Université PSL



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Dynamics of CRISPR-Cas9 genome interrogation in living cells
Knight SC, Xie L, Deng W, Guglielmi B, Witkowsky LB, Bosanac L, Zhang ET, El Beheiry M, Masson J-B, Dahan M, Liu Z, Doudna JA, Tjian R
Science - 350(6262) 823-6 - DOI: 10.1126/science.aac6572 - 2015
The RNA-guided CRISPR-associated protein Cas9 is used for genome editing, transcriptional modulation, and live-cell imaging. Cas9-guide RNA complexes recognize and cleave double-stranded DNA sequences on the basis of 20-nucleotide RNA-DNA complementarity, but the mechanism of target searching in mammalian cells is unknown. Here, we use single-particle tracking to visualize diffusion and chromatin binding of Cas9 in living cells. We show that three-dimensional diffusion dominates Cas9 searching in vivo, and off-target binding events are, on average, short-lived (<1 second). Searching is dependent on the local chromatin environment, with less sampling and slower movement within heterochromatin. These results reveal how the bacterial Cas9 protein interrogates mammalian genomes and navigates eukaryotic chromatin structure.
Droplets in Microchannels: Dynamical Properties of the Lubrication Film
Axel Huerre, Olivier Theodoly, Alexander M. Leshansky, Marie-Pierre Valignat, Isabelle Cantat and Marie-Caroline Jullien
Phys. Rev. Lett. - 115 (064501) 064501 - http://dx.doi.org/10.1103/PhysRevLett.115.064501 - 2015
We study the motion of droplets in a confined, micrometric geometry, by focusing on the lubrication film between a droplet and a wall. When capillary forces dominate, the lubrication film thickness evolves nonlinearly with the capillary number due to the viscous dissipation between the meniscus and the wall. However, this film may become thin enough (tens of nanometers) that intermolecular forces come into play and affect classical scalings. Our experiments yield highly resolved topographies of the shape of the interface and allow us to bring new insights into droplet dynamics in microfluidics. We report the novel characterization of two dynamical regimes as the capillary number increases: (i) at low capillary numbers, the film thickness is constant and set by the disjoining pressure, while (ii) above a critical capillary number, the interface behavior is well described by a viscous scenario. At a high surfactant concentration, structural effects lead to the formation of patterns on the interface, which can be used to trace the interface velocity, that yield direct confirmation of the boundary condition in the viscous regime.
Human Upf1 is a highly processive RNA helicase and translocase with RNP remodelling activities
Francesca Fiorini, Debjani Bagchi, Hervé Le Hir and Vincent Croquette
Nature Communications - Volume 6 (2015) Article number: 7581 - doi:10.1038/ncomms8581 - 2015
RNA helicases are implicated in most cellular RNA-dependent events. In eukaryotes however, only few have been functionally characterized. Upf1 is a RNA helicase essential for nonsense-mediated mRNA decay (NMD). Here, using magnetic tweezers and bulk assays, we observe that human Upf1 is able to translocate slowly over long single-stranded nucleic acids with a processivity >10 kb. Upf1 efficiently translocates through double-stranded structures and protein-bound sequences, demonstrating that Upf1 is an efficient ribonucleoprotein complex remodeler. Our observation of processive unwinding by an eukaryotic RNA helicase reveals that Upf1, once recruited onto NMD mRNA targets, can scan the entire transcript to irreversibly remodel the mRNP, facilitating its degradation by the NMD machinery.
Two-step local functionalization of fluoropolymer Dyneon THV microfluidic materials by scanning electrochemical microscopy combined to click reaction
Cyrine Slim, Eva Ratajovà, Sophie Griveau, Frédéric Kanoufi, David Ferraro, Camille Perréard, Fanny d’Orlyé, Anne Varenne and Fethi Bedioui
Electrochemistry Communications - 60 (5–8) - doi:10.1016/j.elecom.2015.07.019 - 2015
We propose an original two-step strategy combining the use of scanning electrochemical microscopy (SECM) and molecular chemistry via a “click” reaction (copper (I)-catalyzed azide alkyne cycloaddition (CuAAC)) to locally functionalize Dyneon THV surfaces, an attractive fluoropolymer for microfluidic applications. The first step consists in the local reduction of THV using a SECM tip to activate the surface by the creation of a locally carbonized zone and notably the formation of surface alkyne functions. This is then followed by a direct CuAAC reaction with an azide-bearing ligand for its local immobilization. The proof of concept is demonstrated by efficient local functionalization of the substrate with a fluorescent dye stable up to 6 months. Surface modifications were characterized by IR-ATR, XPS, and fluorescence microscopy.
ESCRT Machinery Is Required for Plasma Membrane Repair
Ana Joaquina Jimenez, Paolo Maiuri, Julie Lafaurie-Janvore, Séverine Divoux, Matthieu Piel and Franck Perez
Science - Vol.343(n°6174) 1247136 - DOI: 10.1126/science.1247136 - 2014
Plasma membrane damage can be triggered by numerous phenomena, and efficient repair is essential for cell survival. Endocytosis, membrane patching, or extracellular budding can be used for plasma membrane repair. We found that endosomal sorting complex required for transport (ESCRT), involved previously in membrane budding and fission, plays a critical role in plasma membrane repair. ESCRT proteins were recruited within seconds to plasma membrane wounds. Quantitative analysis of wound closure kinetics coupled to mathematical modeling suggested that ESCRTs are involved in the repair of small wounds. Real-time imaging and correlative scanning electron microscopy (SEM) identified extracellular buds and shedding at the site of ESCRT recruitment. Thus, the repair of certain wounds is ensured by ESCRT-mediated extracellular shedding of wounded portions.
