. The fluorescence properties of these substrates were characterized over a wide pH range, and their specificity was studied on a panel of relevant glycosidases (cellulases and xylanases) in microtiter plates. Finally, the β-d-cellobioside-6,8-difluoro-7-hydroxycoumarin-4-methanesulfonate substrate was used to assay cellobiohydrolase activity on model bacterial strains (Escherichia coli and Bacillus subtilis) in a droplet-based microfluidic format. These new substrates can be used to assay glycosidase activities in a wide pH range (4–11) and with incubation times of up to 24 h in droplet-based microfluidic systems.
Mitotic Rounding Alters Cell Geometry to Ensure Efficient Bipolar Spindle Formation
Laboratoire Biologie cellulaire systémique de la polarité et de la division - 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
Laboratoire Biologie cellulaire systémique de la polarité et de la division - 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
Laboratoire Biologie cellulaire systémique de la polarité et de la division - 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.
Cellular capsules as a tool for multicellular spheroid production and for investigating the mechanics of tumor progression in vitro
Laboratoire Colloïdes et Matériaux Divisés - K. Alessandri, Bibhu Ranjan Sarangi, V. Valérïévitch Gurchenkov, B. Sinha, T. Kissling, L. Fetler, F. Rico, Simon Scheuring, Christophe Lamaze, S. Geraldo, D. Vignjević, H. Doméjean, L. Rolland, A. Funfak, Jérôme Bibette, Nicolas Bremond, Pierre Nas
Proc. Nat. Acad. Sci. USA - vol.110(n°37) 14843–48 - DOI: 10.1073/pnas.1309482110 - 2013
Deciphering the multifactorial determinants of tumor progression requires standardized high-throughput preparation of 3D in vitro cellular assays. We present a simple microfluidic method based on the encapsulation and growth of cells inside permeable, elastic, hollow microspheres. We show that this approach enables mass production of size-controlled multicellular spheroids. Due to their geometry and elasticity, these microcapsules can uniquely serve as quantitative mechanical sensors to measure the pressure exerted by the expanding spheroid. By monitoring the growth of individual encapsulated spheroids after confluence, we dissect the dynamics of pressure buildup toward a steady-state value, consistent with the concept of homeostatic pressure. In turn, these confining conditions are observed to increase the cellular density and affect the cellular organization of the spheroid. Postconfluent spheroids exhibit a necrotic core cemented by a blend of extracellular material and surrounded by a rim of proliferating hypermotile cells. By performing invasion assays in a collagen matrix, we report that peripheral cells readily escape preconfined spheroids and cell–cell cohesivity is maintained for freely growing spheroids, suggesting that mechanical cues from the surrounding microenvironment may trigger cell invasion from a growing tumor. Overall, our technology offers a unique avenue to produce in vitro cell-based assays useful for developing new anticancer therapies and to investigate the interplay between mechanics and growth in tumor evolution.
Influence of Size, Surface Coating and Fine Chemical Composition on the In Vitro Reactivity and In Vivo Biodistribution of Lipid Nanocapsules Versus Lipid Nanoemulsions in Cancer Models
Laboratoire Colloïdes et Matériaux Divisés - Samuli Hirsjärvi, Sandrine Dufort, Julien Gravier, Isabelle Texier, Qiao Yan, Jérome Bibette, Lucie Sancey, Véronique Josserand, Catherine Passirani, Jean-Pierre Benoit and Jean-Luc Coll
Nanomed. Nanotech. Biol. and Med. - 9(3) 375-87 - DOI: 10.1016/j.nano.2012.08.005 - 2013
Lipid nanocapsules (LNCs) and lipid nanoemulsions (LNEs) are biomimetic synthetic nanocarriers. Their in vitro and in vivo performance was evaluated as a function of their size (25, 50 and 100 nm) and the surface PEG chain length. Analysis methods included complement activation test, particle uptake in macrophage and HEK293(β3) cells and biodistribution studies with tumor-grafted mice by fluorescence imaging. A particular attention was paid to keep the concentration of each nanocarrier and to the amount of fluorescent dye in comparable conditions between the in vitro and in vivo studies. Under these conditions, no significant differences were found among the three tested particle sizes and the two nanocarrier types. Longer PEG chains on the LNE surface provided better stealth properties, whereas PEG modification on the LNC formulations inhibited the production of stable nanocarriers. Passive accumulation of LNCs and LNEs in different tumor types depended on the degree of tumor vascularization.
