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Photosensitive Polyamines for High-Performance Photocontrol of DNA Higher-Order Structure Venancio-Marques
Venancio-Marques, Anna, Bergen Anna, Rossi-Gendron Caroline, Rudiuk Sergii, and Baigl Damien
American Chemical Society Nano (ACS Nano) - Volume 8 (4) 3654–3663 - DOI: 10.1021/nn500266b - 2014
Polyamines are small, ubiquitous, positively charged molecules that play an essential role in numerous biological processes such as DNA packaging, gene regulation, neuron activity, and cell proliferation. Here, we synthesize the first series of photosensitive polyamines (PPAs) and demonstrate their ability to photoreversibly control nanoscale DNA higher-order structure with high efficiency. We show with fluorescence microscopy imaging that the efficiency of the PPAs as DNA-compacting agents is directly correlated to their molecular charge. Micromolar concentration of the most efficient molecule described here, a PPA containing three charges at neutral pH, compacts DNA molecules from a few kilobase pairs to a few hundred kilobase pairs, while subsequent 3 min UV illuminations at 365 nm triggers complete unfolding of DNA molecules. Additional application of blue light (440 nm for 3 min) induces the refolding of DNA into the compact state. Atomic force microscopy reveals that the compaction involves a global folding of the whole DNA molecule, whereas UV-induced unfolding is a modification initiated from the periphery of the compacted DNA, resulting in the occurrence of intermediate flower-like structures prior to the fully unfolded state.

Keywords: polyamines; DNA compaction; photocontrol; DNA; AFM; light
RecG and UvsW catalyse robust DNA rewinding critical for stalled DNA replication fork rescue
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
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.
New Glycosidase Substrates for Droplet-Based Microfluidic Screening
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.
Droplet-based microfluidic platform for ultra-high-throughput bioprospecting of cellulolytic microorganisms
Najah M, Calbrix R, Mahendra-Wijaya IP, Beneyton T, Griffiths A.D and Drevelle A
Chem. Biol. - 21(12) 1722-32 - DOI: 10.1016/j.chembiol.2014.10.020. - 2013
Discovery of microorganisms producing enzymes that can efficiently hydrolyze cellulosic biomass is of great importance for biofuel production. To date, however, only a miniscule fraction of natural biodiversity has been tested because of the relatively low throughput of screening systems and their limitation to screening only culturable microorganisms. Here, we describe an ultra-high-throughput droplet-based microfluidic system that allowed the screening of over 100,000 cells in less than 20 min. Uncultured bacteria from a wheat stubble field were screened directly by compartmentalization of single bacteria in 20 pl droplets containing a fluorogenic cellobiohydrolase substrate. Sorting of droplets based on cellobiohydrolase activity resulted in a bacterial population with 17- and 7-fold higher cellobiohydrolase and endogluconase activity, respectively, and very different taxonomic diversity than when selected for growth on medium containing starch and carboxymethylcellulose as carbon source.
Abnormal recruitment of extracellular matrix proteins by excess Notch3ECD: a new pathomechanism in CADASIL
Monet-Leprêtre M, Haddad I, Baron-Menguy C, Fouillot-Panchal M, Riani M, Domenga-Denier V, Dussaule C, Cognat E, Vinh J and Joutel A
Brain Oxford - (Pt 6) 1830-45 - DOI: 10.1093/brain/awt092 - 2013
Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy, or CADASIL, one of the most common inherited small vessel diseases of the brain, is characterized by a progressive loss of vascular smooth muscle cells and extracellular matrix accumulation. The disease is caused by highly stereotyped mutations within the extracellular domain of the NOTCH3 receptor (Notch3(ECD)) that result in an odd number of cysteine residues. While CADASIL-associated NOTCH3 mutations differentially affect NOTCH3 receptor function and activity, they all are associated with early accumulation of Notch3(ECD)-containing aggregates in small vessels. We still lack mechanistic explanation to link NOTCH3 mutations with small vessel pathology. Herein, we hypothesized that excess Notch3(ECD) could recruit and sequester functionally important proteins within small vessels of the brain. We performed biochemical, nano-liquid chromatography-tandem mass spectrometry and immunohistochemical analyses, using cerebral and arterial tissue derived from patients with CADASIL and mouse models of CADASIL that exhibit vascular lesions in the end- and early-stage of the disease, respectively. Biochemical fractionation of brain and artery samples demonstrated that mutant Notch3(ECD) accumulates in disulphide cross-linked detergent-insoluble aggregates in mice and patients with CADASIL. Further proteomic and immunohistochemical analyses identified two functionally important extracellular matrix proteins, tissue inhibitor of metalloproteinases 3 (TIMP3) and vitronectin (VTN) that are sequestered into Notch3(ECD)-containing aggregates. Using cultured cells, we show that increased levels or aggregation of Notch3 enhances the formation of Notch3(ECD)-TIMP3 complex, promoting TIMP3 recruitment and accumulation. In turn, TIMP3 promotes complex formation including NOTCH3 and VTN. In vivo, brain vessels from mice and patients with CADASIL exhibit elevated levels of both insoluble cross-linked and soluble TIMP3 species. Moreover, reverse zymography assays show a significant elevation of TIMP3 activity in the brain vessels from mice and patients with CADASIL. Collectively, our findings lend support to a Notch3(ECD) cascade hypothesis in CADASIL disease pathology, which posits that aggregation/accumulation of Notch3(ECD) in the brain vessels is a central event, promoting the abnormal recruitment of functionally important extracellular matrix proteins that may ultimately cause multifactorial toxicity. Specifically, our results suggest a dysregulation of TIMP3 activity, which could contribute to mutant Notch3(ECD) toxicity by impairing extracellular matrix homeostasis in small vessels.
