Université PSL

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RACK1 controls IRES-mediated translation of viruses
Majzoub K., Hafirassou M.L, Meignin C., Goto A., Marzi S., Fedorova A., Verdier Y., Vinh J., Hoffmann J.A, Martin F., Baumert T.F, Schuster C., Imler J.L
Cell - 159(5) 1086-95 - DOI: 10.1016/j.cell.2014.10.041 - 2014
Fighting viral infections is hampered by the scarcity of viral targets and their variability, resulting in development of resistance. Viruses depend on cellular molecules-which are attractive alternative targets-for their life cycle, provided that they are dispensable for normal cell functions. Using the model organism Drosophila melanogaster, we identify the ribosomal protein RACK1 as a cellular factor required for infection by internal ribosome entry site (IRES)-containing viruses. We further show that RACK1 is an essential determinant for hepatitis C virus translation and infection, indicating that its function is conserved for distantly related human and fly viruses. Inhibition of RACK1 does not affect Drosophila or human cell viability and proliferation, and RACK1-silenced adult flies are viable, indicating that this protein is not essential for general translation. Our findings demonstrate a specific function for RACK1 in selective mRNA translation and uncover a target for the development of broad antiviral intervention.
Interplay of RhoA and mechanical forces in collective cell migration driven by leader cells
M. Reffay, M. C. Parrini, O. Cochet-Escartin, B. Ladoux, A. Buguin, S. Coscoy, F. Amblard, J. Camonis and P. Silberzan
Nat. Cell Biol. - 16 (2014) 217–223 - DOI:10.1038/ncb2917 - 2014
The leading front of a collectively migrating epithelium often destabilizes into multicellular migration fingers where a cell initially similar to the others becomes a leader cell while its neighbours do not alter. The determinants of these leader cells include mechanical and biochemical cues, often under the control of small GTPases. However, an accurate dynamic cartography of both mechanical and biochemical activities remains to be established. Here, by mapping the mechanical traction forces exerted on the surface by MDCK migration fingers, we show that these structures are mechanical global entities with the leader cells exerting a large traction force. Moreover, the spatial distribution of RhoA differential activity at the basal plane strikingly mirrors this force cartography. We propose that RhoA controls the development of these fingers through mechanical cues: the leader cell drags the structure and the peripheral pluricellular acto-myosin cable prevents the initiation of new leader cells.
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.
Multicolor superresolution imaging using volumetric multifocus microscopy
Bassam Hajja, Jan Wisniewskib, Mohamed El Beheiry,, Jiji Chen, Andrey Revyakin, Carl Wu, and Maxime Dahan
Proc. Nat. Acad. Sci. USA - vol.111(n°49) 17480–17485 - DOI: 10.1073/pnas.1412396111 - 2014
Single molecule-based superresolution imaging has become an essential tool in modern cell biology. Because of the limited depth of field of optical imaging systems, one of the major challenges in superresolution imaging resides in capturing the 3D nanoscale morphology of the whole cell. Despite many previous attempts to extend the application of photo-activated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM) techniques into three dimensions, effective localization depths do not typically exceed 1.2 µm. Thus, 3D imaging of whole cells (or even large organelles) still demands sequential acquisition at different axial positions and, therefore, suffers from the combined effects of out-of-focus molecule activation (increased background) and bleaching (loss of detections). Here, we present the use of multifocus microscopy for volumetric multicolor superresolution imaging. By simultaneously imaging nine different focal planes, the multifocus microscope instantaneously captures the distribution of single molecules (either fluorescent proteins or synthetic dyes) throughout an ∼4-µm-deep volume, with lateral and axial localization precisions of ∼20 and 50 nm, respectively. The capabilities of multifocus microscopy to rapidly image the 3D organization of intracellular structures are illustrated by superresolution imaging of the mammalian mitochondrial network and yeast microtubules during cell division.
Single-molecule tracking in live cells reveals distinct target-search strategies of transcription factors in the nucleus
Izeddin I, Récamier V, Bosanac L, Cissé II, Boudarene L, Dugast-Darzacq C, Proux F, Bénichou O, Voituriez R, Bensaude O, Dahan M ans Darzacq X
e-Life - -3 e02230 - DOI: 10.7554/eLife.02230 - 2014
Gene regulation relies on transcription factors (TFs) exploring the nucleus searching their targets. So far, most studies have focused on how fast TFs diffuse, underestimating the role of nuclear architecture. We implemented a single-molecule tracking assay to determine TFs dynamics. We found that c-Myc is a global explorer of the nucleus. In contrast, the positive transcription elongation factor P-TEFb is a local explorer that oversamples its environment. Consequently, each c-Myc molecule is equally available for all nuclear sites while P-TEFb reaches its targets in a position-dependent manner. Our observations are consistent with a model in which the exploration geometry of TFs is restrained by their interactions with nuclear structures and not by exclusion. The geometry-controlled kinetics of TFs target-search illustrates the influence of nuclear architecture on gene regulation, and has strong implications on how proteins react in the nucleus and how their function can be regulated in space and time.
Imaging Cse4 histone fate reveals stable residence at centromeres after de novo replacement in S phase
Jan Wisniewski, Bassam Hajj, Jiji Chen, Gaku Mizuguchi, Hua Xiao, Debbie Wei, Maxime Dahan, and Carl Wu
e-Life - -3 e02203 - DOI:10.7554/eLife.02203 - 2014
The budding yeast centromere contains Cse4, a specialized histone H3 variant. Fluorescence pulse-chase analysis of an internally tagged Cse4 reveals that it is replaced with newly synthesized molecules in S phase, remaining stably associated with centromeres thereafter. In contrast, C-terminally-tagged Cse4 is functionally impaired, showing slow cell growth, cell lethality at elevated temperatures, and extra-centromeric nuclear accumulation. Recent studies using such strains gave conflicting findings regarding the centromeric abundance and cell cycle dynamics of Cse4. Our findings indicate that internally tagged Cse4 is a better reporter of the biology of this histone variant. Furthermore, the size of centromeric Cse4 clusters was precisely mapped with a new 3D-PALM method, revealing substantial compaction during anaphase. Cse4-specific chaperone Scm3 displays steady-state, stoichiometric co-localization with Cse4 at centromeres throughout the cell cycle, while undergoing exchange with a nuclear pool. These findings suggest that a stable Cse4 nucleosome is maintained by dynamic chaperone-in-residence Scm3.
ß-amyloid induces a dying-back process and remote trans-synaptic alterations in a microfluidic-based reconstructed neuronal network
Deleglise B, Magnifico S, Duplus E, Vaur P, Soubeyre V, Belle M, Vignes M, Viovy JL, Jacotot E, Peyrin JM and Brugg B
Acta Neuropathologica - 2 145 - DOI: 10.1186/s40478-014-0145-3 - 2014
INTRODUCTION: Recent histopathological studies have shown that neurodegenerative processes in Alzheimer's and Parkinson's Disease develop along neuronal networks and that hallmarks could propagate trans-synaptically through neuronal pathways. The underlying molecular mechanisms are still unknown, and investigations have been impeded by the complexity of brain connectivity and the need for experimental models allowing a fine manipulation of the local microenvironment at the subcellular level.