A review of microfabrication and hydrogel engineering for micro-organs on chips
Laboratoire Macromolécules et Microsystèmes en Biologie et Médecine - Verhulsel M, Vignes M, Descroix S, Malaquin L, Vignjevic DM, Viovy JL
Biomaterials - 35(6) 1816-32 - DOI:10.1016/j.biomaterials.2013.11.021 - 2013
This review highlights recent trends towards the development of in vitro multicellular systems with definite architectures, or “organs on chips”. First, the chemical composition and mechanical properties of the scaffold have to be consistent with the anatomical environment in vivo. In this perspective, the flourishing interest in hydrogels as cellular substrates has highlighted the main parameters directing cell differentiation that need to be recapitulated in artificial matrix. Another scaffold requirement is to act as a template to guide tissue morphogenesis. Therefore specific microfabrication techniques are required to spatially pattern the environment at microscale. 2D patterning is particularly efficient for organizing planar polarized cell types such as endothelial cells or neurons. However, most organs are characterized by specific sub units organized in three dimensions at the cellular level. The reproduction of such 3D patterns in vitro is necessary for cells to fully differentiate, assemble and coordinate to form a coherent micro-tissue. These physiological microstructures are often integrated in microfluidic devices whose controlled environments provide the cell culture with more life-like conditions than traditional cell culture methods. Such systems have a wide range of applications, for fundamental research, as tools to accelerate drug development and testing, and finally, for regenerative medicine.
A low cost and high throughput magnetic bead-based immuno-agglutination assay in confined droplets
Laboratoire Macromolécules et Microsystèmes en Biologie et Médecine - Teste B, Ali-Cherif A, Viovy JL and Malaquin L
Lab. Chip - 13(12) 2344-9 - DOI: 10.1039/c3lc50353d - 2013
Although passive immuno-agglutination assays consist of one step and simple procedures, they are usually not adapted for high throughput analyses and they require expensive and bulky equipment for quantitation steps. Here we demonstrate a low cost, multimodal and high throughput immuno-agglutination assay that relies on a combination of magnetic beads (MBs), droplets microfluidics and magnetic tweezers. Antibody coated MBs were used as a capture support in the homogeneous phase. Following the immune interaction, water in oil droplets containing MBs and analytes were generated and transported in Teflon tubing. When passing in between magnetic tweezers, the MBs contained in the droplets were magnetically confined in order to enhance the agglutination rate and kinetics. When releasing the magnetic field, the internal recirculation flows in the droplet induce shear forces that favor MBs redispersion. In the presence of the analyte, the system preserves specific interactions and MBs stay in the aggregated state while in the case of a non-specific analyte, redispersion of particles occurs. The analyte quantitation procedure relies on the MBs redispersion rate within the droplet. The influence of different parameters such as magnetic field intensity, flow rate and MBs concentration on the agglutination performances have been investigated and optimized. Although the immuno-agglutination assay described in this work may not compete with enzyme linked immunosorbent assay (ELISA) in terms of sensitivity, it offers major advantages regarding the reagents consumption (analysis is performed in sub microliter droplet) and the platform cost that yields to very cheap analyses. Moreover the fully automated analysis procedure provides reproducible analyses with throughput well above those of existing technologies. We demonstrated the detection of biotinylated phosphatase alkaline in 100 nL sample volumes with an analysis rate of 300 assays per hour and a limit of detection of 100 pM.