Landscape of protein–protein interactions in Drosophila immune deficiency signaling during bacterial challenge
Hidehiro Fukuyamaa, Yann Verdierb, Yongsheng Guana, Chieko Makino-Okamuraa, Victoria Shilovaa, Xi Liua, E. Maksouda, J. Matsubayashia, I. Haddadb, K. Spirohne, K. Onof, C. Hetruc, J. Rossierb, Trey Idekerf, M. Boutrose, Joëlle Vinh and Jules A. Hoffmann
Proc. Nat. Acad. Sci. USA - vol.110(n°26) 10717–22 - DOI: 10.1073/pnas.1304380110 - 2013
The Drosophila defense against pathogens largely relies on the activation of two signaling pathways: immune deficiency (IMD) and Toll. The IMD pathway is triggered mainly by Gram-negative bacteria, whereas the Toll pathway responds predominantly to Gram-positive bacteria and fungi. The activation of these pathways leads to the rapid induction of numerous NF-κB–induced immune response genes, including antimicrobial peptide genes. The IMD pathway shows significant similarities with the TNF receptor pathway. Recent evidence indicates that the IMD pathway is also activated in response to various noninfectious stimuli (i.e., inflammatory-like reactions). To gain a better understanding of the molecular machinery underlying the pleiotropic functions of this pathway, we first performed a comprehensive proteomics analysis to identify the proteins interacting with the 11 canonical members of the pathway initially identified by genetic studies. We identified 369 interacting proteins (corresponding to 291 genes) in heat-killed Escherichia coli-stimulated Drosophila S2 cells, 92% of which have human orthologs. A comparative analysis of gene ontology from fly or human gene annotation databases points to four significant common categories: (i) the NuA4, nucleosome acetyltransferase of H4, histone acetyltransferase complex, (ii) the switching defective/sucrose nonfermenting-type chromatin remodeling complex, (iii) transcription coactivator activity, and (iv) translation factor activity. Here we demonstrate that sumoylation of the IκB kinase homolog immune response-deficient 5 plays an important role in the induction of antimicrobial peptide genes through a highly conserved sumoylation consensus site during bacterial challenge. Taken together, the proteomics data presented here provide a unique avenue for a comparative functional analysis of proteins involved in innate immune reactions in flies and mammals.
Emergence of collective modes and tridimensional structures from epithelial confinement
M. Deforet, V. Hakim, H.G. Yevick, G. Duclos and P. Silberzan
Nature Communications - Vol.5 (n°3747) - DOI:10.1038/ncomms4747 - 2013
Many in vivo processes, including morphogenesis or tumour maturation, involve small populations of cells within a spatially restricted region. However, the basic mechanisms underlying the dynamics of confined cell assemblies remain largely to be deciphered and would greatly benefit from well-controlled in vitro experiments. Here we show that confluent epithelial cells cultured on finite population-sized domains, exhibit collective low-frequency radial displacement modes as well as stochastic global rotation reversals. A simple mathematical model, in which cells are described as persistent random walkers that adapt their motion to that of their neighbours, captures the essential characteristics of these breathing oscillations. As these epithelia mature, a tri-dimensional peripheral cell cord develops at the domain edge by differential extrusion, as a result of the additional degrees of freedom of the border cells. These results demonstrate that epithelial confinement alone can induce morphogenesis-like processes including spontaneous collective pulsations and transition from 2D to 3D.
Collective Cell Motion in an Epithelial Sheet Can Be Quantitatively Described by a Stochastic Interacting
Particle Model, Nestor Sepu lveda, Laurence Petit-jean, Olivier Cochet, Erwan Grasland-Mongrain, Pascal Silberzan and Vincent Hakim
PLoS Comp. Biol. - 10(6) e1003717 - DOI:10.1371/journal.pcbi.1003717 - 2013
Modelling the displacement of thousands of cells that move in a collective way is required for the simulation and the theoretical analysis of various biological processes. Here, we tackle this question in the controlled setting where the motion of Madin-Darby Canine Kidney (MDCK) cells in a confluent epithelium is triggered by the unmasking of free surface. We develop a simple model in which cells are described as point particles with a dynamic based on the two premises that, first, cells move in a stochastic manner and, second, tend to adapt their motion to that of their neighbors. Detailed comparison to experimental data show that the model provides a quantitatively accurate description of cell motion in the epithelium bulk at early times. In addition, inclusion of model “leader” cells with modified characteristics, accounts for the digitated shape of the interface which develops over the subsequent hours, providing that leader cells invade free surface more easily than other cells and coordinate their motion with their followers. The previously-described progression of the epithelium border is reproduced by the model and quantitatively explained.
Cellular capsules as a tool for multicellular spheroid production and for investigating the mechanics of tumor progression in vitro
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.

289 publications.