RESULTS: In this study, we have grown primary cortical mouse neurons in microfluidic (μFD) devices to separate soma from axonal projections in fluidically isolated microenvironments, and applied β-amyloid (Aβ) peptides locally to the different cellular compartments. We observed that Aβ application to the somato-dendritic compartment triggers a "dying-back" process, involving caspase and NAD(+) signalling pathways, whereas exposure of the axonal/distal compartment to Aβ deposits did not induce axonal degeneration. In contrast, co-treatment with somatic sub-toxic glutamate and axonal Aβ peptide triggered axonal degeneration. To study the consequences of such subcellular/local Aβ stress at the network level we developed new μFD multi-chamber devices containing funnel-shaped micro-channels which force unidirectional axon growth and used them to recreate in vitro an oriented cortico-hippocampal pathway. Aβ application to the cortical somato-dendritic chamber leads to a rapid cortical pre-synaptic loss. This happens concomitantly with a post-synaptic hippocampal tau-phosphorylation which could be prevented by the NMDA-receptor antagonist, MK-801, before any sign of axonal and somato-dendritic cortical alteration.

CONCLUSION: Thanks to μFD-based reconstructed neuronal networks we evaluated the distant effects of local Aβ stress on neuronal subcompartments and networks. Our data indicates that distant neurotransmission modifications actively take part in the early steps of the abnormal mechanisms leading to pathology progression independently of local Aβ production. This offers new tools to decipher mechanisms underlying Braak's staging. Our data suggests that local Aβ can play a role in remote tauopathy by distant disturbance of neurotransmission, providing a putative mechanism underlying the spatiotemporal appearance of pretangles.
High Spatiotemporal Control of Spontaneous Reactions Using Ultrasound-Triggered Composite Droplets
M. Bezagu, C. Errico, V. Chaulot-Talmon, F. Monti, M. Tanter, . Tabeling, J. Cossy, T. Arseniyadis and O. Couture
JACS - 136 (20) 7205–7208 - DOI: 10.1021/ja5019354 - 2014
Achieving high spatial and temporal control over a spontaneous reaction is a particularly challenging task with potential breakthroughs in various fields of research including surface patterning and drug delivery. We report here an exceptionally effective method that allows attaining such control. This method relies on a remotely triggered ultrasound-induced release of a reactant encapsulated in a composite microdroplet of liquid perfluorohexane. More specifically, the demonstration was achieved by locally applying a focused 2.25 MHz transducer onto a microfluidic channel in which were injected composite microdroplets containing a solution of an azidocoumarin and an external flow containing a reactive alkyne.

326 publications.