New non-covalent strategies for stable surface treatment of thermoplastic chips
Laboratoire Macromolécules et Microsystèmes en Biologie et Médecine - Karla Perez-Toralla, Jérôme Champ, Mohamad Reza Mohamadi, Olivier Braun, Laurent Malaquin, Jean-Louis Viovy and Stéphanie Descroix
Lab. Chip - 13(22) 4409-18 - DOI: 10.1039/c3lc50888a - 2013
In order to be more extensively used outside of research laboratories, lab-on-chip technologies must be mass-produced using low-cost materials such as thermoplastics. Thermoplastics, however, are generally hydrophobic in their native state, which makes them unsuitable for direct use with biological samples in aqueous solution, and thus require surface coating. This coating should be robust, inexpensive and simple to implement, in order not to hinder the industrial advantage of thermoplastic chips. Cyclic Olefin Copolymer (COC) is a particularly appealing polymer, but it is also difficult to functionalize due to its chemical inertness. Here we introduce and compare the performance of two new approaches for COC coating. One relies on the use of a commercial triblock copolymer, Pluronic® F127. The second approach uses new copolymers synthesized by radical polymerization, and consisting of a dimethylacrylamide (DMA) backbone carrying aliphatic side chains (C22). Two DMA-C22 copolymers were synthesized with various C22/DMA ratios: DMA-S at 0.175% and DMA-M at 0.35%. Different physicochemical properties of the polymers such as critical micellar concentration (CMC), water contact angle, electroosmosis were investigated. Coated COC chips were then tested for their ability to reduce the adsorption of proteins, microparticles, and for protein electrophoresis. For each application we found an optimal treatment protocol to considerably improve the performance of the thermoplastic chip. These treatments use physisorption in situ which requires no photografting or chemical reaction and can be performed by a simple incubation either after chip production, or just prior to use.
FISH in chips: turning microfluidic fluorescence in situ hybridization into a quantitative and clinically reliable molecular diagnosis tool
Laboratoire Macromolécules et Microsystèmes en Biologie et Médecine - Perez-Toralla Karla, Mottet Guillaume, Guneri Ezgi Tulukcuoglu, Champ Jérôme, Bidard François-Clément, Jean-Yves Pierga, Jerzy Klijanienko, Irena Draskovic, Laurent Malaquin, Jean-Louis Viovya and Stéphanie Descroix*
Lab. Chip - 15 (2015) 811-22 - DOI: 10.1039/c4lc01059k - 2013
Microfluidic systems bear promise to provide new powerful tools for the molecular characterization of cancer cells, in particular for the routine detection of multiple cancer biomarkers using a minute amount of the sample. However, taking miniaturized cell-based assays into the clinics requires the implementation and validation of complex biological protocols on chip, as well as the development of disposable microdevices produced at a low cost. Based on a recently developed microfluidic chip made of Cyclic Olefin Copolymer for cell immobilization with minimal dead volume and controlled shear stress, we developed a protocol performed entirely in the liquid phase, allowing the immobilization and fixation of cells and their quantitative characterization by fluorescence in situ hybridization. We demonstrated first in cell lines and then in two clinical case studies the potential of this method to perform quantitative copy number measurement and clinical scoring of the amplification of the ERBB2 gene, a decisive biomarker for the prescription of HER2+ related targeted therapies. This validation was performed in a blind protocol in two clinical case studies, in reference to the gold standard and clinically used method based on glass slides. We obtained a comparable reproducibility and a minor difference in apparent amplification, which can be corrected by internal calibration. The method thus reaches the standard of robustness needed for clinical use. The protocol can be fully automated, and its consumption of samples and DNA probes is reduced as compared to glass slide protocols by a factor of at least 10. The total duration of the assay is divided by two.
Supershear Rayleigh Waves at a Soft Interface
Laboratoire Microfluidique MEMS et nanostructures - Anne Le Goff, Pablo Cobelli, and Guillaume Lagubeau
Phys. Rev. Lett. - Vol.110 236101 - DOI: http://dx.doi.org/10.1103/PhysRevLett.110.236101 - 2013
We report on the experimental observation of waves at a liquid foam surface propagating faster than the bulk shear waves. The existence of such waves has long been debated, but the recent observation of supershear events in a geophysical context has inspired us to search for their existence in a model viscoelastic system. An optimized fast profilometry technique allows us to observe on a liquid foam surface the waves triggered by the impact of a projectile. At high impact velocity, we show that the expected subshear Rayleigh waves are accompanied by faster surface waves that can be identified as supershear Rayleigh waves.