Exploring the function of cell shape and size during mitosis
Clotilde Cadart, Ewa Zlotek-Zlotkiewicz, Maël Le Berre, Matthieu Piel and Helen K. Matthews
Dev Cell - Vol.29(2) 159–169 - DOI: http://dx.doi.org/10.1016/j.devcel.2014.04.009 - 2014
Dividing cells almost always adopt a spherical shape. This is true of most eukaryotic cells lacking a rigid cell wall and is observed in tissue culture and single-celled organisms, as well as in cells dividing inside tissues. While the mechanisms underlying this shape change are now well described, the functional importance of the spherical mitotic cell for the success of cell division has been thus far scarcely addressed. Here we discuss how mitotic rounding contributes to spindle assembly and positioning, as well as the potential consequences of abnormal mitotic cell shape and size on chromosome segregation, tissue growth, and cancer.
ESCRT-III assembly and cytokinetic abscission are induced by tension release in the intercellular bridge
Julie Lafaurie-Janvore, Paolo Maiuri, Irène Wang, Mathieu Pinot, Jean-Baptiste Manneville, Timo Betz, Martial Balland and Matthieu Piel
Science - Vol.339 (n°6127) 1625-1629 - DOI: 10.1126/science.1233866 - 2014
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.
Power transduction of actin filaments ratcheting in vitro against a load
Démoulin D., Carlier M.F, Bibette J. and Baudry J.
Proc. Nat. Acad. Sci. USA - Vol.111(n°50) 17845-50 - DOI: 10.1073/pnas.1414184111 - 2014
The actin cytoskeleton has the unique capability of producing pushing forces at the leading edge of motile cells without the implication of molecular motors. This phenomenon has been extensively studied theoretically, and molecular models, including the widely known Brownian ratchet, have been proposed. However, supporting experimental work is lacking, due in part to hardly accessible molecular length scales. We designed an experiment to directly probe the mechanism of force generation in a setup where a population of actin filaments grows against a load applied by magnetic microparticles. The filaments, arranged in stiff bundles by fascin, are constrained to point toward the applied load. In this protrusion-like geometry, we are able to directly measure the velocity of filament elongation and its dependence on force. Using numerical simulations, we provide evidence that our experimental data are consistent with a Brownian ratchet-based model. We further demonstrate the existence of a force regime far below stalling where the mechanical power transduced by the ratcheting filaments to the load is maximal. The actin machinery in migrating cells may tune the number of filaments at the leading edge to work in this force regime.
Single-molecule dynamics of enhanceosome assembly in embryonic stem cells
Chen J., Zhang Z., Li L., Chen B.C, Revyakin A., Hajj B., Legant W., Dahan M., Lionnet T., Betzig E., Tjian R. and Liu Z.
Cell - 156(6) 1274-85 - DOI: 10.1016/j.cell.2014.01.062 - 2014
Enhancer-binding pluripotency regulators (Sox2 and Oct4) play a seminal role in embryonic stem (ES) cell-specific gene regulation. Here, we combine in vivo and in vitro single-molecule imaging, transcription factor (TF) mutagenesis, and ChIP-exo mapping to determine how TFs dynamically search for and assemble on their cognate DNA target sites. We find that enhanceosome assembly is hierarchically ordered with kinetically favored Sox2 engaging the target DNA first, followed by assisted binding of Oct4. Sox2/Oct4 follow a trial-and-error sampling mechanism involving 84-97 events of 3D diffusion (3.3-3.7 s) interspersed with brief nonspecific collisions (0.75-0.9 s) before acquiring and dwelling at specific target DNA (12.0-14.6 s). Sox2 employs a 3D diffusion-dominated search mode facilitated by 1D sliding along open DNA to efficiently locate targets. Our findings also reveal fundamental aspects of gene and developmental regulation by fine-tuning TF dynamics and influence of the epigenome on target search parameters.
Magneto-fluorescent core-shell supernanoparticles
Ou Chen, Lars Riedemann, Fred Etoc, Hendrik Herrmann, Mathieu Coppey, Mariya Barch, Christian T. Farrar, Jing Zhao, Oliver T. Bruns, He Wei, Peng Guo, Jian Cui, Russ Jensen, Yue Chen, Daniel K. Harris, Jose M. Cordero, Zhongwu Wang, Alan Jasanoff, Dai Fu
Nature Communications - 5 Article number:5093 - DOI:10.1038/ncomms6093 - 2014
Magneto-fluorescent particles have been recognized as an emerging class of materials that exhibit great potential in advanced applications. However, synthesizing such magneto-fluorescent nanomaterials that simultaneously exhibit uniform and tunable sizes, high magnetic content loading, maximized fluorophore coverage at the surface and a versatile surface functionality has proven challenging. Here we report a simple approach for co-assembling magnetic nanoparticles with fluorescent quantum dots to form colloidal magneto-fluorescent supernanoparticles. Importantly, these supernanoparticles exhibit a superstructure consisting of a close-packed magnetic nanoparticle ‘core’, which is fully surrounded by a ‘shell’ of fluorescent quantum dots. A thin layer of ​silica coating provides high colloidal stability and biocompatibility, and a versatile surface functionality. We demonstrate that after surface pegylation, these ​silica-coated magneto-fluorescent supernanoparticles can be magnetically manipulated inside living cells while being optically tracked. Moreover, our ​silica-coated magneto-fluorescent supernanoparticles can also serve as an in vivo multi-photon and magnetic resonance dual-modal imaging probe.

278 publications.