Foam Drainage Control Using Thermocapillary Stress in a Two-Dimensional Microchamber
Laboratoire Microfluidique MEMS et nanostructures - V. Miralles, B. Selva, I. Cantat, and M.-C. Jullien
Phys. Rev. Lett. - Vol.112(23) 238302 - DOI: 10.1103/PhysRevLett.112.238302 - 2013
We investigate the drainage of a 2D microfoam in a vertical Hele-Shaw cell, and show that the Marangoni stress at the air-water interface generated by a constant temperature gradient applied in situ can be tuned to control the drainage. The temperature gradient is applied in such a way that thermocapillarity and gravity have an antagonistic effect. We characterize the drainage over time by measuring the liquid volume fraction in the cell and find that thermocapillarity can overcome the effect of gravity, effectively draining the foam towards the top of the cell, or exactly compensate it, maintaining the liquid fraction at its initial value over at least 60 s. We quantify these results by solving the mass balance in the cell, and provide insight into the interplay between gravity, thermocapillarity, and capillary pressure governing the drainage dynamics.
Electronic hybridization detection in microarray format and DNA genotyping
Laboratoire Nanobiophysiques - Antoine Blin, Ismaïl Cissé and Ulrich Bockelmann
Scientific Reports - 4(n°4194) - DOI: 10.1038/srep04194 - 2013
We describe an approach to substituting a fluorescence microarray with a surface made of an arrangement of electrolyte-gated field effect transistors. This was achieved using a dedicated blocking of non-specific interactions and comparing threshold voltage shifts of transistors exhibiting probe molecules of different base sequence. We apply the approach to detection of the 35delG mutation, which is related to non-syndromic deafness and is one of the most frequent mutations in humans. The process involves barcode sequences that are generated by Tas-PCR, a newly developed replication reaction using polymerase blocking. The barcodes are recognized by hybridization to surface attached probes and are directly detected by the semiconductor device.
Revealing the competition between peeled DNA, melting bubbles, and S-DNA during DNA overstretching using fluorescence microscopy
Laboratoire Nanobiophysiques - Graeme A. Kinga, Peter Grossa, Ulrich Bockelmannb, Mauro Modestic, Gijs J. L. Wuitea, and Erwin J. G. Petermana
Proc. Nat. Acad. Sci. USA - vol.110 (n°10) 3859–64 - DOI: 10.1073/pnas.1213676110 - 2013
Mechanical stress plays a key role in many genomic processes, such as DNA replication and transcription. The ability to predict the response of double-stranded (ds) DNA to tension is a cornerstone of understanding DNA mechanics. It is widely appreciated that torsionally relaxed dsDNA exhibits a structural transition at forces of ∼65 pN, known as overstretching, whereby the contour length of the molecule increases by ∼70%. Despite extensive investigation, the structural changes occurring in DNA during overstretching are still generating considerable debate. Three mechanisms have been proposed to account for the increase in DNA contour length during overstretching: strand unpeeling, localized base-pair breaking (yielding melting bubbles), and formation of S-DNA (strand unwinding, while base pairing is maintained). Here we show, using a combination of fluorescence microscopy and optical tweezers, that all three structures can exist, uniting the often contradictory dogmas of DNA overstretching. We visualize and distinguish strand unpeeling and melting-bubble formation using an appropriate combination of fluorescently labeled proteins, whereas remaining B-form DNA is accounted for by using specific fluorescent molecular markers. Regions of S-DNA are associated with domains where fluorescent probes do not bind. We demonstrate that the balance between the three structures of overstretched DNA is governed by both DNA topology and local DNA stability. These findings enhance our knowledge of DNA mechanics and stability, which are of fundamental importance to understanding how proteins modify the physical state of DNA.
Microfluidic device with integrated microfilter of conical-shaped holes for high efficiency and high purity capture of circulating tumor cells
Laboratoire Nanobioscience et Microsystèmes group - Yadong Tang, Jian Shi, Sisi Li, Li Wang, Yvon E. Cayre and Yong Chen
Scientific Reports - 4 (n°6052) - DOI:10.1038/srep06052 - 2013
Capture of circulating tumor cells (CTCs) from peripheral blood of cancer patients has major implications for metastatic detection and therapy analyses. Here we demonstrated a microfluidic device for high efficiency and high purity capture of CTCs. The key novelty of this approach lies on the integration of a microfilter with conical-shaped holes and a micro-injector with cross-flow components for size dependent capture of tumor cells without significant retention of non-tumor cells. Under conditions of constant flow rate, tumor cells spiked into phosphate buffered saline could be recovered and then cultured for further analyses. When tumor cells were spiked in blood of healthy donors, they could also be recovered at high efficiency and high clearance efficiency of white blood cells. When the same device was used for clinical validation, CTCs could be detected in blood samples of cancer patients but not in that of healthy donors. Finally, the capture efficiency of tumor cells is cell-type dependent but the hole size of the filter should be more closely correlated to the nuclei size of the tumor cells. Together with the advantage of easy operation, low-cost and high potential of integration, this approach offers unprecedented opportunities for metastatic detection and cancer treatment monitoring.
RecG and UvsW catalyse robust DNA rewinding critical for stalled DNA replication fork rescue
Laboratoire Physique des biomolécules - Maria Manosas, Senthil K. Perumal, Piero R. Bianco, Felix Ritort, Stephen J. Benkovic and Vincent Croquette
Nature Communications - -4 2368 - DOI: 10.1038/ncomms3368 - 2013
Helicases that both unwind and rewind DNA have central roles in DNA repair and genetic recombination. In contrast to unwinding, DNA rewinding by helicases has proved difficult to characterize biochemically because of its thermodynamically downhill nature. Here we use single-molecule assays to mechanically destabilize a DNA molecule and follow, in real time, unwinding and rewinding by two DNA repair helicases, bacteriophage T4 UvsW and Escherichia coli RecG. We find that both enzymes are robust rewinding enzymes, which can work against opposing forces as large as 35 pN, revealing their active character. The generation of work during the rewinding reaction allows them to couple rewinding to DNA unwinding and/or protein displacement reactions central to the rescue of stalled DNA replication forks. The overall results support a general mechanism for monomeric rewinding enzymes.
Cell–cell contacts confine public goods diffusion inside Pseudomonas aeruginosa clonal microcolonies
Laboratoire Physique des biomolécules - Thomas Julou, Thierry Mora, Laurent Guillon, Vincent Croquette, Isabelle J. Schal, David Bensimon, and Nicolas Desprat
Proc. Nat. Acad. Sci. USA - vol.110 (n°31) 12577–82 - DOI: 10.1073/pnas.1301428110 - 2013
he maintenance of cooperation in populations where public goods are equally accessible to all but inflict a fitness cost on individual producers is a long-standing puzzle of evolutionary biology. An example of such a scenario is the secretion of siderophores by bacteria into their environment to fetch soluble iron. In a planktonic culture, these molecules diffuse rapidly, such that the same concentration is experienced by all bacteria. However, on solid substrates, bacteria form dense and packed colonies that may alter the diffusion dynamics through cell–cell contact interactions. In Pseudomonas aeruginosa microcolonies growing on solid substrate, we found that the concentration of pyoverdine, a secreted iron chelator, is heterogeneous, with a maximum at the center of the colony. We quantitatively explain the formation of this gradient by local exchange between contacting cells rather than by global diffusion of pyoverdine. In addition, we show that this local trafficking modulates the growth rate of individual cells. Taken together, these data provide a physical basis that explains the stability of public goods production in packed colonies.

638 publications.